Summary of the 2019 alcohol and immunology research interest group (AIRIG) meeting: Alcohol-mediated mechanisms of multiple organ injury.

On November 15, 2019, the 24th annual Alcohol and Immunology Research Interest Group (AIRIG) meeting was held as a satellite conference during the annual Society for Leukocyte Biology meeting in Boston, Massachusetts. The 2019 meeting focused on alcohol, immunity, and organ damage, and included two plenary sessions. The first session highlighted new research exploring the mechanisms of alcohol-induced inflammation and liver disease, including effects on lipidomics and lipophagy, regulatory T cells, epigenetics, epithelial cells, and age-related changes in the gut. The second session covered alcohol-induced injury of other organs, encompassing diverse areas of research ranging from neurodegeneration, to lung barrier function, to colon carcinogenesis, to effects on viral infection. The discussions also highlighted current laboratory and clinical research used to identify biomarkers of alcohol use and disease.

Inhibition controls for qualitative real-time PCR assays: are they necessary for all specimen matrices?

A retrospective analysis of 386,706 specimens representing a variety of matrix types used in qualitative real-time PCR assays determined the overall inhibition rate to be 0.87% when the inhibition control was added preextraction to 5,613 specimens and 0.01% when the inhibition control was added postextraction but preamplification in 381,093 specimens. Inhibition rates of ≤ 1% were found for all specimen matrix types except urine and formalin-fixed, paraffin-embedded tissue.

Real-time PCR in clinical microbiology: applications for routine laboratory testing.

Real-time PCR has revolutionized the way clinical microbiology laboratories diagnose many human microbial infections. This testing method combines PCR chemistry with fluorescent probe detection of amplified product in the same reaction vessel. In general, both PCR and amplified product detection are completed in an hour or less, which is considerably faster than conventional PCR detection methods. Real-time PCR assays provide sensitivity and specificity equivalent to that of conventional PCR combined with Southern blot analysis, and since amplification and detection steps are performed in the same closed vessel, the risk of releasing amplified nucleic acids into the environment is negligible. The combination of excellent sensitivity and specificity, low contamination risk, and speed has made real-time PCR technology an appealing alternative to culture- or immunoassay-based testing methods for diagnosing many infectious diseases. This review focuses on the application of real-time PCR in the clinical microbiology laboratory.

Comparison of the Roche LightCycler vanA/vanB detection assay and culture for detection of vancomycin-resistant enterococci from perianal swabs.

We compared the performance characteristics of a real-time PCR method, the LightCycler vanA/vanB detection assay (Roche Diagnostics Corporation, Indianapolis, Ind.) to that of Enterococcosel agar (BBL, Sparks, Md.) for direct detection of vancomycin-resistant enterococci (VRE) from 894 perianal stool swabs. For 421 of 894 swabs, the result for LightCycler PCR was compared to an Enterococcosel plate containing vancomycin at 6 microg/ml; for the remaining 473 swabs, the result for LightCycler PCR was compared to an Enterococcosel plate containing 8 microg/ml vancomycin. The LightCycler method produced considerably more positive results than either the Enterococcosel plate containing vancomycin at 6 microg/ml (n = 25 versus n = 11; sensitivity, 100%; specificity, 97%; positive predictive value [PPV], 42%; negative predictive value [NPV], 100%) or the Enterococcosel plate containing vancomycin at 8 microg/ml (n = 31 versus n = 10; sensitivity, 100%; specificity, 95%; PPV, 32%; NPV, 100%). When possible, additional testing, including culture, LightCycler PCR, and/or a conventional PCR method (PCR-restriction fragment length polymorphism assay), were performed on either the original specimens or original cultures or subsequent specimens for cases in which the original specimen was positive by LightCycler PCR but the Enterococcosel plate was negative. This additional testing demonstrated positive results for 7 of 14 (50%) evaluable discordant specimens which initially tested as LightCycler PCR positive but culture negative using the Enterococcosel plate containing vancomycin at 6 microg/ml and 12 of 17 (71%) evaluable discordant specimens which initially tested as LightCycler positive but culture negative using the Enterococcosel plate containing vancomycin at (8 microg/ml). These results demonstrate that the LightCycler VRE detection assay is considerably more sensitive than the standard culture method for detecting VRE directly from perianal swab specimens. The LightCycler assay also provides results much faster than culture (approximately 3.5 versus > or =72 h). The use of this test could have important implications for the effective control and prevention of nosocomial outbreaks of VRE.

