Search

In this study we hypothesized that the alleviation of neurological symptoms long after internal carotid artery (ICA) stenting may be related to sustained improvement of cerebral perfusion. Thirty-four subjects (F/M; 15/19) with >70% stenosis of a single internal carotid artery and neurological symptoms, who underwent a carotid artery stenting procedure, were studied. Brain computed tomography perfusion (CTP) imaging was performed before and 3 years after ICA stenting. The following relative variables were compared: cerebral blood flow (rCBF), cerebral blood volume (rCBV), mean transit time (rMTT), time to peak (rTTP), and permeability surface area product (rPS). A survey also was conducted to compare the patients’ clinical symptoms. Overall, we found that a trend toward rMTT decline was the only persisting change after ICA stenting. We then stratified the patients into the subgroups of 5% rMTT decline and found that those with a rMTT decline >2% reported a prominent reduction in subjective clinical symptoms such as headache, dizziness, tinnitus, blurred vision, transient blindness, a sense of gravity of the head, and pain in the eyeballs. We conclude that a shortened mean rMTT, likely reflecting improved cerebral microcirculation, underlies the improvement of neurological symptoms in patients with ICA stenosis.

Percutaneous vertebroplasty is a treatment option in vertebral compression fractures (VCF). The aim of the study was to propose the mathematical calculation of the “optimum volume” of acrylic cement filling of the vertebral body, depending on the severity of a fracture. Two hundred computed tomography (CT) scans of vertebral columns in healthy adult Caucasians were analyzed. Vertebral body width (VBW), vertebral body depth (VBD), vertebral body height (VBH), and vertebral body volume (VBV) were measured. The “optimum volume” of cement injections in mild (25% collapse) and moderate (40% collapse) VCF were calculated. We found that moving caudally from Th11 to L2, the mean values of the examined parameters increased: VBH from 22.6 to 26.0 mm, VBW from 34.0 to 39.5 mm, VBD from 28.1 to 30.9 mm, and VBV from 17.1 to 24.8 cm3. The calculated hypothetical “optimum volume” of cement injection increased from 7.4 to 10.0 cm3 in mild VCF and from 5.9 to 7.8 cm3 in moderate VCF, with some variability depending on the vertebral level and gender. These values are akin to those present in other past studies. We conclude that morphometric measurements, based on CT images, are a reliable source of practical anatomical savvy, which may be of help in spine surgery.

The aim of the study was to assess blood pressure-subarachnoid space (BP-SAS) width coupling properties using time-frequency bispectral analysis based on wavelet transforms during handgrip and cold tests. The experiments were performed on a group of 16 healthy subjects (F/M; 7/9) of the mean age 27.2 ± 6.8 years and body mass index of 23.8 ± 4.1 kg/m

The objective of our study was to estimate the seroprevalence of six pathogens transmitted by ticks in HIV-infected persons and blood donors in Poland (B. burgdorferi s.l., A. phagocytophilum, Ehrlichia spp., Babesia spp., Rickettsia spp. Bartonella henselae) to assess the frequency of exposure to such microorganisms in immunocompetent and immunocompromised individuals in endemic regions for I. ricinus ticks. Serum samples were collected from 227 HIV-infected patients and 199 blood donors. All samples were analyzed for antibodies against six tick-borne pathogens and seroprevalence rates were statistically compared between two tested group as well as age, sex and lymphocyte T CD4+ level in HIV infected patients. The seroprevalence of tick-borne infections in HIV-infected patients is higher than that of the healthy population in Poland, although no association between serological status of patients and lymphocyte CD4+ T cell level has been observed. The frequency of tick-borne coinfections and doubtful results of serological tests were significantly higher in HIV-positive individuals. In Poland, the possibility of tick-borne diseases transmission with blood is rather negligible.

This study seeks to define the role of predictive values of the motor speed, inhibition control, and fluid and crystallized intelligence in estimating the cortical thickness in healthy elderly. Forty-six older healthy subjects (37 women, 9 men) over 60 years of age were included in the study. The participants were examined on 3.0 T MRI scanners. The protocol included standard anatomical sequences, to exclude brain pathology, and a high-resolution T1-weighted sequence used to estimate the cortical thickness. The neuropsychological protocol included fluid intelligence assessment (Raven Progressive Matrices), crystalized intelligence assessment (information or vocabulary subtest of the Wechsler Adult Intelligence Scale-Revised (WAIS-R)), and executive functioning (Color Traits Test). The findings unraveled several interdependencies. The higher the intelligence, the thicker was the grey matter in nine regions of both hemispheres, but also some paradoxical reversed associations were found in four areas; all of them were localized along different sections of the cingulate gyrus in both hemispheres. An inverse association was found between crystallized intelligence and the thickness of the pars opecularis of the right hemisphere. The better the executive functioning, the thicker was the grey matter of a given region. The better the motor performance, the thicker was the grey matter of the rostral middle frontal area of the left hemisphere and the lingual gyrus of both hemispheres. In conclusion, the associations unraveled demonstrate that the neural mechanisms underlying healthy aging are complex and heterogenic across different cognitive domains and neuroanatomical regions. No brain aging theory seems to provide a suitable interpretative framework for all the results. A novel, more integrative approach to the brain aging should be considered.

The infection with more than one hepatitis C virus (HCV) genotype especially in subjects with a high risk of multiple HCV exposures has been demonstrated. The role of HCV mixed-genotype infection in viral persistence and treatment effect is not fully understood. The prevalence of such infection varies greatly depending on the technique used for genotype determination and studied population. Next-generation sequencing (NGS) which is suitable for extensive analysis of complex viral populations is a method of choice for studying mixed infections. The aim of the present study was to determine the prevalence of mixed-genotype HCV infections in the Polish seronegative, HCV-RNA-positive blood donors (n = 76). Two-step PCR was used for amplification of 5'-UTR of HCV. Using pyrosequencing altogether, 381,063 reads were obtained. The raw reads were trimmed and subjected to similarity analysis against the entire unfiltered NCBI nt database. Results obtained from NGS were compared with the standard genotyping. One (1.3%) mixed-genotype [3a, 2989 reads (94.8%); 1b, 164 reads (5.2%)] infection was found in a sample diagnosed as genotype 3a only by routine testing. Two samples were identified with different genotypes, compared to routine testing. In conclusion, NGS is a sensitive method for HCV genotyping. The prevalence of mixed-genotype HCV infections in blood donors is low.

In the cerebrospinal fluid (CSF) circulation, two components can be distinguished: bulk flow (circulation) and pulsatile flow (back and forth motion). CSF pulsatile flow is generated by both cardiac and respiratory cycles. Recent years have seen increased interest in cardiac- and respiratory-driven CSF pulsatility as an important component of cerebral homeostasis. CSF pulsatility is affected by cerebral arterial inflow and jugular outflow and potentially linked to white matter abnormalities in various diseases, such as multiple sclerosis or hypertension. In this review, we discuss the physiological mechanisms associated with CSF pulsation and its clinical significance. Finally, we explain the concept of using the oscillations of subarachnoid space width as a surrogate for CSF pulsatility.

