Loop-mediated isothermal amplification assay for the diagnosis of retinitis caused by herpes simplex virus-1.

A loop-mediated isothermal amplification (LAMP) assay was developed for the detection of herpes simplex virus 1 (HSV-1). The specificity of the assay was tested using DNA extracted from HSV-1-infected rabbit corneal epithelium cultures, HSV-2 grown on Vero cell line, cytomegalovirus (CMV) (AD-169), varicella zoster virus (VZV) (Oka-vaccine), adenovirus, Aspergillus flavus and Staphylococcus aureus. The specificity of LAMP was confirmed by bidirectional sequencing of the amplicons. The sensitivity of the LAMP assay was tested using different concentrations of HSV-1 DNA. To evaluate the application of the LAMP assay in clinical diagnosis, we tested vitreous samples from 20 patients with suspected viral retinitis using LAMP and real-time PCR for HSV-1. The LAMP primers amplified only HSV-1 DNA; no LAMP products were detected with the DNAs of HSV-2, CMV, VZV, adenovirus A. flavus and S. aureus. The sequences of the positive HSV-1 LAMP products perfectly (99-100%) matched the HSV-1 sequences deposited in the GenBank database. LAMP is as sensitive as real-time PCR, with the lowest detection limit being 10 copies/µL of HSV-1 DNA. Of the 20 patients with suspected viral retinitis, four tested positive for HSV-1 using real- time PCR and LAMP. A 100% concordance was observed across the two methods. The LAMP assay is a rapid, highly specific and sensitive method for the diagnosis of retinitis caused by HSV-1.

Categorization of environmental sounds.

Sounds in the natural environment are non-stationary, in that their spectral dynamics is time-dependent. We develop measures to analyze the spectral dynamics of environmental sound signals and find that they fall into two categories-simple sounds with slowly varying spectral dynamics and complex sounds with rapidly varying spectral dynamics. Based on our results and those from auditory processing we suggest rate of spectral dynamics as a possible scheme to categorize sound signals in the environment.

Effects of Wetness Duration and Grain Development Stages on Sorghum Grain Mold Infection.

Grain mold caused by a complex of fungi is an economically important disease of sorghum worldwide. Little is known about the epidemiology of sorghum grain mold, which is essential for its management. Studies were conducted to quantify the effects of wetness duration on grain mold development under controlled conditions at ICRISAT. Six major sorghum grain mold fungi determined from previous field experiments, Curvularia lunata, Cladosporium oxysporum, Bipolaris australiensis, Fusarium moniliforme, F. pallidoroseum, and Phoma sorghina, were used. Panicles of a pot-grown mold-susceptible sorghum line, IS 10513, were spray inoculated with each fungus at five growth stages: flowering (F), milk (M), soft dough (S), hard dough (H), and physiological maturity (P), and were incubated in dew chambers for 0, 16, 24, 40, 48, and 72 h. Then, the plants were placed on greenhouse benches at 25 ± 1°C to allow grain mold infection to develop. Eight days after treatments, grains from F, M, and S stages were plated onto potato dextrose agar, while those from H and P stages were incubated in blotter paper humid chambers at 28 ± 1°C. Fungal colonization of grains were scored after 7 days. Results indicated a significant (P < 0.01) correlation between wetness duration and grain mold development at different stages of inoculation. Generally, with increasing wetness duration, there was an increase in grain infection by all six fungi. However, infection frequency varied among fungi and grain development stages, indicating that individual fungi might have different windows for maximum infection during the grain development stages.

Peginterferon alfa-2a in patients with chronic hepatitis C and cirrhosis.

