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PubMed2026

Biologic-biologic and biologic-JAK inhibitor combination therapy in refractory systemic autoinflammatory diseases.

OBJECTIVES: Systemic autoinflammatory diseases (SAIDs) arise from genetic defects in innate immunity, leading to dysregulated activation of inflammatory pathways, including interleukin (IL)-1, IL-6, TNF, and JAK/STAT. Clinical manifestations range from recurrent fever to severe complications such as encephalitis and AA amyloidosis. Management aims to control inflammation using immunosuppressive agents and targeted monotherapies (biologics or JAK inhibitors). Advanced combination therapy (ACT), defined as the use of biologics and/or JAK inhibitors in combination, has emerged as a strategy for refractory disease. METHODS: In this observational retrospective longitudinal cohort study, patients with SAIDs treated with ACT were included. Demographic, clinical, treatment, and safety data were collected. Treatment response was assessed using a composite outcome incorporating corticosteroid dose, C-reactive protein (CRP), and clinical improvement and categorized as non-response, partial response, or complete response. RESULTS: Thirty-eight patients (median age 30 years [range 4-76]) were included. The most common indications for ACT were pyogenic arthritis, pyoderma gangrenosum and acne (PAPA), mevalonate kinase deficiency (MKD), and undifferentiated SAIDs. Most patients had disease-related complications and were dependent on glucocorticoids and/or opioids to control inflammation and pain, respectively. Following multiple ACT trials, complete response was observed in 21 patients (55.3%), partial response in 12 (31.6%), and no response in 5 (13.1%). Overall, 65 ACT regimens were administered, most commonly combining IL-1 and TNF inhibitors. Thirty-nine regimens were discontinued because of lack of efficacy, secondary loss of response, or adverse events. At the final follow-up, 26 patients (68%) remained on ACT, with a median treatment duration of 60 months (range, 11-186). CONCLUSIONS: ACT offers significant clinical benefits for patients with difficult-to-treat SAIDs, though challenges such as secondary loss of efficacy and infection risks remain.

Humans↗
PubMed1998

Basics of flow cytometry.

In summary, a beginner requires fundamental knowledge about flow cytometric instrumentation in order to effectively use this technology. It is important to remember that flow cytometers are very complex instruments that are composed of four closely related systems. The fluidic system transports particles from a suspension through the cytometer for interrogation by an illumination system. The resulting light scattering and fluorescence is collected, filtered, and converted into electrical signals by the optical and electronics system. The data storage and computer control system saves acquired data and is also the user interface for controlling most instrument functions. These four systems provide a very unique and powerful analytical tool for researchers and clinicians. This is because they analyze the properties of individual particles, and thousands of particles can be analyzed in a matter of seconds. Thus, data for a flow cytometric sample are a collection of many measurements instead of a single bulk measurement. Basic knowledge of instrumentation is a tremendous aid to designing experiments that can be successfully analyzed using flow cytometry. For example, it is important to know the emission wavelength of the laser in the instrument that will be used for analysis. This wavelength is critical knowledge for selecting probes. It is also important to understand that a different range of wavelengths is detected for each fluorescent channel. This will aid selection of probes that are compatible with the flow cytometer. Understanding the complication that emission spectra overlap contributes to detection can be used to guide fluorochrome selections for multicolor analysis. All of these experiment design considerations that rely on knowledge of how flow cytometers work are a very practical and effective means of avoiding wasted time, energy, and costly reagents. Data analysis is a paramount issue in flow cytometry. Analysis includes interpreting as well as presenting data that has been stored in list-mode files. Data analysis is very graphically oriented. There are a number of types of graphic representation that are available to visually aid data analysis. Two standard types of displays are used. These data plots are one-parameter histograms and bivariate plots. A user must be familiar with these two fundamental types of display in order to effectively analyze data. Histograms are the most simple modes of data representation. Histograms allow visualization of a single acquired parameter. Mean fluorescence and distributional statistics can be obtained based on markers that the user can graphically set on the plot. Percentages of positively expressing particles relative to a control sample can also obtained in a similar manner. In addition, multiple histograms can be overlayed on one another to depict qualitative and quantitative differences in two or more samples. Two-parameter data plots are somewhat more complicated than histograms; however, they can yield more information. Two-parameter dot plots of FSC vs SSC allow visualization of both light-scattering parameters that are important for identifying populations of interest. Bivariate fluorescent plots allow discrimination of dual-labeled populations that might remain hidden if histograms were used to display fluorescent data. Two-parameter plots that combine one light-scattering parameter and a fluorescent parameter are useful for analyzing control samples to elucidate the origin of nonspecific binding. Data analysis is very graphically oriented. Experience and pattern recognition become important when using two-parameter data plots for qualitative as well as quantitative analysis. The technique of gating or drawing regions on dual parameter light-scatter plots allows one to exclude information and examine the population of interest by disallowing particles that might confound or interfere with analysis. This is one of the fundamental uses for gating. (ABSTRACT TRUNCATED)