Altres ajuts: This work was funded by grants from the National Institutes of Health (RO1AI46995 to P.G.) and the Wellcome Trust (WT104748MA to P.G.). This project has been funded in whole or in part with federal funds from the Frederick National Laboratory for Cancer Research under contract no. HHSN261200800001E (to M.C.). The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government. This research was supported in part by the Intramural Research Program of the NIH, Frederick National Lab, Center for Cancer Research. The MACS is funded primarily by the National Institute of Allergy and Infectious Diseases (NIAID), U01-AI35042 (Johns Hopkins University Bloomberg School of Public Health; Joseph Margolick, rincipal investigator [PI]), U01-AI35039 (Northwestern University; Steven Wolinsky, PI), U01-AI35040 (University of California, Los Angeles; Roger Detels and Oto Martinez, multiple principal investigators [MPI]), U01-AI35041 (University of Pittsburgh; Charles Rinaldo, PI), and UM1-AI35043 (Johns Hopkins University Bloomberg School of Public Health; Lisa Jacobson, PI). The SCOPE cohort was supported by the UCSF/Gladstone Institute of Virology and Immunology CFAR (P30 AI027763) and the CFAR Network of Integrated Systems (R24 AI067039). Additional support was provided by the Delaney AIDS Research Enterprise (DARE; AI096109 and A127966) and the amfAR Institute for HIV Cure Research (amfAR 109301). P.B. is a Jenner Investigator. I.W. and P.P. are funded by MRC Programme grant MR/K012037. The well-characterized association between HLA-B*27:05 and protection against HIV disease progression has been linked to immunodominant HLA-B*27:05-restricted CD8 + T-cell responses toward the conserved Gag KK10 (residues 263 to 272) and polymerase (Pol) KY9 (residues 901 to 909) epitopes. We studied the impact of the 3 amino acid differences between HLA-B*27:05 and the closely related HLA-B*27:02 on the HIV-specific CD8 + T-cell response hierarchy and on immune control of HIV. Genetic epidemiological data indicate that both HLA-B*27:02 and HLA-B*27:05 are associated with slower disease progression and lower viral loads. The effect of HLA-B*27:02 appeared to be consistently stronger than that of HLA-B*27:05. In contrast to HLA-B*27:05, the immunodominant HIV-specific HLA-B*27:02-restricted CD8 + T-cell response is to a Nef epitope (residues 142 to 150 [VW9]), with Pol KY9 subdominant and Gag KK10 further subdominant. This selection was driven by structural differences in the F pocket, mediated by a polymorphism between these two HLA alleles at position 81. Analysis of autologous virus sequences showed that in HLA-B*27:02-positive subjects, all three of these CD8 + T-cell responses impose selection pressure on the virus, whereas in HLA-B*27:05-positive subjects, there is no Nef VW9-mediated selection pressure. These studies demonstrate that HLA-B*27:02 mediates protection against HIV disease progression that is at least as strong as or stronger than that mediated by HLA-B*27:05. In combination with the protective Gag KK10 and Pol KY9 CD8 + T-cell responses that dominate HIV-specific CD8 + T-cell activity in HLA-B*27:05-positive subjects, a Nef VW9-specific response is additionally present and immunodominant in HLA-B*27:02-positive subjects, mediated through a polymorphism at residue 81 in the F pocket, that contributes to selection pressure against HIV. IMPORTANCE CD8 + T cells play a central role in successful control of HIV infection and have the potential also to mediate the eradication of viral reservoirs of infection. The principal means by which protective HLA class I molecules, such as HLA-B*27:05 and HLA-B*57:01, slow HIV disease progression is believed to be via the particular HIV-specific CD8 + T cell responses restricted by those alleles. We focus here on HLA-B*27:05, one of the best-characterized protective HLA molecules, and the closely related HLA-B*27:02, which differs by only 3 amino acids and which has not been well studied in relation to control of HIV infection. We show that HLA-B*27:02 is also protective against HIV disease progression, but the CD8 + T-cell immunodominance hierarchy of HLA-B*27:02 differs strikingly from that of HLA-B*27:05. These findings indicate that the immunodominant HLA-B*27:02-restricted Nef response adds to protection mediated by the Gag and Pol specificities that dominate anti-HIV CD8 + T-cell activity in HLA-B*27:05-positive subjects.

Background Risk of depression and suicide in patients on interferon remains also after the treatment, the pathogenesis of which is still unclear. We aimed to determine the influence of the PEG-IFN-α2a on tryptophan metabolism along the kynurenine pathway during treatment and up to 6 months after the end of treatment. Methods We evaluated 101 patients with chronic hepatitis C treated with PEG-IFN-α2a, and 40 controls, so as to determine the activation of indolamine 2,3-dioxygenase (IDO) and tryptophan (TRP) and their metabolites’ concentrations/levels: kynurenine (KYN), kynurenic acid (KYNA) and anthranilic acid (AA). The subjects were evaluated before and after weeks 2, 4, 8, 12, 24, 48, as well as 6 months after the end of the treatment. Results In the group of patients treated 24 weeks, six months after the end of treatment IDO activity was significantly higher compared to baseline (69.5 vs 57.2 β = 0.21 P = 0.000); TRP concentration was significantly lower compared to baseline (30.0 vs 35.6 β=-0.21 P = 0.001); KYNA concentration was significantly higher compared to baseline (37.2 nmol/L vs 29.4 nmol/L β = 0.22 P = 0.02), and AA concentration was significantly higher compered to baseline (51.0 nmol/L vs 38.4 nmol/L β = 0.22 P = 0.05) In the group of patients treated 48 weeks six months, after the end of treatment both the IDO activity and KYNA concentration were significantly higher compared to baseline (respective values – IDO: 78.8 vs 56.2 β = 0.14 P = 0.02; KYNA: 39.2 nmol/L vs 27.0 nmol/L β = 0.26 P = 0.000). Conclusions This is the first report of a prolonged activation of IDO six months after the end of PEG-IFN-α2a treatment. The clinical significance of the finding can be implicated in the pathophysiology of depressive episodes.

This study seeks to define the role of predictive values of the motor speed, inhibition control, and fluid and crystallized intelligence in estimating the cortical thickness in healthy elderly. Forty-six older healthy subjects (37 women, 9 men) over 60 years of age were included in the study. The participants were examined on 3.0 T MRI scanners. The protocol included standard anatomical sequences, to exclude brain pathology, and a high-resolution T1-weighted sequence used to estimate the cortical thickness. The neuropsychological protocol included fluid intelligence assessment (Raven Progressive Matrices), crystalized intelligence assessment (information or vocabulary subtest of the Wechsler Adult Intelligence Scale-Revised (WAIS-R)), and executive functioning (Color Traits Test). The findings unraveled several interdependencies. The higher the intelligence, the thicker was the grey matter in nine regions of both hemispheres, but also some paradoxical reversed associations were found in four areas; all of them were localized along different sections of the cingulate gyrus in both hemispheres. An inverse association was found between crystallized intelligence and the thickness of the pars opecularis of the right hemisphere. The better the executive functioning, the thicker was the grey matter of a given region. The better the motor performance, the thicker was the grey matter of the rostral middle frontal area of the left hemisphere and the lingual gyrus of both hemispheres. In conclusion, the associations unraveled demonstrate that the neural mechanisms underlying healthy aging are complex and heterogenic across different cognitive domains and neuroanatomical regions. No brain aging theory seems to provide a suitable interpretative framework for all the results. A novel, more integrative approach to the brain aging should be considered.