BACKGROUND: Chronic hepatitis C virus (HCV) infection in patients with cirrhosis is difficult to treat. In patients with chronic hepatitis C but without cirrhosis, once-weekly administration of interferon modified by the attachment of a 40-kd branched-chain polyethylene glycol moiety (peginterferon alfa-2a) is more efficacious than a regimen of unmodified interferon. We examined the efficacy and safety of peginterferon alfa-2a in patients with HCV-related cirrhosis or bridging fibrosis. METHODS: We randomly assigned 271 patients with cirrhosis or bridging fibrosis to receive subcutaneous treatment with 3 million units of interferon alfa-2a three times weekly (88 patients), 90 microg of peginterferon alfa-2a once weekly (96), or 180 microg of peginterferon alfa-2a once weekly (87). Treatment lasted 48 weeks and was followed by a 24-week follow-up period. We assessed efficacy by measuring HCV RNA and alanine aminotransferase and by evaluating liver-biopsy specimens. A histologic response was defined as a decrease of at least 2 points on the 22-point Histological Activity Index. RESULTS: In an intention-to-treat analysis, HCV RNA was undetectable at week 72 in 8 percent, 15 percent, and 30 percent of the patients treated with interferon alfa-2a and with 90 microg and 180 microg of peginterferon alfa-2a, respectively (P=0.001 for the comparison between 180 microg of peginterferon alfa-2a and interferon alfa-2a). At week 72, alanine aminotransferase concentrations had normalized in 15 percent, 20 percent, and 34 percent of patients, respectively (P=0.004 for the comparison between 180 microg of peginterferon alfa-2a and interferon alfa-2a). In the subgroup of 184 patients with paired liver-biopsy specimens, the rates of histologic response at week 72 were 31 percent, 44 percent, and 54 percent, respectively (P=0.02 for the comparison between 180 microg of peginterferon alfa-2a and interferon alfa-2a). All three treatments were similarly tolerated. CONCLUSIONS: In patients with chronic hepatitis C and cirrhosis or bridging fibrosis, 180 microg of peginterferon alfa-2a administered once weekly is significantly more effective than 3 million units of standard interferon alfa-2a administered three times weekly.

Iron reduction as an adjuvant to interferon therapy in patients with chronic hepatitis C who have previously not responded to interferon: a multicenter, prospective, randomized, controlled trial.

Hepatic iron concentration has consistently been observed as being directly correlated with the response to interferon therapy in chronic hepatitis C virus (HCV). We therefore conducted a randomized, controlled trial comparing iron reduction by phlebotomy with iron reduction followed by retreatment with interferon in 96 patients with chronic hepatitis C who had previously not responded to a course of interferon. During the initial phase when all patients were undergoing phlebotomy, we found that serum alanine transaminase (ALT) activities decreased but by less than 50% from baseline in 67 patients (89%), decreased by more than 50% in 12 patients (13%) and became normal in 9 patients (9%) with no overall change in HCV-RNA levels. Subsequently no patient in either treatment group achieved a sustained virologic response. Improvements in necroinflammatory changes were noted in liver biopsy specimens in those patients receiving phlebotomy plus interferon (mean index 8.59 vs. 7.37, P <. 05). A slight but not statistically significant decrease in histologic activity index was noted in those subjects treated by phlebotomy alone (mean index 8.4 vs. 7.75, P not significant). We conclude that, although prior phlebotomy therapy does not improve the rate of sustained response to interferon retreatment, it does result in less liver injury manifested by a decrease in serum transaminase activity and a slight improvement in liver histopathology.

High-dose bolus lidocaine for chemical cardioversion of atrial fibrillation: a prospective, randomized, double-blind crossover trial.

BACKGROUND: Most drugs used for chemical cardioversion of atrial fibrillation have significant proarrhythmia risk and require close monitoring after administration. Lidocaine has few of the proarrhythmic concerns of most antiarrhythmic drugs and, at high bolus doses, prolongs the atrial refractory period well enough to be effective in converting atrial fibrillation to sinus rhythm. This finding has been previously demonstrated in a dog model. We sought to confirm the animal findings in human beings with lidocaine doses of 1.5 to 2.5 mg/kg. METHODS: Twenty patients with atrial fibrillation scheduled for elective cardioversion were enrolled in this study. In a randomized, double-blind, crossover study design, each patient received intravenous bolus lidocaine or saline. Patients were observed for 10 minutes after the initial bolus to assess efficacy. The second test drug was then delivered if the first was unsuccessful at cardioversion. RESULTS: All 20 patients received both lidocaine and saline placebo therapy in a crossover manner. None of the 20 patients converted to sinus rhythm with either therapy. The 95% confidence interval for effectiveness of lidocaine in this population was 0% to 14%. CONCLUSION: In this population of patients referred for elective cardioversion of atrial fibrillation, high-dose bolus lidocaine was ineffective in converting patients to sinus rhythm. Although this study was not sufficiently powered to rule out a low efficacy of lidocaine (<15%) or a higher efficacy in certain subgroups of atrial fibrillation, routine use of lidocaine for this indication is not warranted.