Animals↗
PubMedDate not supplied

Issues of women dually diagnosed with HIV infection and substance use problems in the Carolinas.

A growing number of women are being dually diagnosed with HIV infection and substance use problems. Forty-two percent of all women diagnosed with AIDS have been infected through injection drug use. Many more women with HIV are exposed to nonintravenous drugs that potentially affect their quality of life and illness experience. This study sought to identify from the perspective of women factors that most influenced their ability to obtain treatment for their HIV infection and control their substance use. A focus group approach was used for data collection. Twenty-five HIV-infected women participated in one of four focus groups. Women were asked to identify and discuss their concerns and needs related to HIV/AIDS and substance use. Twenty-four women were African-American; one was white. All the women reside in South Carolina or North Carolina. Each focus group session was audiotaped and transcribed. Content analysis, following Krippendorff's (1980) methodology, was used to analyze the data. Five themes emerged: 1) AIDS as a life-altering event; 2) spirituality; 3) mental health issues; 4) barriers to health care services; and 5) environmental influences. It was concluded that the coexistence of HIV and substance abuse adds to the complexity of women's treatment needs. For these women, an HIV diagnosis can serve to alter their lives either positively or negatively. Dually diagnosed women have unique needs that require integration of physical and psychosocial interventions. These women may benefit from the services of psychiatric or mental health nurse practitioners who have the skills necessary to address the many psychosocial issues women face as well as provide physical treatment. Additionally, drug treatment services need to be expanded and made more comprehensive. Drug treatment programs need to be developed specifically for women, and these services need to be made accessible to poor women with substance abuse problems. Further, drug treatment programs need to provide comprehensive services that can appropriately integrate the treatment of HIV disease and substance abuse.

Adult↗
PubMed2000

Genetically and epidemiologically related "non-syncytium-inducing" isolates of HIV-1 display heterogeneous growth patterns in macrophages.

The objective of this study was to identify phenotypic parameters that could distinguish among seemingly homogeneous non-syncytium-inducing (NSI) viruses and that might provide a surrogate marker for clinical progression in pediatric human immunodeficiency virus type 1 (HIV-1) infection. We undertook a pilot analysis of 15 independent HIV-1 isolates collected prospectively from two mothers and their four children who displayed a spectrum of disease stages ranging from CDC categories A1 to C3. Viruses were evaluated for their ability to replicate in primary cells (including monocyte-derived macrophages [MDM]) and cell lines, for their co-receptor preference and for genetic features of the V3 hypervariable domain of env. Virtually all isolates displayed NSI phenotypes that were restricted in their capacity to replicate in cell lines and displayed V3 loops with uniformly low net positive charges. NSI viruses from two symptomatic children and one mother were macrophage-tropic, whereas NSI isolates from two asymptomatic children were unable to replicate in MDM and were designated primary lymphotropic viruses. Only one isolate was syncytium-inducing (SI), replicated in a variety of cell lines and in MDM, used multiple co-receptors, and was dual tropic, rather than a mixture of T-cell tropic and M-tropic viruses, as assessed by genetic analysis. Phenotypic heterogeneity among NSI viruses is revealed in the ability of isolates to replicate in MDM. This characteristic is related to disease stage and provides a potentially new in vitro criterion to distinguish among NSI isolates that is unlinked to other surrogate markers.

Adult↗
PubMed1999

Linking the integrated management of childhood illness (IMCI) and health information system (HIS) classifications: issues and options.