The aim of the study was to assess blood pressure–subarachnoid space (BP–SAS) width coupling properties using time–frequency bispectral analysis based on wavelet transforms during handgrip and cold tests. The experiments were performed on a group of 16 healthy subjects (F/M; 7/9) of the mean age 27.2 ± 6.8 years and body mass index of 23.8 ± 4.1 kg/m2. The sequence of challenges was first handgrip and then cold test. The handgrip challenge consisted of a 2-min strain, indicated by oral communication from the investigator, at 30% of maximum strength. The cold test consisted of 2 min of hand immersion to approximately wrist level in cold water of 4 °C, verified by a digital thermometer. Each test was preceded by 10 min at baseline and was followed by 10-min recovery recordings. BP and SAS were recorded simultaneously. Three 2-min stages of the procedure, baseline, test, and recovery, were analyzed. We found that BP–SAS coupling was present only at cardiac frequency, while at respiratory frequency both oscillators were uncoupled. Handgrip and cold test failed to affect BP–SAS cardiac–respiratory coupling. We showed similar handgrip and cold test cardiac bispectral coupling for individual subjects. Further studies are required to establish whether the observed intersubject variability concerning the BP–SAS coupling at cardiac frequency has any potential clinical predictive value.

The infection with more than one hepatitis C virus (HCV) genotype especially in subjects with a high risk of multiple HCV exposures has been demonstrated. The role of HCV mixed-genotype infection in viral persistence and treatment effect is not fully understood. The prevalence of such infection varies greatly depending on the technique used for genotype determination and studied population. Next-generation sequencing (NGS) which is suitable for extensive analysis of complex viral populations is a method of choice for studying mixed infections. The aim of the present study was to determine the prevalence of mixed-genotype HCV infections in the Polish seronegative, HCV-RNA-positive blood donors (n = 76). Two-step PCR was used for amplification of 5′-UTR of HCV. Using pyrosequencing altogether, 381,063 reads were obtained. The raw reads were trimmed and subjected to similarity analysis against the entire unfiltered NCBI nt database. Results obtained from NGS were compared with the standard genotyping. One (1.3%) mixed-genotype [3a, 2989 reads (94.8%); 1b, 164 reads (5.2%)] infection was found in a sample diagnosed as genotype 3a only by routine testing. Two samples were identified with different genotypes, compared to routine testing. In conclusion, NGS is a sensitive method for HCV genotyping. The prevalence of mixed-genotype HCV infections in blood donors is low.

In the cerebrospinal fluid (CSF) circulation, two components can be distinguished: bulk flow (circulation) and pulsatile flow (back and forth motion). CSF pulsatile flow is generated by both cardiac and respiratory cycles. Recent years have seen increased interest in cardiac- and respiratory-driven CSF pulsatility as an important component of cerebral homeostasis. CSF pulsatility is affected by cerebral arterial inflow and jugular outflow and potentially linked to white matter abnormalities in various diseases, such as multiple sclerosis or hypertension. In this review, we discuss the physiological mechanisms associated with CSF pulsation and its clinical significance. Finally, we explain the concept of using the oscillations of subarachnoid space width as a surrogate for CSF pulsatility.

BackgroundEncephalitis is a severe neurological syndrome usually caused by viruses. Despite significant progress in diagnostic techniques, the causative agent remains unidentified in the majority of cases. The aim of the present study was to test an alternative approach for the detection of putative pathogens in encephalitis using next-generation sequencing (NGS).MethodsRNA was extracted from cerebrospinal fluid (CSF) from a 60-year-old male patient with encephalitis and subjected to isothermal linear nucleic acid amplification (Ribo-SPIA, NuGen) followed by next-generation sequencing using MiSeq (Illumina) system and metagenomics data analysis.ResultsThe sequencing run yielded 1,578,856 reads overall and 2579 reads matched human herpesvirus I (HHV-1) genome; the presence of this pathogen in CSF was confirmed by specific PCR. In subsequent experiments we found that the DNAse I treatment, while lowering the background of host-derived sequences, lowered the number of detectable HHV-1 sequences by a factor of 4. Furthermore, we found that the routine extraction of total RNA by the Chomczynski method could be used for identification of both DNA and RNA pathogens in typical clinical settings, as it results in retention of a significant amount of DNA.ConclusionIn summary, it seems that NGS preceded by nucleic acid amplification could supplement currently used diagnostic methods in encephalitis.

Hepatitis C virus (HCV) is the etiological agent of chronic hepatitis C and a major cause of liver cirrhosis and hepatocellular carcinoma. Only a minority of infected individuals can clear the virus spontaneously. The knowledge of the determinants of virus clearance would allow the development of effective methods preventing its further spread and optimizing treatment regimens. Viral factors associated with spontaneous virus clearance in the acute phase of infection, such as HCV genotype, virus heterogeneity, and the impact of viral proteins on the immune system have been characterized. Likewise, host genetic markers, such as the interleukin genotypes, HLA alleles, and factors affecting the T lymphocyte response appear to play an important role. Studies have revealed that natural clearance of HCV infection in the chronic phase is rare and its mechanisms are not well understood. In this review, we present the state-of-the art knowledge on the viral and host factors affecting the spontaneous elimination of HCV infection.

BACKGROUND Genetic alterations of TGF-β pathway members, including its transmembrane receptor, TGFBR1, may influence the course of HCV infection. Rs868 is a single-nucleotide polymorphism of the 3'UTR region of TGFBR1, located in a binding site for the conserved let-7/miR98 microRNA family. Previously, we demonstrated a favorable course of hepatitis C recurrence after liver transplantation in rs868 AG genotype of the transplanted liver when compared to rs868 AA. The aim of the present study was to confirm the biological effect of rs868. MATERIAL AND METHODS HepG2 cell line was transfected with luciferase vectors cloned with 3'UTR of TGFBR1 gene encompassing different rs868 alleles. Post-transplant liver biopsies from 61 patients with HCV-related end-stage liver disease were evaluated histopathologically and analyzed for the expression of TGFBR1 mRNA, let-7/miR98 microRNAs, HCV RNA load, and rs868 genotype. RESULTS Luciferase expression was significantly lower in the A allele-containing vector. TGFBR1 mRNA and HCV RNA load were correlated negatively with let-7/miR98 microRNAs and this correlation was significantly stronger for rs868 AG compared to AA genotype. A strong positive correlation was demonstrated between TGFBR1 and HCV in both genotypes. In AG heterozygotes, let-7/miR98 microRNAs showed a strong negative correlation with periportal or periseptal interface hepatitis (Ishak A score). CONCLUSIONS There is a negative correlation between let-7/miR98 microRNAs and HCV viral load and TGFBR1 mRNA after liver transplantation. In the rs868 AG heterozygotes, this correlation was stronger and there was a negative correlation between let-7/miR98 and Ishak A score, which is in concordance with the previously demonstrated protective role of this genotype in post-transplant hepatitis C recurrence.