The endoplasmic reticulum chaperone glycoprotein GRP94 with Ca(2+)-binding and antiapoptotic properties is a novel proteolytic target of calpain during etoposide-induced apoptosis.

GRP94 is a 94-kDa chaperone glycoprotein with Ca(2+)-binding properties. We report here that during apoptosis induced by the topoisomerase II inhibitor etoposide, a fraction of GRP94 associated with the endoplasmic reticulum membrane undergoes specific proteolytic cleavage, coinciding with the activation of the caspase CPP32 and initiation of DNA fragmentation. In vivo, inhibitors of caspases able to block etoposide-induced apoptosis can only partially protect GRP94 from proteolytic cleavage, whereas complete inhibition is observed with calpain inhibitor I but not with the proteasome inhibitor. In vitro, GRP94 is not a substrate for CPP32; rather, it can be completely cleaved by calpain, a Ca(2+)-regulated protease. The cleavage of GRP94 by calpain is Ca(2+)-dependent and generates a discrete polypeptide of 80 kDa. In contrast, calpain has no effect on other stress proteins such as GRP78 or HSP70. Further, immunohistochemical staining reveals specific co-localization of GRP94 with calpain in the perinuclear region following etoposide treatment. We further showed that reduction of GRP94 by antisense decreased cell viability in etoposide-treated Jurkat cells. Our studies provide new evidence that the cytoprotective GRP94, as in the case of the antiapoptotic protein Bcl-2, can be targets of proteolytic cleavage themselves during the apoptotic process.

The significance of hepatitis G virus in serum of patients with sporadic fulminant and subfulminant hepatitis of unknown etiology.

Excluding acute hepatic failure caused by drugs, the etiology of fulminant hepatitis (FH) remains unknown in many patients. There are conflicting data about a possible pathogenic role for the hepatitis G virus (HGV) in patients with cryptogenic fulminant hepatitis (non-A-E FH). We investigated the presence of circulating HGV in 36 patients with well-documented non-A-E fulminant and 5 patients with subfulminant hepatitis from 3 geographic locations in the United States. Serum HGV RNA was determined by reverse transcriptase-polymerase chain reaction using primers from the NS5 region of the HGV genome. HGV RNA was also measured before and after liver transplantation in 5 patients and at different time points in 7 patients. Serum samples were recoded and reanalyzed for HGV RNA using different primer sets to assess the validity of the HGV RNA assay. HGV was present in serum of 14 of the 36 patients (38.8%) with non-A-E fulminant hepatitis. Twenty percent of patients from the Northeast, 11% of the patients from the Southeast, and 50% from the Mid-Atlantic regions of the United States had circulating HGV RNA. The use of therapeutic blood products was significantly associated with the presence of serum HGV RNA (P <.02). Retesting for HGV RNA with different primers was positive in all but 1 case. HGV RNA is not causally related to non-A-E fulminant hepatitis. The finding of HGV RNA in serum from these patients is likely related to the administration of blood product transfusion after the onset of fulminant hepatitis.

High-molecular-weight FK506-binding proteins are components of heat-shock protein 90 heterocomplexes in wheat germ lysate.

