Identification, replication, and functional fine-mapping of expression quantitative trait loci in primary human liver tissue.

The discovery of expression quantitative trait loci ("eQTLs") can help to unravel genetic contributions to complex traits. We identified genetic determinants of human liver gene expression variation using two independent collections of primary tissue profiled with Agilent (n = 206) and Illumina (n = 60) expression arrays and Illumina SNP genotyping (550K), and we also incorporated data from a published study (n = 266). We found that ∼30% of SNP-expression correlations in one study failed to replicate in either of the others, even at thresholds yielding high reproducibility in simulations, and we quantified numerous factors affecting reproducibility. Our data suggest that drug exposure, clinical descriptors, and unknown factors associated with tissue ascertainment and analysis have substantial effects on gene expression and that controlling for hidden confounding variables significantly increases replication rate. Furthermore, we found that reproducible eQTL SNPs were heavily enriched near gene starts and ends, and subsequently resequenced the promoters and 3'UTRs for 14 genes and tested the identified haplotypes using luciferase assays. For three genes, significant haplotype-specific in vitro functional differences correlated directly with expression levels, suggesting that many bona fide eQTLs result from functional variants that can be mechanistically isolated in a high-throughput fashion. Finally, given our study design, we were able to discover and validate hundreds of liver eQTLs. Many of these relate directly to complex traits for which liver-specific analyses are likely to be relevant, and we identified dozens of potential connections with disease-associated loci. These included previously characterized eQTL contributors to diabetes, drug response, and lipid levels, and they suggest novel candidates such as a role for NOD2 expression in leprosy risk and C2orf43 in prostate cancer. In general, the work presented here will be valuable for future efforts to precisely identify and functionally characterize genetic contributions to a variety of complex traits.

The role of various immersion liquids at digital dermoscopy in structural analysis.

BACKGROUND: Dermoscopy is a useful method that allows dermal and epidermal structures to be easily analysed non-invasively. AIM: In this study, immersion oil, which is widely used in dermoscopy, and ultrasound gel, which is less preferred, are evaluated comparatively in terms of displaying structural parameters and number of air bubbles in the image. METHODS: A total of 71 nevomelanocytic or non-melanocytic pigmented lesions were taken up for this study. Structural characteristics of the obtained images were assessed by an experienced observer who scored the images in terms of color, pigment network, globule, vascular structure, number of air bubbles and other pigmentation parameters. RESULTS: In the images obtained through immersion oil or ultrasound gel from all of the lesions, no statistical difference was found between the average values of air bubbles and in the evaluation of structural components (t=1.09, P=0.2). In the identification of pigment network in melanocytic lesions, immersion oil was observed to be more appropriate than ultrasound gel (t=0.01, P=0.02). CONCLUSIONS: Ultrasound gel may be preferred in the assessment of mucosa and nail bed lesions. Ultrasound gel is a good alternative compared to immersion oil in pigmented skin lesions as it is cheap and easily removable.

[Utility of molecular biology in the microbiological diagnosis of mycobacterial infections]. / Utilidad de la biología molecular en el diagnóstico microbiológico de las infecciones por micobacterias.

Species within the Mycobacterium genus are of major medical interest, since, together with environmental and opportunistic species, there are two species (Mycobacterium tuberculosis and Mycobacterium leprae) that remain an important public health challenge. Despite efforts to control tuberculosis (TB), this disease remains one of the most prominent health problems worldwide. In the last few years, mycobacteriology has experienced major technological advances. Nevertheless, the early diagnosis of mycobacterial infection and, especially of TB, is still based on microscopic examination of properly stained samples. At present, this procedure is still the simplest, fastest and most cost-effective method for preliminary diagnostic guidance. Effective control of TB is based on rapid detection of M. tuberculosis, followed by immediate implementation of the appropriate antituberculosis therapy. Because of the emergence of multidrug resistant strains, the development of rapid diagnostic methods, both for identification of M. tuberculosis and susceptibility testing, has become a pressing need. The availability of molecular epidemiology methods that are easy to implement and standardized and that would allow identification of related cases is of key importance to identify epidemic outbreaks and control the spread of TB. Despite the evident progress in the molecular diagnosis of mycobacterial infections, the available techniques are still inadequate. In this review, we describe the state of the art of the main molecular techniques for direct detection of mycobacteria in clinical samples, their identification, detection of resistance to the most important antituberculosis agents, and molecular epidemiology. In each case, we describe the advantages and limitations of current techniques. In the near future, clinical mycobacteriology will probably evolve to the universal use of genetic techniques for direct diagnosis and detection of resistance. The molecular epidemiology of TB will be performed, in its various applications, by faster and more automated techniques than those currently available.