The value of immunotherapy in lung cancer has been confirmed. However, there is a need for precise recognition of local immune response before such a treatment. The aim of our study was to analyse a cytokine profile in the lung of lung cancer patients. The soluble factors in bronchoalveolar lavage fluid (BALF) supernatans harvested from the lung affected by cancer (A) and from symmetrical lung free of tumor (B) were analyzed by Luminex assay. It was a panel of 27 cytokines with Th1, Th2, Th0 derived interleukins, chemokines, interferon and hematopoietic cytokines. The group of 35 patients with confirmed primary nonsmall cell lung cancer was investigated (n=14- adenocarcinoma, n=13- squamous cell carcinoma, n=1-large cell carcinoma, n=7- not otherwise specified). We found increased concentration of the majority of interleukins Th1 as well Th2 and growth factors and chemokines in the BALF (A) when compared with (B). However, only the differences in the concentration of proinflammatory cytokines showed significance. A significantly higher concentration of: IL-1ra, IL-6, IL-8, GM CSF, IL-12p70 in the BALF (A) when compared with (B) was found (p

A complex group of mechanisms is involved in the escape of cancer from immunosurveillance. Regulatory cells play an important role in this process. We previously documented the augmentation of regulatory T cells (Tregs) in bronchoalveolar lavage fluid (BALF) of the lung affected by cancer. The aim of this study was to assess the cytokine profile in relation to the mechanisms of immune response regulation. 35 patients with confirmed primary lung cancer were investigated. A panel of 27 cytokines was measured by Luminex assay in the BALF supernatants from: A- BALF from the lung with tumor, B- symmetrically, from “healthy” lung. For the detection of Tregs and Tregs with CTLA-4 expression the flow cytometry method with the panel of monoclonal antibodies CD4⁄CD25⁄CD127/FoxP3/CTLA-4 was used. The median value of Tregs proportion in lung A was 9.1%. So we compared the concentration of Th1, Th2, Th0 derived interleukins, chemokines, interferon and hematopoietic cytokines in the BALF with higher than 9.1% of Tregs with this with lower than 9.1% of Tregs. We found significantly increased concentration of Th2 cytokines and some chemokines in the BALF with high proportion of Tregs (Table). No relation between cytokine concentration and CTLA-4 expression on Tregs was found. Our results may lead to better define the character of immune regulation in lung cancer microenvironment. Median values of cytokine concentration in two groups, differences significant, p

Objective A high-salt diet is considered a cardiovascular risk factor; however, the mechanisms are not clear. Research suggests that gut bacteria-derived metabolites such as trimethylamine N-oxide (TMAO) are markers of cardiovascular diseases. We evaluated the effect of high salt intake on gut bacteria and their metabolites plasma level. Methods Sprague Dawley rats ages 12–14 wk were maintained on either water (controls) or 0.9% or 2% sodium chloride (NaCl) water solution (isotonic and hypertonic groups, respectively) for 2 wk. Blood plasma, urine, and stool samples were analyzed for concentrations of trimethylamine (TMA; a TMAO precursor), TMAO, and indoxyl sulfate (indole metabolite). The gut-blood barrier permeability to TMA and TMA liver clearance were assessed at baseline and after TMA intracolonic challenge test. Gut bacterial flora was analyzed with a 16S ribosomal ribonucleic acid (rRNA) gene sequence analysis. Results The isotonic and hypertonic groups showed a significantly higher plasma TMAO and significantly lower 24-hr TMAO urine excretion than the controls. However, the TMA stool level was similar between the groups. There was no significant difference between the groups in gut-blood barrier permeability and TMA liver clearance. Plasma indoxyl concentration and 24-hr urine indoxyl excretion were similar between the groups. There was a significant difference between the groups in gut bacteria composition. Conclusions High salt intake increases plasma TMAO concentration, which is associated with decreased TMAO urine excretion. Furthermore, high salt intake alters gut bacteria composition. These findings suggest that salt intake affects an interplay between gut bacteria and their host homeostasis.

Over the past two decades, many hypotheses have been put forward to explain the cause of knee osteoarthritis. Scientific reports bring up the role of adipose tissue in the activation of the inflammatory mechanisms, which is a characteristic feature of osteoarthritis natural history. Adipose tissue produces and releases cytokines, interleukins, and growth factors by means of paracrine, endocrine, and autocrine mechanisms. Hoffa's fat pad (infrapatellar adipose tissue) plays a viable role in the initiation and progression of osteoarthritis due to its role in the activation and release of pro-inflammatory mediators. The degenerative joint disease is considered an inflammatory process. Therefore, in this article we overview the importance of Hoffa's fat pad in the development and progression of osteoarthritis.

Stimulating structural and functional adaptation that improves cognitive performance in specific tasks is the major objective of therapeutic exercise training. In this review we briefly summarize central physiological mechanisms activated by exercise. We further discuss the influence of different kinds of exercise on cognitive improvement. In particular, the effects on cognitive function of aerobic endurance, resistance and respiratory exercise, and combinations thereof are presented. The accumulating evidence reinforces the position that regular aerobic, and possibly also resistance training, offers a powerful tool to cope with biologic aging of central nervous system functions. Nevertheless, the potential magnitude of cognition improvement or restrain of age-related cognition deterioration and the quantity of physical activity required to induce meaningful responses remain to be clarified.

Locus coeruleus is a critical component of the brain noradrenergic system. The brain noradrenergic system provides the neural substrate for the architecture supporting the interaction with, and navigation through, an external world complexity. Changes in locus coeruleus tonic and phasic activity and the interplay between norepinephrine and α

Hepatitis C virus (HCV) is the etiological agent of chronic hepatitis C and a major cause of liver cirrhosis and hepatocellular carcinoma. Only a minority of infected individuals can clear the virus spontaneously. The knowledge of the determinants of virus clearance would allow the development of effective methods preventing its further spread and optimizing treatment regimens. Viral factors associated with spontaneous virus clearance in the acute phase of infection, such as HCV genotype, virus heterogeneity, and the impact of viral proteins on the immune system have been characterized. Likewise, host genetic markers, such as the interleukin genotypes, HLA alleles, and factors affecting the T lymphocyte response appear to play an important role. Studies have revealed that natural clearance of HCV infection in the chronic phase is rare and its mechanisms are not well understood. In this review, we present the state-of-the art knowledge on the viral and host factors affecting the spontaneous elimination of HCV infection.

Stimulating structural and functional adaptation that improves cognitive performance in specific tasks is the major objective of therapeutic exercise training. In this review we briefly summarize central physiological mechanisms activated by exercise. We further discuss the influence of different kinds of exercise on cognitive improvement. In particular, the effects on cognitive function of aerobic endurance, resistance and respiratory exercise, and combinations thereof are presented. The accumulating evidence reinforces the position that regular aerobic, and possibly also resistance training, offers a powerful tool to cope with biologic aging of central nervous system functions. Nevertheless, the potential magnitude of cognition improvement or restrain of age-related cognition deterioration and the quantity of physical activity required to induce meaningful responses remain to be clarified.

Locus coeruleus is a critical component of the brain noradrenergic system. The brain noradrenergic system provides the neural substrate for the architecture supporting the interaction with, and navigation through, an external world complexity. Changes in locus coeruleus tonic and phasic activity and the interplay between norepinephrine and α1- and α2-adrenoceptors in the prefrontal cortex are the key elements of this sophisticated architecture. In this narrative review we discuss how the brain noradrenergic system is affected by increased exposure to corticotropin-releasing hormone triggered by stress response. In particular, we present the mechanisms responsible for thinking inflexibility often observed under highly stressful conditions. Finally, the main directions for future research are highlighted.