In animal cell lysates the multiprotein heat-shock protein 90 (hsp90)-based chaperone complexes consist of hsp70, hsp40, and p60. These complexes act to convert steroid hormone receptors to their steroid-binding state by assembling them into heterocomplexes with hsp90, p23, and one of several immunophilins. Wheat germ lysate also contains a hsp90-based chaperone system that can assemble the glucocorticoid receptor into a functional heterocomplex with hsp90. However, only two components of the heterocomplex-assembly system, hsp90 and hsp70, have thus far been identified. Recently, purified mammalian p23 preadsorbed with JJ3 antibody-protein A-Sepharose pellets was used to isolate a mammalian p23-wheat hsp90 heterocomplex from wheat germ lysate (J.K. Owens-Grillo, L.F. Stancato, K. Hoffmann, W.B. Pratt, and P. Krishna [1996] Biochemistry 35: 15249-15255). This heterocomplex was found to contain an immunophilin(s) of the FK506-binding class, as judged by binding of the radiolabeled immunosuppressant drug [3H]FK506 to the immune pellets in a specific manner. In the present study we identified the immunophilin components of this heterocomplex as FKBP73 and FKBP77, the two recently described high-molecular-weight FKBPs of wheat. In addition, we present evidence that the two FKBPs bind hsp90 via tetratricopeptide repeat domains. Our results demonstrate that binding of immunophilins to hsp90 via tetratricopeptide repeat domains is a conserved protein interaction in plants. Conservation of this protein-to-protein interaction in both plant and animal cells suggests that it is important for the biological action of the high-molecular-weight immunophilins.

Small-diameter (2 mm) laparoscopy in the evaluation of liver disease.

BACKGROUND: Percutaneous liver biopsy fails to demonstrate cirrhosis in approximately 32% of cases when compared with laparoscopy with liver biopsy. The aim of this study is to determine the usefulness of small-diameter (2 mm) laparoscopes compared with larger laparoscopes. METHODS: Patients undergoing diagnostic laparoscopy for various liver diseases were evaluated with small-diameter (2 mm) laparoscopes either alone or in combination with a 5 or 10 mm laparoscope. RESULTS: Twenty patients were enrolled in this study. Small-diameter laparoscopes provided appropriate visualization of the abdominal organs and proper guidance to liver biopsy in 9 cases. In the remaining 11 cases a larger laparoscope was used for the following reasons: short length of the trocar/introducer in a morbidly obese patient (1), liver mass located in the anterosuperior aspect of the liver precluding good visualization with forward lenses (1), and inability to properly visualize the anterosuperior aspect of the liver (9). No complications were noted with the use of the small-diameter laparoscopes alone. CONCLUSION: There is a need for an oblique-viewing minilaparoscope that allows visualization comparable to the larger laparoscopes.

A mixed population of immature and mature leucocytes in umbilical cord blood results in a reduced expression and function of CR3 (CD11b/CD18).

Neonatal neutrophils express less membrane and cytoplasmic CR3 (iC3b-receptor, Mac-1, alphaM beta2-integrin) than do adult neutrophils, and it has been suggested that this renders neonatal neutrophils deficient in diapedesis and bactericidal activity. The reason(s) for this deficiency are unknown. In this study, CR3 expression and the CR3-dependent respiratory burst activity of individual neonatal neutrophils are quantified in comparison with adult leucocytes using flow cytometry. Monocytes and neutrophils are defined as CD14highCD15low and CD14lowCD15high, respectively. Although neonatal neutrophils bore less CR3 on average than did adult neutrophils, neonatal neutrophils were more heterogeneous and many neonatal neutrophils expressed adult levels of CR3. Because of higher neutrophil concentrations in cord versus adult blood, the calculated number of neutrophils in cord blood expressing high amounts of CR3 was equivalent to that of adult blood. Similar findings were made with monocytes. The size of the CR3-dependent respiratory burst stimulated by particulate beta-glucan correlated directly with the expression of CR3 by individual neutrophils. With neonatal and adult neutrophils having comparable CR3 densities, the respiratory burst activities were equivalent. Wright-Giemsa differential staining of the subset of neonatal neutrophils with low CR3 levels isolated by fluorescence-activated cell sorting showed a higher proportion of immature cells than the sorted population expressing high CR3 levels. Therefore, higher proportions of immature cells in cord blood probably explain previous reports of deficient CR3 expression and function. The typical neutrophilia of cord blood may compensate for this apparent deficiency by providing adult concentrations of mature neutrophils.

Quantitative reverse transcriptase-polymerase chain reaction measured expression of MDR1 and MRP in primary breast carcinoma.