Real-time PCR method for detection of Encephalitozoon intestinalis from stool specimens.

The prevalence of microsporidiosis is likely underestimated due to the labor-intensive, insensitive, and nonspecific clinical laboratory methods used for the diagnosis of this disease. A real-time PCR assay was designed to assess DNA extraction methods and to detect three Encephalitozoon species in feces. Modifications of the MagNA Pure LC DNA isolation kit protocol (Roche Applied Sciences, Indianapolis, Ind.) were compared by using the automated MagNA Pure LC instrument (Roche) and fecal specimens spiked with Encephalitozoon intestinalis spores. Extracted DNA was amplified by the LightCycler (Roche) PCR assay. Assay sensitivity, reproducibility, and efficiency were assessed by comparing threshold crossover values achieved with different extraction and storage conditions (fresh, refrigerated, frozen, and preserved specimens). Optimal extraction conditions were achieved by using a commercial buffer, tissue lysis buffer (Roche), as the specimen diluent. LightCycler PCR results were compared to those obtained from routine stool microscopy with trichrome blue stain. The lower limit of detection for the LightCycler PCR assay varied by storage conditions from 10(2) to 10(4) spores/ml of feces, a value which represented a significant improvement over that achieved by staining (> or =1.0 x 10(6) spores/ml). Melting temperature analysis of the amplicons allowed for the differentiation of three Encephalitozoon species (E. intestinalis, E. cuniculi, and E. hellem). The assay is readily adaptable to the clinical laboratory and represents the first real-time PCR assay designed to detect Encephalitozoon species.

Electrostatic control of half-site spacing preferences by the cyclic AMP response element-binding protein CREB.

Basic region leucine zipper (bZIP) proteins represent a class of transcription factors that bind DNA using a simple, dimeric, alpha-helical recognition motif. The cAMP response element-binding protein (CREB) is a member of the CREB/ATF subfamily of bZIP proteins. CREB discriminates effectively in vivo and in vitro between the 10 bp cAMP response element (ATGACGTCAT, CRE) and the 9 bp activating protein 1 site (ATGACTCAT, AP-1). Here we describe an alanine scanning mutagenesis study designed to identify those residues within the CREB bZIP element that control CRE/AP-1 specificity. We find that the preference of CREB for the CRE site is controlled in a positive and negative way by acidic and basic residues in the basic, spacer and zipper segments. The CRE/AP-1 specificity of CREB is increased significantly by four glutamic acid residues located at positions 24, 28, 35 and 41; glutamic acid residues at positions 10 and 48 contribute in a more modest way. Specificity is decreased significantly by two basic residues located at positions 21 and 23; basic residues at positions 14, 18, 33 and 34 and V17 contribute in a more modest way. All of the residues that influence specificity significantly are located on the solvent-exposed face of the protein-DNA complex and likely participate in interactions between and among proteins, not between protein and DNA. The finding that the CRE/AP-1 specificity of CREB is dictated by the presence or absence of charged residues has interesting implications for how transcription factors seek and selectively bind sequences within genomic DNA.

Detection of herpes simplex virus DNA in genital and dermal specimens by LightCycler PCR after extraction using the IsoQuick, MagNA Pure, and BioRobot 9604 methods.