The aim of this study was to determine the effects of long-term administration of the oral antidiabetic metformin or the pineal hormone melatonin, and a combination thereof, in preventing oxidative stress in the heart tissue of female Sprague-Dawley rats with mammary tumors induced by N-methyl-N-nitrosourea (NMU) (50 mg/kg) given on the 42nd postnatal day. Metformin and melatonin were administered 12 days before and 16 weeks after the carcinogen. During the experiment, all animals were fed a high fat diet (10% total fat, 2.5% from lard, and 7.5% from palm oil). The findings are that mammary carcinogenesis generated oxidative stress. Reactive oxygen species (ROS) content, estimated from thiobarbituric acid reactive substances (TBARS), oxidatively modified protein content (aldehyde and ketone derivatives), and the activity of the antioxidant enzymes superoxide dismutase, glutathione reductase, and glutathione peroxidase were all augmented. Metformin caused a decrease in oxidative stress in the heart, accompanied by a decrease in diene conjugates, the elimination of ROS (stable total antioxidant status), and the activation of catalase and glutathione reductase. Melatonin caused an increase in total antioxidant status and a substantial reduction in ROS as estimated from aldehyde and ketone derivatives, lipid peroxidation at the initial (diene conjugates) and terminal stages (TBARS), and increased catalase and glutathione peroxidase activities. Metformin and melatonin combined reversed the effects of NMU on oxidative stress. In conclusion, melatonin reduces the level of oxidative stress in the heart tissue, caused by NMU carcinogenesis and a high fat diet, significantly stronger than metformin.

Background Hepatitis C virus (HCV) infection is commonly associated with cognitive dysfunction and depression, which could be related to direct brain infection. Viral sequences and proteins were found in brain macrophage/microglia cells and these cells were reported to be activated. Since blood leukocytes cross blood–brain barrier, activation state of peripheral blood mononuclear cells (PBMC) could reflect the state of brain immune cells. Objective The aim of the study was to determine whether depression and neuroticism in chronic HCV infection correlates with the expression of key cytokines and chemokines in PBMC. Design We studied 24 HCV-positive patients undergoing treatment with interferon and ribavirin. Patients were tested for depression using Beck Depression Inventory (BDI) and Montgomery Asberg Depression Rating Scale (MADRS), while neuroticism was assessed by the Revised Eysenck Personality Inventory (N/EPO-R). Transcripts representing 28 various cytokines and chemokines were measured by real-time quantitative PCR in PBMC. Results Prior to therapy BDI and MADRS positively correlated with viral load while neuroticism correlated with IL-3, IL-8 and M-CSF transcription levels. Six months after therapy there was positive correlation between depression and/or neuroticism scores and the levels of proinflammatory cytokines TNF-α and IL-12 transcripts, as well as IL-8, IL-10, IL-16, MCP-1, MCP-2, MIP-1-alpha, MIP-1-beta, and TGF-beta, and IFN-β transcripts. Conclusion Activation of PBMC, as measured by the level of cytokine and chemokine transcripts, correlates with depression and neuroticism scores. These findings suggest a pivotal role of immune cells activation in depression and possibly neurocognitive dysfunction among chronic hepatitis C patients.

Hepatitis C virus (HCV) highly conserved IRES (internal ribosome entry site) sequence, localized within the 5′-untranslated region (5′UTR), may determine viral properties like replication efficiency and cell tropism. The aim of the present study was to characterize newly emerging 5′UTR variants in serum and peripheral blood mononuclear cells (PBMC) in chronic hepatitis C patients treated with interferon (IFN) and ribavirin and to identify their effect on IRES secondary structures. The study group consisted of 87 patients infected with genotype 1b from whom serum and PBMC samples were collected at 9 time points (before, during, and after treatment). New 5′UTR variants developed in 9 patients. Out of the overall 14 new variants, 9 (64%) were found in PBMC. HCV variants with decreased thermodynamic stability were identified only in PBMC and C183U mutation was the most common one in this compartment. In conclusion, antiviral treatment may favor emergence of new 5′UTR variants both in blood and in PBMC compartments. However, variants developing in the latter compartment were predicted to have lower thermodynamic stability of the IRES secondary structures compared to serum strains. C-U change in position 183, which has not been described previously, might indicate viral adaptation to lymphoid cells.

Inherited encephalopathies include a broad spectrum of heterogeneous disorders. To provide a correct diagnosis, an integrated approach including genetic testing is warranted. We report seven patients with difficult to diagnose inborn paediatric encephalopathies. The diagnosis could not be attained only by means of clinical and laboratory investigations and MRI. Additional genetic testing was required. Cytogenetics, PCR based tests, and array-based comparative genome hybridization were performed. In 4 patients with impaired language abilities we found the presence of microduplication in the region 16q23.1 affecting two dose-sensitive genes:WWOX(OMIM 605131) andMAF(OMIM 177075) (1 case), an interstitial deletion of the 17p11.2 region (2 patients further diagnosed as Smith-Magenis syndrome), and deletion encompassing first three exons of Myocyte Enhancer Factor gene2MEF2C(1 case). The two other cases represented progressing dystonia. Characteristic GAG deletion inDYT1consistently with the diagnosis of torsion dystonia was confirmed in 1 case. Last enrolled patient presented with clinical picture consistent with Krabbe disease confirmed by finding of two pathogenic variants ofGALCgene and the absence of mutations inPSAP. The integrated diagnostic approach including genetic testing in selected examples of complicated hereditary diseases of the brain is largely discussed in this paper.

Hepatitis C virus (HCV) is a major cause of liver disease worldwide. The routine diagnostics identifying HCV infection include testing for specific anti-HCV antibodies by enzyme-linked immnunosorbent assay and viral genetic material in serum or plasma. However, a small proportion of patients persistently infected with HCV, in whom anti-HCV are undetectable, constitute a serious diagnostic and possibly epidemiologic problem, as they could facilitate pathogen spread in the population. This type of infection is termed seronegative or serosilent. Seronegative HCV infection is currently of great interest to both scientists and physicians. The review presents epidemiological data concerning the prevalence of seronegative HCV infection in HIV/HCV co-infected individuals, hemodialysis patients, and blood and organ donors. The possible mechanisms behind this atypical course of infection are discussed. Furthermore, the differences between seronegative and occult infections and prolonged seroconversion are explained.

Summary It has been reported that hepatitis C virus (HCV) RNA may be present in serum and/or lymphoid cells in the absence of specific circulating antibodies. The current study analysed seronegative HCV infection in patients with lymphoproliferative disorders. We studied 77 anti-HCV-negative patients (45 male and 32 female, mean age 54.8 ± 14.2 years) with various lymphoproliferative disorders. HCV-RNA was detected by RT-PCR in plasma, peripheral blood mononuclear cells (PBMC) and bone marrow. Furthermore, the presence of viral nonstructural protein 3 (NS3) was determined in PBMC and bone marrow by immunostaining. HCV-RNA was detectable in at least one compartment in 27 (35.1%) patients. Viral RNA was found in bone marrow in 22 patients (28.6%), in PBMC in 13 (16.9%) and in plasma in 10 (13%) patients. In nine patients, evidence of infection was confined to the bone marrow compartment. Viral load in HCV-RNA-positive plasma ranged from 15 to 1.17 × 103 IU/mL. NS3 was detected in all but two HCV-RNA-positive bone marrow samples and in all but one HCV-RNA-positive PBMC samples. All 27 HCV-RNA-positive patients remained anti-HCV-negative when tested again after 6–12 months, but only four remained HCV-RNA positive. In conclusion, among patients with lymphoproliferative disorders, HCV can be present in plasma, PBMC and bone marrow despite the lack of circulating specific antibodies. Further studies are required to analyse the phenomenon of seronegative infection and to determine whether such patients are infectious.