Analysis of quantitative relationship between viability determination in leprosy by MFP, ATP bioluminescence and gene amplification assay.

Two hundred twenty-one untreated, borderline lepromatous/lepromatous (BL/LL) leprosy patients have been investigated for viability by the mouse foot pad method (MFP), adenosine triphosphate (ATP) and polymerase chain reaction (PCR). The biopsies were collected at the beginning of and 12/24 months after treatment. The patient group was treated with a) immunotherapy (BCG/Mw) + MDT; b) MDT + pyrazinamide; c) control MDT; d) MDT + minocycline 100 mg once a month supervised + ofloxacin 400 mg once a month supervised. Biopsies were divided in three parts for use in the mouse foot pad, molecular and ATP investigations. In untreated and treated patients (at 12 and 24 months), there was a general agreement among all three techniques, and PCR and ATP showed higher positivity as compared to MFP. Further, there was good correlation among the viable biomass estimated by bacillary ATP levels, PCR assay and growth in mouse foot pads. The positivity was observed by MFP as well as PCR assay (18-kDa and 36-kDa) from all of the specimens when the ATP content was more than 3.6 pg/million. When the ATP content was below 3.5 pg/million, the positive takes in MFP decreased but the PCR positivity correlated with ATP bioluminescence up to 0.04 pg/million. When the ATP content was even lower, the uptake in the MFP was possibly a matter of chance, while PCR positivity was observed in 96% of the cases. For specimens with undetectable ATP, positivity was seen in 1% of the cases, showing the inability of ATP bioluminescence method to detect low background due to host ATP. PCR signals in some cases could be due to the higher sensitivity of the method or persistence of DNA after bacterial death in some cases. On the whole, the PCR methods even though targeting DNA have shown good correlations with biomass which confirm their usefulness in monitoring therapeutic responses in leprosy.

ATP content of Mycobacterium tuberculosis grown in vivo and in vitro.

In order to determine the reason for the slow growth of Mycobacterium leprae either in a host or in vitro, the growth characteristics of Mycobacterium tuberculosis were studied. The ATP content of in vitro-grown M. tuberculosis was about 520 pg/10(6) viable organisms. The ATP levels from in vivo-derived organisms obtained from liver and spleen of mice was about 130 pg (in cases of chronic infection) and about 270 pg (in cases of acute infection). When the in vivo-derived organisms were inoculated into culture medium, the growth rates for both types of organisms, acute as well as chronic infection, were the same and the maximum growth was reached during the fifth subculture. Although the maximum ATP content for both types of organism was the same, it was attained during the 4th subculture for organisms obtained during acute infection and during the 6th subculture for those obtained during chronic infection. The comparison between the ATP content of M. leprae and of M. tuberculosis indicates the reason for the slow growth of M. leprae.

Assessment of viability by normal mouse foot-pad and bacillary ATP bioluminescence assay in multibacillary cases treated with an MDT regimen using conventional as well as newer drugs like minocycline and ofloxacin.

The therapeutic effect of a drug regimen of conventional drugs as well as newer drugs like ofloxacin and minocycline in smear-positive multibacillary (MB) leprosy cases was assessed by mouse foot-pad and ATP bioluminiscence methods. Biopsies were taken before starting treatment and after one year of treatment. They were processed for viability assessment by normal mouse foot-pad inoculation and bacillary ATP assay techniques. The test regimen was quite effective in its anti-bacterial effect as it was found to result in loss of bacillary viability in all the cases, as assessed by both methods.

Detection of viable organisms in leprosy patients treated with multidrug therapy.

Cutaneous biopsies were collected from multibacillary leprosy patients who attended the out-patient department of Jalma Institute for treatment at different time intervals, i.e. 6 months, 12 months, 18 months, 24 months, 30 months, 36 months and 42 months after starting multidrug therapy (MDT) when they were still skin smear positive. Biopsies were processed for inoculation into mouse foot pad (MFP) and estimation of bacillary ATP levels by bioluminescent assay (ATP assay) by earlier established procedures. Viable bacilli were detectable after 1 year (25% cases by MFP and 31% cases by ATP assay), 2 years (8% cases by MFP and 12% cases by ATP assay) and 3 years (4% cases by both MFP and ATP assays). Overall, the percentage of the persisters was 10% by MFP and 13% by ATP assay. It would be important to carry out surveillance studies in larger number of BL/LL cases to know the trends and also the resultant relapses.