We evaluated two automated systems, MagNA Pure (Roche Molecular Biochemicals, Indianapolis, Ind.) and BioRobot 9604 (Qiagen, Inc., Chatsworth, Calif.) as effective replacements for the manual IsoQuick method (Orca Research, Inc., Bothell, Wash.) for extraction of herpes simplex virus (HSV) DNA from dermal and genital tract specimens prior to analysis by LightCycler PCR. Of 198 specimens (152 genital, 46 dermal), 92 (46.2%) were positive for HSV DNA by LightCycler PCR after automated extraction of specimens with either the MagNA Pure or BioRobot 9604 instrument. The manual IsoQuick method yielded HSV DNA (total n = 95) from three additional specimens that were negative by the automated method (P = 0.25, sign test). Although the mean numbers of LightCycler PCR cycles required to reach positivity differed statistically significantly among all three of the methods of extraction, the estimated means differed by no more than 1.5 cycles (P < 0.05). Seventy (76%) of the 92 specimens that were LightCycler PCR positive by all three extraction methods were also positive by shell vial cell culture assay. HSV DNA was detected by a lower LightCycler PCR cycle number (26.1 cycles) in specimens culture positive for the virus than in culture-negative samples (33.3 cycles) (P < 0.0001). The manual IsoQuick and automated MagNA Pure and BioRobot 9604 methods provide standardized, reproducible extraction of HSV DNA for LightCycler PCR. The decision to implement a manual versus an automated procedure depends on factors such as costs related to the number of specimens processed rather than on the minimal differences in the technical efficiency of extraction of nucleic acids among these methods.

Trends, rhythms, and aberrations in global climate 65 Ma to present.

Since 65 million years ago (Ma), Earth's climate has undergone a significant and complex evolution, the finer details of which are now coming to light through investigations of deep-sea sediment cores. This evolution includes gradual trends of warming and cooling driven by tectonic processes on time scales of 10(5) to 10(7) years, rhythmic or periodic cycles driven by orbital processes with 10(4)- to 10(6)-year cyclicity, and rare rapid aberrant shifts and extreme climate transients with durations of 10(3) to 10(5) years. Here, recent progress in defining the evolution of global climate over the Cenozoic Era is reviewed. We focus primarily on the periodic and anomalous components of variability over the early portion of this era, as constrained by the latest generation of deep-sea isotope records. We also consider how this improved perspective has led to the recognition of previously unforeseen mechanisms for altering climate.

The impact of alcohol and alcohol expectancies on male perception of female sexual arousal in a date rape analog.

The impact of alcohol and alcohol expectancies on men's perception of female sexual arousal and men's ability to discriminate accurately female sexual intentions in a dating situation was examined. In a 2 (alcohol) x 2 (expectancy) balanced placebo design, men were exposed to an audiotape of a date rape and asked to signal when the man should stop making sexual advances. On 4 occasions during the vignette, participants rated how sexually aroused the woman on the tape was at that moment. Relative to controls, participants who consumed alcohol or expected to consume alcohol took significantly longer to identify the inappropriateness of the man's sexual behavior toward his date. Similarly, alcohol participants also rated the woman's arousal level significantly higher at the first 2 refusals. Implications of the results are discussed.

Immunoserologic evidence of Human Granulocytic Ehrlichiosis in Danish patients with Lyme neuroborreliosis.

Human Granulocytic Ehrlichiosis (HGE) is a recently described human illness in the US which manifests as fever, myalgia and headache combined with pancytopenia and elevated concentrations of hepatic transaminases. Genetic analyses indicate that the agent of HGE appears to be an Ehrlichia species that is closely related to E. equi and E. phagocytophila. Ixodes dammini and I. scapularis were identified as potential vectors of HGE. Ixodes ticks are also the vector of Borrelia burgdorferi, the agent of Lyme borreliosis. The presence of antibodies against Ehrlichia in 132 sera from Danish patients with definite Lyme neuroborreliosis were examined in order to provide immunoserologic evidence of this infection in Denmark. Patients with Lyme neuroborreliosis were chosen as a test cohort, as these patients had been infested by a tick sufficient for transmission of B. burgdorferi. All had cerebrospinal fluid lymphocytic pleocytosis. As controls, serum samples from 50 healthy Danish blood donors were included. Of the 132 patients with Lyme neuroborreliosis, 5 (3.8%) reacted with the E. equi antigen substrate at titres 1:128. None of the blood donors were found seropositive for E. equi. At least 2 of the patients found seropositive for HGE constituted probable cases of HGE with E. equi antibody titres of at least 80 combined with fever, headache and myalgias. However, in no cases were we able to detect the presence of the HGE agent in the serum by PCR. We conclude that human exposure to granulocytic Ehrlichiae species may also occur in Europe, although further studies will be necessary to document active infection with these potential pathogens.