West Nile virus (WNV) infection usually causes mild febrile illness, but in a small proportion of patients it can lead to encephalitis. Epidemiological studies of WNV indicate fast spread of infection worldwide and in Europe, but there have been no comprehensive studies of WNV infection among encephalitis patients in Poland. Here we present the results of WNV RNA and anti-WNV testing in serum and cerebrospinal fluid (CSF) samples in 80 patients with the clinical diagnosis of viral encephalitis. WNV RNA was not detected in any of the analyzed samples. Anti-WNV IgG and IgM were not present in CSF in any of the investigated patients, but anti-WNV IgM were unexpectedly detected in serum of 14 subjects. The latter represented false positive results are probably related to cross reactivity of antibodies. Although there was no evidence of WNV infection in any of our patients, epidemiological situation in the neighbouring countries warrants vigilance and appropriate measures, including introduction of specific diagnostic tools into clinical practice, seem necessary.

Purpose of review Inflammation of forebrain and hindbrain nuclei has recently been highlighted as an emerging factor in the pathogenesis of neurogenic hypertension. The aim of this review is to summarize the state of the art in this field and to discuss recently discovered pathophysiological mechanisms, opening new perspectives for therapeutic application. Recent findings Microglia Toll-like receptor 4 causally links angiotensin II (AngII)-mediated microglia cell activation and oxidative stress within the hypothalamic paraventricular nucleus (PVN). Toll-like receptor 4 can also be activated by lipopolysaccharides. PVN infusion of nuclear factor κB inhibitor lowers the blood pressure and ameliorates cardiac hypertrophy. Ang-(1-7) exerts direct effects on microglia, causing a reduction in both baseline and prorenin-induced release of proinflammatory cytokines. A compromised blood-brain barrier (BBB) constitutes a complementary mechanism that exacerbates AngII-driven neurohumoral activation, contributing to the development of hypertension. Summary PVN and BBB seem to be pivotal targets for therapeutic intervention in hypertension. Recent advances in imaging techniques enable visualization of the inflammatory state in microglia and BBB integrity in humans. AngII type I receptor blockers and AngII-converting enzyme inhibitors are the most likely candidates for controlled randomized trials in humans aimed at amelioration of brain inflammation in the forthcoming years.

Next-generation sequencing (NGS) followed by metagenomic enables the detection and identification of known as well as novel pathogens. It could be potentially useful in the diagnosis of encephalitis, caused by a variety of microorganisms. The aim of the present study was to evaluate the sensitivity of isothermal RNA amplification (Ribo-SPIA) followed by NGS metagenomic analysis in the detection of human immunodeficiency virus (HIV) and herpes simplex virus (HSV) in cerebrospinal fluid (CSF). Moreover, we analyzed the contamination background. We detected 10

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of central nervous system of unknown etiology. However, some infectious agents have been suggested to play a significant role in its pathogenesis. Next-generation sequencing (NGS) and metagenomics can be employed to characterize microbiome of MS patients and to identify potential causative pathogens. In this study, 12 patients with idiopathic inflammatory demyelinating disorders (IIDD) of the central nervous system were studied: one patient had clinically isolated syndrome, one patient had recurrent optic neuritis, and ten patients had multiple sclerosis (MS). In addition, there was one patient with other non-inflammatory neurological disease. Cerebrospinal fluid (CSF) was sampled from all patients. RNA was extracted from CSF and subjected to a single-primer isothermal amplification followed by NGS and comprehensive data analysis. Altogether 441,608,474 reads were obtained and mapped using blastn. In a CSF sample from the patient with clinically isolated syndrome, 11 varicella-zoster virus reads were found. Other than that similar bacterial, fungal, parasitic, and protozoan reads were identified in all samples, indicating a common presence of contamination in metagenomics. In conclusion, we identified varicella zoster virus sequences in one out of the 12 patients with IIDD, which suggests that this virus could be occasionally related to the MS pathogenesis. A widespread bacterial contamination seems inherent to NGS and complicates the interpretation of results.

Major congenital anomalies are detectable in 2-3 % of the newborn population. Some of their genetic causes are attributable to copy number variations identified by array comparative genomic hybridization (aCGH). The value of aCGH screening as a first-tier test in children with multiple congenital anomalies has been studied and consensus adopted. However, array resolution has not been agreed upon, specifically in the newborn or infant population. Moreover, most array studies have been focused on mixed populations of intellectual disability/developmental delay with or without multiple congenital anomalies, making it difficult to assess the value of microarrays in newborns. The aim of the study was to determine the optimal quality and clinical sensitivity of high-resolution array comparative genomic hybridization in neonates with multiple congenital anomalies. We investigated a group of 54 newborns with multiple congenital anomalies defined as two or more birth defects from more than one organ system. Cytogenetic studies were performed using OGT CytoSure 8 × 60 K microarray. We found ten rearrangements in ten newborns. Of these, one recurrent syndromic microduplication was observed, whereas all other changes were unique. Six rearrangements were definitely pathogenic, including one submicroscopic and five that could be seen on routine karyotype analysis. Four other copy number variants were likely pathogenic. The candidate genes that may explain the phenotype were discussed. In conclusion, high-resolution array comparative hybridization can be applied successfully in newborns with multiple congenital anomalies as the method detects a significant number of pathogenic changes, resulting in early diagnoses. We hypothesize that small changes previously considered benign or even inherited rearrangements should be classified as potentially pathogenic at least until a subsequent clinical assessment would exclude a developmental delay or dysmorphism.

Identification of submicroscopic chromosomal aberrations, as a cause of structural malformations, is currently performed by MLPA (multiplex ligation-dependent probe amplification) or array CGH (array comparative genomic hybridization) techniques. The aim of this study was the evaluation of diagnostic usefulness of MLPA and array CGH in patients with congenital malformations or abnormalities (at least one major or minor birth defect, including dysmorphism) with or without intellectual disability or developmental delay and the optimization of genetic counseling in the context of the results obtained. The MLPA and array CGH were performed in 91 patients diagnosed with developmental disorders and major or minor congenital anomalies. A total of 49 MLPA tests toward common microdeletion syndromes, 42 MLPA tests for subtelomeric regions of chromosomes, two tests for common aberrations in autism, and five array CGH tests were performed. Eight (9 %) patients were diagnosed with microdeletion MLPA, four (4 %) patients with subtelomeric MLPA, one (1 %) patient with autism MLPA. Further three (3 %) individuals had rearrangements diagnosed by array CGH. Altogether, chromosomal microaberrations were found in 16 patients (17 %). All the MLPA-detected rearrangements were found to be pathogenic, but none detected with array CGH could unequivocally be interpreted as pathogenic. In patients with congenital anomalies, the application of MLPA and array CGH techniques is efficient in detecting syndromic and unique microrearrangements. Consistent pre-MLPA test phenotyping leads to better post-test genetic counseling. Incomplete penetrance and unknown inheritance of detected variants are major issues in clinical interpretation of array CGH data.