Detection of borna disease virus-reactive antibodies from patients with psychiatric disorders and from horses by electrochemiluminescence immunoassay.

The prevalence of Borna disease virus (BDV)-specific antibodies among patients with psychiatric disorders and healthy individuals has varied in several reports using several different serological assay methods. A reliable and specific method for anti-BDV antibodies needs to be developed to clarify the pathological significance of BDV infections in humans. We developed a new electrochemiluminescence immunoassay (ECLIA) for the antibody to BDV that uses two recombinant proteins of BDV, p40 and p24 (full length). Using this ECLIA, we examined 3,476 serum samples from humans with various diseases and 917 sera from blood donors in Japan for the presence of anti-BDV antibodies. By ECLIA, 26 (3.08%) of 845 schizophrenia patients and 9 (3.59%) of 251 patients with mood disorders were seropositive for BDV. Among 323 patients with other psychiatric diseases, 114 with neurological diseases, 75 with chronic fatigue syndrome, 85 human immunodeficiency virus-infected patients, 50 with autoimmune diseases including rheumatoid arthritis and systemic lupus erythematosis and 17 with leprosy, there was no positive case except one case each with alcohol addiction, AIDS, and dementia. Although 19 (1.36%) of 1,393 patients with various ocular diseases, 10 (1.09%) of 917 blood donors, and 3 (4.55%) of 66 multitransfused patients were seropositive for BDV-specific antigen, high levels of seroprevalence in schizophrenia patients and young patients (16 to 59 years old) with mood disorders were statistically significant. The immunoreactivity of seropositive sera could be verified for specificity by blocking with soluble p40 and/or p24 recombinant protein. Anti-p24 antibody was more frequent than p40 antibody in most cases, and in some psychotic patients antibody profiles showed only p40 antibody. Although serum positive for both p40 and p24 antibodies was not found in this study, the p40 ECLIA count in schizophrenia patients was higher than that of blood donors. Furthermore, we examined 90 sera from Japanese feral horses. Antibody profiles of control human samples are similar to that of naturally BDV-infected feral horses. We concluded that BDV infection was associated in some way with psychiatric disorders.

Factors influencing the in vitro growth of Mycobacterium leprae: effect of inoculum.

Factors responsible for the in vitro growth of Mycobacterium leprae in Dhople-Hanks (DH) medium, and also to improve the technique devised earlier, and the source of the M. leprae used as inoculum, were investigated. M. leprae were obtained from armadillos and nude mice, both inoculated earlier with human- or armadillo-derived M. leprae. The growth of M. leprae in DH medium was monitored using two biochemical indicators. Normal growth was obtained when inocula were from livers and spleens of M. leprae-infected armadillos. The M. leprae harvested from the footpads of nude mice failed to multiply in the same medium. Using inocula from livers and spleens of infected armadillos, a gradual decrease in inoculum size resulted in a proportionately slower multiplication of M. leprae. When the DH medium was supplemented with whole M. leprae, or cell-free extracts of M. leprae, from irradiated livers and spleens of infected armadillos, nude mouse-derived M. leprae exhibited growth in the DH medium in accord with that obtained using armadillo-derived M. leprae. Similar results were obtained with cell-free extracts of M. leprae harvested from non-irradiated livers and spleens of infected armadillos, but no growth was obtained when the medium was supplemented with extracts from livers or spleens of normal armadillos. These results indicate the possible existence of a growth factor in armadillo-derived M. leprae.

Viability determination of M.leprae: comparison of normal mouse foot pad and bacillary ATP bioluminescence assay.

Correlation between viability assessment by mouse foot pad and ATP bioluminescence was studied in biopsy specimens from multibacillary leprosy cases. Biopsies were processed for inoculation into mouse foot pad and estimation of bacillary ATP levels by bioluminescent assay by earlier established procedures. ATP content as pg/million bacilli was estimated and correlation was assessed with growth in the mouse foot pad. It was observed that when the ATP content was > 36 pg/million bacterial cells, (> 1% probable viables) there was growth in the mouse foot pad from all the specimens. Similar results were observed when the ATP content was in the range of 3.6 to 35.99 pg/million cells (0.1 to 1% probable viables). The positivity rates in the mouse foot pad decreased when the ATP content decreased further. No positive growth in the specimens below 0.04 pg/million bacilli (< 0.001% viable organisms) was observed. These findings show an overall correlation between viability assessed by mouse foot pad and ATP bioluminescence. These observations validate the concept of ATP content of viable unit of M.leprae being in the order of 10(-15) g/live cell which is in the same order of magnitude as a colony forming unit of cultivable mycobacteria.