Sequence determinants of the intrinsic bend in the cyclic AMP response element.

The cyclic AMP response element (CRE site, ATGACGTCAT) is the DNA target for transcription factors whose activities are regulated by cyclic AMP (1). Recently, we discovered that the CRE site is bent by 10-13 degrees toward the major groove (2). Little or no bend is detected in the related AP-1 site (ATGACTCAT), which differs from the CRE site by loss of a single, central, C.G base pair (2, 3). Here we describe experiments designed to identify which base pairs within the CRE site induce the bent structure in an attempt to understand the origins of the dramatically different conformations of the CRE and AP-1 sites. Our data indicate that the intrinsic CRE bend results from distortion within the TGA sequence found in each CRE half site (ATGAC). These two TGA sequences are located in phase with one another in the CRE sequence but are not (completely) in phase in the AP-1 sequence. This difference in phasing leads to the overall difference in bend as detected by gel (2) and cyclization methods (S. C. Hockings, J. D. Kahn, and D. M. Crothers, unpublished results; M. A. Fabian and A. Schepartz, unpublished results). Our results confirm earlier predictions of altered structure within TG steps, provide insight into the structural reorganizations induced in DNA by bZIP proteins, and lead to a revision of the relationship between the structures of the free and bZIP-bound forms of the CRE and AP-1 sites.

Social workers' knowledge of and experience with sexual exploitation by psychotherapists.

A survey of licensed social workers in Texas using self-administered questionnaires revealed that, during the previous 12 months, 17 percent had worked with at least one victim of sexual exploitation by a psychotherapist. Social workers licensed as advanced clinical practitioners, licensed social workers with master's degrees, and those working in private practice were most likely to have worked with a victim of such sexual exploitation. Most respondents were not familiar with the provisions of new Texas legislation concerning sexual exploitation by psychotherapists, including definitions and reporting requirements. The majority indicated they would be interested in additional training.

Does inflammation contribute to the eradication of basal cell carcinoma following curettage and electrodesiccation?

BACKGROUND: Curettage and electrodesiccation (C&D) is probably the technique most frequently utilized by dermatologists to treat basal cell carcinomas (BCC). From histologic studies, it appears C&D does not completely mechanically remove all nests of BCC in a substantial number of cases. Nevertheless, the reported 5-year reoccurrence rate following C&D is significantly less than this histologically observed residual tumor frequency immediately following C&D. Among the multiple possibilities that exist to explain why these residual nests do not appear as recurrent tumor more frequently is the theory that inflammation developing after C&D clears residual tumor. OBJECTIVE: To test the hypothesis that inflammation developing after C&D clears residual tumor not mechanically removed by the procedure. METHODS: The frequency of residual BCC detected histologically immediately following C&D was compared with the frequency 1 month after the C&D, an amount of time in which an effect (if any) of inflammation could occur. RESULTS: Twenty-two of 29 primary BCC < 1 cm treated by C&D were tumor free immediately following the procedure (clearance rate, 75.9%). Eleven of 14 primary BCC < 1 cm treated by C&D then allowed to granulate 1 month before excision and histologic analysis were tumor free, for a clearance rate of 78.6%. Examination of larger tumors immediately following C&D revealed size is a significant variable for clearance rates. Eleven primary BCC > 1 cm but < 2 cm were examined histologically immediately following C&D; only three were tumor free for a clearance rate of 27.3%. Only one of five tumors > 2 cm thus treated was tumor free, for a clearance rate of 20%. Nine recurrent BCC of various sizes were treated by C&D and immediately examined histologically. Two were tumor free for a clearance rate of 22.2%. Two recurrent BCC were allowed to heal 1 month following C&D; one of these was tumor free when excised. CONCLUSION: For primary BCC < 1 cm, no evidence was found that inflammation occurring over 1 month following C&D clears residual tumor. It was also noted that C&D fails to completely remove tumor in a large majority of primary BCC > 1 cm, and in recurrent BCC.