Hepatitis C virus (HCV) transmission between spouses remains poorly characterized, largely due to the limited availability of samples from the early stage of infection, as well as methodological constraints. A fifty-eight year-old male developed acute hepatitis C infection and his 53-year old spouse has been HCV-positive for over 10 years. Serum samples were collected from both at the time of acute hepatitis C diagnosis in male (baseline) and then at 9 and 13 months. Hypervariable region 1 (HVR1) and 5' untranslated region (5'UTR) sequences were amplified and subjected to next generation sequencing (NGS) using a pyrosequencing platform. Genetic variants were inferred by Shorah reconstruction method and compared by phylogenetic and sequence diversity analysis. As the sequencing error of the procedure was previously determined to be ≤ 1.5%, the analysis was conducted with and without the 1.5% cut-off with regard to the frequency of variants. No identical HVR1 variants were identified in spouses at baseline and follow-up samples regardless whether the cut-off was applied or not. However, there was high similarity (98.3%) between a minor baseline donor variant (1.7% frequency) and the most abundant baseline recipient variant (62.5% frequency). Furthermore, donor and recipient strains clustered together when compared to 10 control subjects from the same area and infected with the same HCV subtype. There was an increase in HVR1 complexity (number of genetic variants) over time in both spouses. In contrast, the 5'UTR region was stable and of low complexity throughout the study. In conclusion, intrafamilial HCV transmission may be established by a very minor variant and investigation of this phenomenon requires high-sensitivity assays, such as NGS.

The role of mixed infections with different hepatitis C virus (HCV) genotypes in viral persistence, treatment effects, and tissue tropism is unclear. Next-generation sequencing (NGS), which is suitable for analysis of large, genetically diverse populations offers unparalleled advantages for the study of mixed infections. The aim of the study was to determine, using two different deep sequencing strategies (pyrosequencing - 454 Life Sciences/Roche and reversible terminator sequencing-by-synthesis by Illumina), the origin of a novel HCV genotype transiently detectable during antiviral therapy (pre-existing minor population vs. de novo superinfection). Secondly, we compared 5' untranslated region (5'-UTR) variants obtained by the two NGS approaches. 5' UTR amplification products from 9 samples collected from genotype 1b infected patient before, during, and after treatment (4 serum and 5 peripheral blood mononuclear cell - PBMC - samples) were subjected to the next-generation sequencing. The sequencing revealed the presence of two (454/Roche) and one (Illumina) genotype 4 variants in PBMC at Week 16. None of these variants were present either in the preceding or following samples as revealed by both platforms. 454/Roche sequencing detected 24 different 5'-UTR variants: 8 were present in serum and PBMC, 4 only in serum and 12 only in PBMC. Illumina sequencing detected 11 different 5'-UTR variants: 5 in serum and PBMC, 4 only in serum and 2 only in PBMC. Six variants were identical for both sequencing platforms. The difference in variants number was primarily due to variability in two 5'-UTR homopolymeric regions. In conclusion, longitudinal analysis of HCV variants, employing two independent deep sequencing methods, suggests that the transient presence of a different genotype strain in PBMC was a result of superinfection and not a selection of pre-existing minor variant.

Major congenital anomalies are detectable in 2–3 % of the newborn population. Some of their genetic causes are attributable to copy number variations identified by array comparative genomic hybridization (aCGH). The value of aCGH screening as a first-tier test in children with multiple congenital anomalies has been studied and consensus adopted. However, array resolution has not been agreed upon, specifically in the newborn or infant population. Moreover, most array studies have been focused on mixed populations of intellectual disability/developmental delay with or without multiple congenital anomalies, making it difficult to assess the value of microarrays in newborns. The aim of the study was to determine the optimal quality and clinical sensitivity of high-resolution array comparative genomic hybridization in neonates with multiple congenital anomalies. We investigated a group of 54 newborns with multiple congenital anomalies defined as two or more birth defects from more than one organ system. Cytogenetic studies were performed using OGT CytoSure 8 × 60 K microarray. We found ten rearrangements in ten newborns. Of these, one recurrent syndromic microduplication was observed, whereas all other changes were unique. Six rearrangements were definitely pathogenic, including one submicroscopic and five that could be seen on routine karyotype analysis. Four other copy number variants were likely pathogenic. The candidate genes that may explain the phenotype were discussed. In conclusion, high-resolution array comparative hybridization can be applied successfully in newborns with multiple congenital anomalies as the method detects a significant number of pathogenic changes, resulting in early diagnoses. We hypothesize that small changes previously considered benign or even inherited rearrangements should be classified as potentially pathogenic at least until a subsequent clinical assessment would exclude a developmental delay or dysmorphism.

Identification of submicroscopic chromosomal aberrations, as a cause of structural malformations, is currently performed by MLPA (multiplex ligation-dependent probe amplification) or array CGH (array comparative genomic hybridization) techniques. The aim of this study was the evaluation of diagnostic usefulness of MLPA and array CGH in patients with congenital malformations or abnormalities (at least one major or minor birth defect, including dysmorphism) with or without intellectual disability or developmental delay and the optimization of genetic counseling in the context of the results obtained. The MLPA and array CGH were performed in 91 patients diagnosed with developmental disorders and major or minor congenital anomalies. A total of 49 MLPA tests toward common microdeletion syndromes, 42 MLPA tests for subtelomeric regions of chromosomes, two tests for common aberrations in autism, and five array CGH tests were performed. Eight (9 %) patients were diagnosed with microdeletion MLPA, four (4 %) patients with subtelomeric MLPA, one (1 %) patient with autism MLPA. Further three (3 %) individuals had rearrangements diagnosed by array CGH. Altogether, chromosomal microaberrations were found in 16 patients (17 %). All the MLPA-detected rearrangements were found to be pathogenic, but none detected with array CGH could unequivocally be interpreted as pathogenic. In patients with congenital anomalies, the application of MLPA and array CGH techniques is efficient in detecting syndromic and unique microrearrangements. Consistent pre-MLPA test phenotyping leads to better post-test genetic counseling. Incomplete penetrance and unknown inheritance of detected variants are major issues in clinical interpretation of array CGH data.

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of central nervous system of unknown etiology. However, some infectious agents have been suggested to play a significant role in its pathogenesis. Next-generation sequencing (NGS) and metagenomics can be employed to characterize microbiome of MS patients and to identify potential causative pathogens. In this study, 12 patients with idiopathic inflammatory demyelinating disorders (IIDD) of the central nervous system were studied: one patient had clinically isolated syndrome, one patient had recurrent optic neuritis, and ten patients had multiple sclerosis (MS). In addition, there was one patient with other non-inflammatory neurological disease. Cerebrospinal fluid (CSF) was sampled from all patients. RNA was extracted from CSF and subjected to a single-primer isothermal amplification followed by NGS and comprehensive data analysis. Altogether 441,608,474 reads were obtained and mapped using blastn. In a CSF sample from the patient with clinically isolated syndrome, 11 varicella-zoster virus reads were found. Other than that similar bacterial, fungal, parasitic, and protozoan reads were identified in all samples, indicating a common presence of contamination in metagenomics. In conclusion, we identified varicella zoster virus sequences in one out of the 12 patients with IIDD, which suggests that this virus could be occasionally related to the MS pathogenesis. A widespread bacterial contamination seems inherent to NGS and complicates the interpretation of results.