Role of reactive oxygen species in renal damage in experimental leprosy.

Renal involvement is known to occur in leprosy. In the present study the possible role of reactive oxygen species (ROS) in causation of renal damage in mice infected with Mycobacterium leprae has been investigated. At least six animals from each group (control and infected) were killed at 0 day, 3, 6 and 9 months postinfection. The results showed a significant increase in the chemiluminescence (CL) response of peritoneal macrophages which was maximum between 3 and 6 months. No significant increase was observed in CL response of blood neutrophils. A significant increase in lipid peroxidation was observed at 3 and 6 months as evident by an increase in malondialdehyde levels. The increased ROS production might be the cause of lipid peroxidation. The renal damage is alos evident by decrease in the activity of renal brush border membrane enzymes, namely, alkaline phosphatase, leucine aminopeptidase and r-glutamyl transpeptidase. Thus ROS might play a role during early stages of M. leprae infection but in the later stages other immunological mechanisms may overpower the effect of ROS.

Evaluation of chemiluminescence, procoagulant activity and antigen presentation by monocytes from lepromatous leprosy patients with or without reactional episodes.

In this study, we evaluated the activity of peripheral blood mononuclear cells (PBMC), isolated from treated and untreated lepromatous leprosy patients, from lepromatous leprosy patients during and after reactional episodes (erythema nodosum leprosum (ENL) and reversal reaction (RR)), and from normal healthy individuals. We determined reactive oxygen intermediate (ROI) production, procoagulant activity (PCA) and HLA-DR antigen expression of monocytes, besides lymphoproliferation, both in the presence and absence of various stimulatory agents. Phorbol myristate acetate (PMA) stimulated ROI production by monocytes from all the groups studied, with patients during reactional episodes (ENL and RR) showing a significantly higher response (p < 0.009 and p < 0.00001). Irradiated Mycobacterium leprae, although having little effect when added alone, strongly suppressed PMA-stimulated ROI production. Muramyl dipeptide (MDP) had no influence on either basal or on PMA-induced ROI production. Basal monocyte PCA, as well as M. leprae or concanavalin A (ConA)-induced monocyte PCA was comparable in monocytes from all the groups studied. ConA was able to induce mitogenic activity in mononuclear cells isolated from all the groups studied. M. leprae, although stimulatory for normal individuals, did not induce lymphoproliferation in lepromatous leprosy patients, except for cells from patients during RR, which responded equally to M. leprae and to ConA. The absence of M. leprae-induced lymphoproliferation in lepromatous leprosy patients is not caused by the lack of basal HLA-DR expression, as PBMC from all individuals studied showed the same level of this antigen. Our results suggest an increase of spontaneous or PMA-induced monocyte activity, as detected by ROI production, during the reactional episode; addition of M. leprae suppressed this response. The increase in monocyte activity could be correlated with the increase of lymphoproliferation response to M. leprae during RR, but not during ENL. The importance of a possible immune suppressive action of M. leprae is discussed.

Heat-stable opsonins in tuberculosis and leprosy.

We have examined heat-stable opsonins to 4 species of gamma-irradiated mycobacteria (M. tuberculosis (H37Rv), M. avium (28A), M. scrofulaceum and M. leprae (cd 103)) in complement-depleted sera collected from Indonesian subjects with tuberculosis (106 patients),-leprosy (24 patients) and controls (40 hospital workers and 41 factory workers) indirectly by microtitre plate chemiluminescence (CL) assay and compared the results with antibody levels. The results indicate that there is a wide range of opsonic capacity for mycobacteria in complement-depleted sera. There was a poor correlation between the opsonic capacity as measured by CL and the anti-mycobacterial antibody content of sera measured by ELISA, suggesting that anti-mycobacterial antibody has little influence on the uptake of mycobacteria. However, a non-specific heat-stable opsonin appears to be present in some sera. Conversely, some sera from tuberculosis or leprosy patients suppress the production of reactive oxygen species from normal phagocytes in vitro when stimulated with M. tuberculosis. The relevance of this inhibition and the presence of heat-stable opsonins to the pathogenesis of tuberculosis have yet to be determined, but it is possible that the presence of opsonins may inhibit dissemination of tubercle bacilli to other organs.

Human phagocyte respiratory burst by Mycobacterium bovis BCG and M. leprae: functional activation by BCG is mediated by complement and its receptors on monocytes.