To evaluate the clinical significance of drug resistance mechanisms in breast cancer, we examined the expression of MDR1 and MRP in primary breast carcinoma and normal adjacent tissue using a highly quantitative and reproducible reverse transcription-PCR assay. Expression of both genes was observed in all specimens examined, both tumor (n = 74) and normal adjacent tissue (n = 55). The expression of MDR1, however, was low, with the level of expression being 25 times less than the drug-resistant control cell line KB 8-5. Immunohistochemical analysis of P-glycoprotein corroborated the PCR results; only 6% (2 of 31) were positive for JSB1 staining, and 0 of 32 were positive for for UIC2. MRP expression did not exceed control cell line levels, and immunohistochemistry detected moderate levels of expression. MDR1 expression was independent of grade, stage, tumor size, nodal status, metastasis, and estrogen receptor and progesterone receptor status. There was, however, a significant correlation of MDR1 expression with age and histology. Approximately twice the expression of MDR1 was observed in the < 50 age group compared to the > 50 age group, and lobular carcinoma had 4 times the expression of MDR1 of other histological types. MRP expression was independent of all other clinical parameters. Thus, these results show that although MDR1 expression is detectable in primary breast carcinoma by PCR, this expression as measured by quantitative reverse transcriptase-PCR is extremely low. The significance of these low levels is yet to be determined. MDR1 expression was higher in < 50 age group and lobular carcinoma, which may contribute to poor prognosis associated with young age and lobular histology.

Biphasic transthoracic defibrillation causes fewer ECG ST-segment changes after shock.

STUDY OBJECTIVE: Electrocardiographic abnormalities are common after transthoracic defibrillation. ECG ST-segment changes are especially problematic after defibrillation and may indicate ischemic or shock-induced cardiac dysfunction after resuscitation. Biphasic defibrillation waveforms, compared with monophasic waveforms, diminish shock-induced cardiac dysfunction in laboratory preparations. This effect has not been validated in human subjects. We therefore evaluated in a prospective, blinded fashion the effect of biphasic and monophasic transthoracic defibrillation on the ECG ST segment in 30 consecutive patients during surgery for the implantation of a cardioverter-defibrillator. METHODS: In each patient two low-energy truncated biphasic transthoracic defibrillation shocks (115 and 130 J) were compared with a standard clinical 200 J monophasic damped-sine wave shock. The biphasic shocks and the damped-sine wave shock have been demonstrated to have equal defibrillation efficacy of 97%. Fifteen-second ECG signals recorded across transthoracic defibrillation electrodes were digitized before ventricular fibrillation induction and immediately after each defibrillation attempt. The ST segments 80 msec after the J point were analyzed in a blinded fashion by two reviewers. The ST-segment deflection, QRS-interval duration, QT interval, and heart rate after each therapy were compared with baseline values. RESULTS: ECG ST-segment elevation was significantly greater with the 200-J damped-sine waveform than with either biphasic waveform. The ECG ST-segment levels were -.55 +/- .36 at baseline, -.76 +/- .36 mm after internal shock, -.02-.36 mm after 115-J biphasic shock, .21 +/- .38 mm after 130-J biphasic shock, and 2.09 +/- .37 mm after 200-J damped-sine wave shock (P<.0001). QRS-interval duration, QT interval, and heart rate did not change significantly with any waveform. CONCLUSION: Transthoracic defibrillation with biphasic waveforms results in less postshock ECG evidence of myocardial dysfunction (injury or ischemia) than standard monophasic damped sine waveforms without compromise of defibrillation efficacy.

Analysis of expression and function of topoisomerase I and II during meiosis in male mice.

Topoisomerases are nuclear enzymes that remove torsional stress in DNA. Their function is important for replication, transcription, chromosome condensation, and chromosome segregation during mitosis and meiosis. The goal of this work is to analyze both expression and function of topoisomerases during the meiotic stages of mammalian spermatogenesis. The patterns of expression of topoisomerase I and topoisomerase II alpha genes were followed on Northern blots of RNA from testes of mice of different ages and from specific germ cell populations. The transcript of the topoisomerase I gene was highest in somatic cells of the testis and in the mitotically proliferating spermatogonia and meiotic prophase spermatocytes, with the level of transcript decreasing dramatically in postmeiotic spermatids. In contrast, the levels of topoisomerase II alpha transcript were negligible in germ-cell free testes and highest in late meiotic prophase cells and round spermatids. Enzyme activity for both topoisomerase I and topoisomerase II was detected in both pachytene spermatocytes and in round spermatids; topoisomerase II exhibited a higher level of activity in meiotic spermatocytes than in round spermatids. In cultured cells, camptothecin, an inhibitor of topoisomerase I, caused some abnormalities of paired meiotic homologs, but did not inhibit the transition to metaphase. In contrast, teniposide and ICRF-193, inhibitors of topoisomerase II, dramatically inhibited the formation of metaphase chromosomes in cells induced to progress from prophase to metaphase. However, the disassembly of the synaptonemal complex was not inhibited, indicating that this process could be uncoupled from condensation of chromatin to form chromosomes. These studies constitute evidence for a functional requirement for topoisomerase II activity in the transition from meiotic prophase to meiotic metaphase I in mammalian spermatocytes.