Next-generation sequencing (NGS) followed by metagenomic analysis enables the detection and identification of known as well as novel pathogens. It could be potentially useful in the diagnosis of encephalitis, caused by a variety of microorganisms. The aim of the present study was to evaluate the sensitivity of isothermal RNA amplification (Ribo-SPIA) followed by NGS metagenomic analysis in the detection of human immunodeficiency virus (HIV) and herpes simplex virus (HSV) in cerebrospinal fluid (CSF). Moreover, we analyzed the contamination background. We detected 102 HIV copies and 103 HSV copies. The analysis of control samples (two water samples and one CSF sample from an uninfected patient) revealed the presence of human DNA in the CSF sample (91 % of all reads), while the dominating sequences in water were qualified as ‘other’, related to plants, plant viruses, and synthetic constructs, and constituted 31 % and 60 % of all reads. Bacterial sequences represented 5.9 % and 21.4 % of all reads in water samples and 2.3 % in the control CSF sample. The bacterial sequences corresponded mainly to Psychrobacter, Acinetobacter, and Corynebacterium genera. In conclusion, Ribo-SPIA amplification followed by NGS metagenomic analysis is sensitive for detection of RNA and DNA viruses. Contamination seems common and thus the results should be confirmed by other independent methods such as RT-PCR and PCR. Despite these reservations, NGS seems to be a promising method for the diagnosis of viral infections.

Purpose Genetic variability of hepatitis C virus (HCV) is considered to be an important factor defining viral pathogenesis, persistence and resistance to treatment. The aim of the present study was to characterize HCV genetic heterogeneity within a hypervariable region 1 (HVR-1) before and during the early period of pegylated interferon alfa (PEG-IFN-α) and ribavirin treatment in correlation with treatment outcome. Material and Methods The study involved 24 patients treated with PEG-IFN-α and ribavirin whose sera were collected before (baseline) and at 7, 14, 21 28 and 56 day of treatment. HCV HVR-1 region was amplified by nested RT- PCR and subjected to SSCP (single strand conformational polymorphism) analysis. SSCP changes of HCV HVR-1 over time in each patient were compared to treatment outcome results. Results In 2/11 (18%) SVR+ and 8/13 (62%) SVR- treated patients, HVR-1 genetic changes manifested by new SSCP bands (new genetic variants) and were significantly more frequent in nonresponders (P Conclusions Our results indicate that HCV HVR-1 variability during the early phase of PEG-IFN-α and ribavirin therapy may be predictive of treatment outcome.

Objectives Hepatitis C virus (HCV) infection is commonly associated with cognitive dysfunction. Viral sequences and proteins were previously found in brain macrophage/microglia cells. The aim of the current study was to determine whether HCV infection affects the expression of key cytokines and chemokines in these cells. Methods Autopsy brain tissue from 15 patients was studied; 7 patients were HCV positive and 8 were HCV negative. Cryostat sections of frontal cortex and subcortical white matter were stained with monoclonal antibodies specific for microglia/macrophages (anti-CD68) and separated by laser capture microscopy. Transcripts representing 25 various cytokines and chemokines were measured by real-time quantitative PCR. Results Compared with HCV-negative controls, HCV-positive patients demonstrated significantly higher levels of proinflammatory cytokines interleukin 1α (IL-1α), IL-1β, tumour necrosis factor α (TNFα), IL-12 and IL-18. HCV infection was also associated with increased transcription of chemokines IL-8, IL-16 and interferon-inducible protein 10 (IP-10). Type 1 interferon (IFN) activation was suggested by increased concentrations of IFNβ and myxovirus resistance protein A (MxA) transcripts. Similar results were obtained when CD68-positive/HCV-positive cells were compared with CD68-positive/HCV-negative cells in each of the 7 HCV-infected patients. Conclusion Evidence was found for activation of brain macrophages/microglia cells in autopsy brain tissue from HCV-positive patients. These findings could relate to the common presence of neurocognitive dysfunction among patients with chronic hepatitis C.

Molecular characterization of early hepatitis C virus (HCV) infection remains rare. Ten out of 78 patients of a hematology/oncology center were found to be HCV RNA positive two to four months after hospitalization. Only two of the ten patients were anti-HCV positive. HCV hypervariable region 1 (HVR1) was amplified in seven patients (including one anti-HCV positive) and analyzed by next generation sequencing (NGS). Genetic variants were reconstructed by Shorah and an empirically established 0.5% variant frequency cut-off was implemented. These sequences were compared by phylogenetic and diversity analyses. Ten unrelated blood donors with newly acquired HCV infection detected at the time of donation (HCV RNA positive and anti-HCV negative) served as controls. One to seven HVR1 variants were found in each patient. Sequences intermixed phylogenetically with no evidence of clustering in individual patients. These sequences were more similar to each other (similarity 95.4% to 100.0%) than to those of controls (similarity 64.8% to 82.6%). An identical predominant variant was present in four patients, whereas other closely related variants dominated in the remaining three patients. In five patients the HCV population was limited to a single variant or one predominant variant and minor variants of less than 10% frequency. In conclusion, NGS analysis of a cluster of HCV infections acquired in the hospital setting revealed the presence of low diversity, very closely related variants in all patients, suggesting an early-stage infection with the same virus. NGS combined with phylogenetic analysis and classical epidemiological analysis could help in tracking of HCV outbreaks.

Hepatitis C virus (HCV) infection often is associated with cognitive dysfunction and depression. HCV sequences and replicative forms were detected in autopsy brain tissue and cerebrospinal fluid from infected patients, suggesting direct neuroinvasion. However, the phenotype of cells harboring HCV in brain remains unclear. We studied autopsy brain tissue from 12 HCV-infected patients, 6 of whom were coinfected with human immunodeficiency virus. Cryostat sections of frontal cortex and subcortical white matter were stained with monoclonal antibodies specific for microglia/macrophages (CD68), oligodendrocytes (2′,3′-cyclic nucleotide 3′-phosphodiesterase), astrocytes (glial fibrillary acidic protein [GFAP]), and neurons (neuronal-specific nuclear protein); separated by laser capture microscopy (LCM); and tested for the presence of positive- and negative-strand HCV RNA. Sections also were stained with antibodies to viral nonstructural protein 3 (NS3), separated by LCM, and phenotyped by real-time PCR. Finally, sections were double stained with antibodies specific for the cell phenotype and HCV NS3. HCV RNA was detected in CD68-positive cells in eight patients, and negative-strand HCV RNA, which is a viral replicative form, was found in three of these patients. HCV RNA also was found in astrocytes from three patients, but negative-strand RNA was not detected in these cells. In double immunostaining, 83 to 95% of cells positive for HCV NS3 also were CD68 positive, while 4 to 29% were GFAP positive. NS3-positive cells were negative for neuron and oligodendrocyte phenotypic markers. In conclusion, HCV infects brain microglia/macrophages and, to a lesser extent, astrocytes. Our findings could explain the biological basis of neurocognitive abnormalities in HCV infection.