Conformation of Tax-response elements in the human T-cell leukemia virus type I promoter.

BACKGROUND: HTLV-I Tax is believed to activate viral gene expression by binding bZIP proteins (such as CREB) and increasing their affinities for proviral TRE target sites. Each 21 bp TRE target site contains an imperfect copy of the intrinsically bent CRE target site (the TRE core) surrounded by highly conserved flanking sequences. These flanking sequences are essential for maximal increases in DNA affinity and transactivation, but they are not, apparently, contacted by protein. Here we employ non-denaturing gel electrophoresis to evaluate TRE conformation in the presence and absence of bZIP proteins, and to explore the role of DNA conformation in viral transactivation. RESULTS: Our results show that the TRE-1 flanking sequences modulate the structure and modestly increase the affinity of a CREB bZIP peptide for the TRE-1 core recognition sequence. These flanking sequences are also essential for a maximal increase in stability of the CREB-DNA complex in the presence of Tax. CONCLUSIONS: The CRE-like TRE core and the TRE flanking sequences are both essential for formation of stable CREB-TRE-1 and Tax-CREB-TRE-1 complexes. These two DNA segments may have co-evolved into a unique structure capable of recognizing Tax and a bZIP protein.

Evaluation of enzyme-linked immunoassay for serological diagnosis of cysticercosis.

We evaluated a commercially available enzyme-linked immunoassay (ELISA) from LMD Laboratories, Inc., Carlsbad, Calif., for the detection of antibodies in serum to the cysticercus of Taenia solium. The ELISA was performed on 308 serum samples; 198 from a pool of healthy individuals, 42 from patients who had antibodies against a variety of parasites other than T. solium, and 68 from patients suspected of having cysticercosis. All of these 68 specimens were tested both by the ELISA and by an immunoblot method (enzyme-linked immunoelectrotransfer blot assay [EITB]) developed at the Parasitic Serology Laboratory of the Centers for Disease Control and Prevention. Twenty-seven of the 68 serum samples from patients suspected of having cysticercosis were positive by both EITB and ELISA, while 31 were negative by both assays. ELISA results for three and two samples were considered false positive and false negative, respectively, when compared with the results of EITB. Results for an additional five samples were considered equivocal but were technically positive because their optical density readings were slightly above the cutoff value. Three of the 198 serum samples from the bank of serum samples from healthy individuals were also false positive by ELISA (the EITB result for the samples was negative). Six other serum samples from healthy individuals which had equivocal results and the five serum samples from patients with equivocal results were EITB negative. Serum samples containing antibodies against Echinococcus spp. frequently cross-reacted with the cysticercus ELISA antigen (13 of 16 specimens), but serum samples with antibodies against other parasites did not (2 of 26 specimens); all of these serum samples were EITB negative. The commercially available ELISA that we describe is a simple and rapid test. Considering all 308 specimens, the ELISA had a specificity of 93% (when samples with equivocal results were considered negative) or 89% (when samples with equivocal results were considered positive); the sensitivity was 93%.

Evaluation of an enzyme-linked immunoassay for the detection in serum of antibodies to Entamoeba histolytica.