Analysis of the native forms of the 90 kDa heat shock protein (hsp90) in plant cytosolic extracts.

A polyclonal antibody, R2, was raised against a fusion protein consisting of a portion of plant hsp90 fused to the trpE protein of Escherichia coli. This antibody was found to be specific towards plant hsp90, showing little or no cross-reactivity with mouse and human hsp90 proteins. The R2 antibody identified an 83 kDa protein as the hsp90 homologue in cytosolic extracts of several dicot and monocot plants. Two-dimensional gel electrophoresis indicated that at least two different isoforms of hsp90 are expressed in Brassica napus seedlings. An examination of the native state of hsp90 by non-denaturing gel electrophoresis showed that this protein exists as a monomer, dimer and as a high-molecular-mass complex of ca. 680 kDa in cell extracts of spinach cotyledons and leaves, B. napus seedlings and wheat germ. Native gel analysis and cross-linking studies of purified hsp90 showed that plant hsp90 exists predominantly as a monomer. When 35S-labelled B. napus cytosolic extracts were immunoprecipitated with the R2 antiserum, hsp90 and two additional proteins with approximate molecular masses of 49 and 45 kDa were detected in the immunoprecipitates. These results are consistent with the idea that hsp90:protein heterocomplexes exist in plant cells.

Unipolar pectoral defibrillation systems.

Over the past 15 years, the implantation of automatic defibrillations has evolved from an obscure, impractical, and often morbid procedure to nearly a routine therapy. Initial large abdominally implanted generators with multiple epicardial leads have given way to much smaller, pectorally implanted systems utilizing only a single lead. These systems are better accepted by physicians and patients and rival recent-generation pacemakers in their implantation simplicity. Outcomes with single lead defibrillator implantation have been excellent. They are 99% effective at eliminating sudden death in large cohorts of patients, with overall survival of 94.4% at 18 months. Previously significant perioperative complications and mortality associated with epicardial systems have been virtually eliminated. Transvenous single lead systems now provide defibrillation efficacy at a level that makes epicardial leads unnecessary in most patients. Although inappropriate shocks are not a morbid complication, they still occur in approximately 15%-30% of patients. This is an area for improvement in defibrillator therapy, which, though invisible in total mortality statistics, is significant in terms of patient comfort and acceptance. Incremental improvements in pulse generator design and defibrillator lead technology are being made. Perhaps the most interesting new development will be the dual chamber device, incorporating and atrial electrode for sensing, pacing, and perhaps, atrial defibrillation. Such improvements will continue to make device therapy of all arrhythmias more versatile and improve patient comfort both in terms of device size and inappropriate shocks. It is unlikely, however, that further technological advances can further diminish the already small complication rate or improve the already excellent efficacy of current single lead systems. Defibrillator technology has already reached a maturity where technological improvements are less significant than efforts to better define the patient population who will benefit from the therapy.

Experience with unipolar pectoral defibrillation.

With simple, single lead unipolar pectoral defibrillators, ICD technology has reached a level of ease and safety comparable to pacemaker implantation. It will be difficult to further decrease the morbidity associated with ICD implantation; just as it will be difficult to improve upon current device treatment of sudden cardiac death. Even as further incremental improvements in devices and leads will undoubtedly occur, at this point in ICD evolution, it is investigating the expanded use of this therapy as a prevention tool that is likely to have the largest overall impact on cardiac arrest survival.