We have measured the role of serum components on two parameters of the phagocytosis reaction: a) the chemiluminescence (CL) response associated with the oxidative respiratory burst in response to Mycobacterium bovis BCG and M. leprae, and b) the uptake of these two mycobacteria by healthy human monocytes. Pre-incubations of fresh or heat-inactivated serum or serum containing EGTA or EDTA indicate that these two mycobacteria activate the alternative complement pathway. Monoclonal antibodies against CR1 and CR3 inhibit the responses of M. bovis BCG and M. leprae, demonstrating that complement receptors mediate the phagocytosis of these two mycobacteria. Thus, complement and its receptors on the surface of the monocytes (CR1 and CR3) are important in the functional activation of phagocytosis of M. bovis BCG and M. leprae.

TNF-alpha failed to reverse the M. leprae-induced defective chemiluminescence response of human mononuclear cells.

The effect of phagocyte activation by TNF-alpha on the ability to trigger a chemiluminescence (CL) response, associated with the release of oxidizing species was evaluated in healthy human mononuclear cells in the presence of Mycobacterium leprae. Recombinant TNF-alpha (r-TNF-alpha) increased the CL response of unstimulated M. bovis BCG- and PMA-stimulated cells but did not reverse the M. leprae defective activation of the human phagocyte oxidative burst. M. leprae was less well phagocytosed than M. bovis BCG but phagocytosis of mycobacteria was not altered by addition of r-TNF-alpha. The failure of activation of oxygen-free radical production might have some relevance to the pathogenesis of leprosy.

Phenolic glycolipid-1 from M. leprae inhibits oxygen free radical production by human mononuclear cells.

We studied the effect of PGL1, a phenolic glycolipid unique to Mycobacterium leprae, on the activation of the phagocyte oxidative respiratory burst, by measuring the chemiluminescence (CL) generated by normal mononuclear cells. PGL1 induced a decrease in oxygen free radical production stimulated by mycobacteria (M. leprae, BCG and M. kansasii) or by phorbol myristate acetate, but did not prevent the binding or ingestion of fluorescein-conjugated mycobacteria. In contrast, mycoside A from M. kansasii, a structurally related compound, did not alter the CL response. In addition, treatment of M. leprae with anti-PGL1 antibodies failed to restore the response to this microorganism. PGL1 could act as an oxygen species scavenger and protect M. leprae from killing by toxic oxygen metabolites.

Human phagocyte oxidative burst activation by BCG, M. leprae, and atypical mycobacteria: defective activation by M. leprae is not reversed by interferon gamma.

The activation of the phagocyte oxidative respiratory burst by various mycobacteria was evaluated in an in vitro assay, by measuring the chemiluminescence, associated to the release of oxidizing species, generated by normal human whole blood phagocytes. All mycobacterium species, except Mycobacterium leprae, induced a significant chemiluminescence response. The strongest stimulus was provided by BCG, followed by M. triviale, M. chelonei, and M. fortuitum. M. kansasii, M. intracellulare, and M. lepraemurium elicited a weak response, although higher than that triggered by M. leprae. Both polymorphonuclear and mononuclear cells contributed to the whole blood cell chemiluminescence stimulated by mycobacteria, mononuclear cells being more efficient on a per cell basis. Phagocyte activation by recombinant interferon gamma did not improve M. leprae ability to trigger a significant chemiluminescence response. The failure of M. leprae and of some atypical mycobacteria to stimulate a strong phagocyte oxidative respiratory burst may have some relevance to their pathogenicity.

Measurement of phagocyte chemiluminescence using a microtitre plate luminometer.

The use of chemiluminescence techniques to study the interaction between bacteria and phagocytes has been useful for examining the extent to which serum factors, such as opsonins, are important in internalization of the organisms and the response of the cell to phagocytosed bacteria. However, such methods have been limited by the number of experiments which can be performed at one time using most commercial luminometers. However, the recent introduction of the Amerlite microtitre plate luminometer allows the measurement of chemiluminescence responses in 96-well microtitre plates. Using this instrument, lucigenin-enhanced chemiluminescence can be detected from as few as 5000 cells (polymorphonuclear leukocytes or monocytes) per well with a 1:10 ratio of cells to zymosan particles opsonized with 10% serum. The opsonic capacity of up to 100 sera can be measured in triplicate wells in a single experiment using four microtitre plates and polymorphonuclear leukocytes prepared from less than 40 ml freshly obtained venous blood. We are currently using this technique to investigate the effect of serum opsonins on the interaction between normal human polymorphonuclear leukocytes and monocytes with mycobacteria of three species (Mycobacterium leprae, M. tuberculosis, and M. avium-intracellulare). Other possible applications of this method are discussed.