A commercially available enzyme-linked immunoassay (ELISA) was compared to an indirect hemagglutination test (IHA) for the detection of antibodies to Entamoeba histolytica on 225 patients' serums. All of the 107 serums that had IHA titers of < 32 (interpreted as excluding the presence of invasive amebiasis) were ELISA negative. Sixty-three of 68 (93%) serums that had IHA titers of > or = 128 (interpreted as indicative of the presence of active or recent infection) were ELISA positive. Fifty serums had IHA titers of 32 or 64 that were considered "equivocal," and the ELISA results for these were: six positive, 35 negative, and nine "intermediate" (optical density values between the arbitrary positive cutoff and the low positive control). It was concluded that "intermediate" (and negative) ELISA results should be interpreted as excluding the presence of invasive amebiasis. Using these criteria, the results obtained with this ELISA appear to compare favorably with those of the "gold standard" IHA. Therefore, this ELISA provides a reliable alternative to the IHA for the serologic diagnosis of amebiasis, which may be advantageous for some laboratories in terms of lower cost, shorter test time, and improved efficiency.

Possible role of oceanic heat transport in early Eocene climate.

Increased oceanic heat transport has often been cited as a means of maintaining warm high-latitude surface temperatures in many intervals of the geologic past, including the early Eocene. Although the excess amount of oceanic heat transport required by warm high latitude sea surface temperatures can be calculated empirically, determining how additional oceanic heat transport would take place has yet to be accomplished. That the mechanisms of enhanced poleward oceanic heat transport remain undefined in paleoclimate reconstructions is an important point that is often overlooked. Using early Eocene climate as an example, we consider various ways to produce enhanced poleward heat transport and latitudinal energy redistribution of the sign and magnitude required by interpreted early Eocene conditions. Our interpolation of early Eocene paleotemperature data indicate that an approximately 30% increase in poleward heat transport would be required to maintain Eocene high-latitude temperatures. This increased heat transport appears difficult to accomplish by any means of ocean circulation if we use present ocean circulation characteristics to evaluate early Eocene rates. Either oceanic processes were very different from those of the present to produce the early Eocene climate conditions or oceanic heat transport was not the primary cause of that climate. We believe that atmospheric processes, with contributions from other factors, such as clouds, were the most likely primary cause of early Eocene climate.

Revictimization by polygraph: the practice of polygraphing survivors of sexual assault.

Sexual assault survivors are scrutinized in a manner unlike that meted out to any other victims of crime. Law enforcement officers or prosecutors may subject the survivor to a polygraph exam in an attempt to ascertain the truth or as a prerequisite to further investigation of the case. In a survey conducted with rape crisis centres across the United States, 63 centres in 17 states reported working with survivors of sexual assault who had been polygraphed. Rape crisis centres in 11 states reported that children had been polygraphed. This article examines the practice of polygraphing survivors of sexual assault.

Hemodialysis: evidence of enhanced molecular clearance and ultrafiltration volume by using pulsatile flow.

We describe several in vitro experiments showing evidence that pulsatile flow hemodialysis enhances ultrafiltration volume and molecular clearance as compared with steady flow hemodialysis. A new pulsatile pump and a conventional roller pump were compared using different hollow fiber dialyzers and a simulated blood solution containing urea, aspartame and vitamin B-12 at different flow rates and configurations. Ultrafiltration volume and concentration of urea, aspartame and B-12 were measured and molecular clearance (K) calculated. Ultrafiltration volume markedly increased with pulsatile flow. After 10 min K for urea with pulsatile flow was higher in all experiments even when ultrafiltration was prevented. Clearance of aspartame and B-12 also increased with pulsatile flow. We propose three mechanisms by which pulsatile flow is more efficient than steady flow hemodialysis: greater fluid energy, avoidance of molecular channeling and avoidance of membrane layering. We hypothesize that using pulsatile flow in hemodialysis can significantly shorten the duration of dialysis sessions for most of the patients, and consequently reduce the duration of the procedure and its cost.