Differences in the terms used to classify diseases in the Integrated Management of Childhood Illness (IMCI) guidelines and for health information system (HIS) disease surveillance could easily create confusion among health care workers. If the equivalent terms in the two classifications are not clear to health workers who are following the guidelines, they may have problems in performing the dual activities of case management and disease surveillance. These difficulties could adversely affect an individual's performance as well as the overall effectiveness of the IMCI strategy or HIS surveillance, or both. We interviewed key informants to determine the effect of these differences between the IMCI and HIS classifications on the countries that were implementing the IMCI guidelines. Four general approaches for addressing the problem were identified: translating the IMCI classifications into HIS classifications; changing the HIS list to include the IMCI classifications; using both the IMCI and HIS classification systems at the time of consultations; and doing nothing. No single approach can satisfy the needs of all countries. However, if the short-term or medium-term goal of IMCI planners is to find a solution that will reduce the problem for health workers and is also easy to implement, the approach most likely to succeed is translation of IMCI classifications into HIS classifications. Where feasible, a modification of the health information system to include the IMCI classifications may also be considered.

Case Management↗
PubMed2005

The Italian Implantable Cardioverter-Defibrillator Registry. A survey of the national activity during the years 2001-2003.

BACKGROUND: In recent years several trials demonstrated the efficacy of implantable cardioverter-defibrillator (ICD) therapy for sudden cardiac death prevention and total mortality reduction in particular high-risk groups of patients. The aim of this review was to report the main epidemiological data and the most important clinical characteristics of patients enrolled in the Italian ICD Registry in the years 2001-2003. METHODS: The Italian ICD Registry--official member of the Italian Association of Arrhythmology and Cardiac Pacing (AIAC)--collects 85% of the data concerning the national ICD implantation activity, based on the European Implantable Defibrillator form (EURID). Data are validated for quality of information and uniqueness at the moment of data entry and in successive steps at the time of the annual analysis. RESULTS: The number of ICDs implanted in Italy has been continuing to increase during the last years according to the general trend in European and non-European countries: 2400 in the year 2001, 3934 in the year 2002, and 5318 in the year 2003. The number of ICDs per million of inhabitants in Italy was 42.1 in the year 2001 (+11.8% with respect to 2000), 69.0 in the year 2002 (+63.9% with respect to 2001), and 93.3 in the year 2003 (+35.2% with respect to 2002). The number of implanting centers increased progressively from 273 in the year 2001 to 304 in the year 2002, and 340 in the year 2003. The median age of patients treated with ICD implantation was 67 years in the years 2001-2002, 68 years in the year 2003. The prevalence of male patients was significantly higher (79.3% in 2001, 82.3% in 2002, and 81.4% in 2003). The main indication was syncope (25.5, 29.3, and 32.9% in the years 2001, 2002, and 2003, respectively), followed by palpitations (17.7, 18.5, and 16.4% in the years 2001, 2002, and 2003, respectively), and cardiac arrest (10.0, 13.1, and 16.5% in the years 2001, 2002, and 2003, respectively). The use of ICD in patients considered at risk but without history of sustained ventricular tachycardia had a 3-fold increase during the 3 years, from 6.4% in 2001 to 18.2% in 2003. Ventricular tachycardia was the main arrhythmia in 50.4 to 55.0% of cases, ventricular fibrillation in 13.5 to 18.1%, both in 4.1 to 6.5%. The vast majority of patients presented at the enrolment either a mild or severe reduction in ejection fraction (30 to 50%, < 30%). Amiodarone was administered alone or in combination with antiarrhythmics in 29.7 to 40.0% of patients. Single-chamber ICDs were implanted in the years 2002 and 2003 in 45.7 and 39.2% of patients, dual-chamber ICDs in 34.9 and 32.4%, biventricular ICDs in 19.4 and 28.4%, respectively. CONCLUSIONS: The ICD implantation rate in Italy increased significantly in the period 2001-2003, similarly to the trend in the other western countries and following the publication of controlled studies in the field of primary and secondary prevention of sudden cardiac death. The Italian ICD Registry showed during the last 3 years an important increase in prophylactic ICD utilization. A sophisticated ICD, including dual-chamber pacing or cardiac resynchronization therapy, was chosen in a high percentage of patients.

Aged↗
PubMed2005

Cloning and molecular modeling of duodenase with respect to evolution of substrate specificity within mammalian serine proteases that have lost a conserved active-site disulfide bond.

Mammalian serine proteases such as the chromosome 14 (Homo sapiens, Mus musculus) located granzymes, chymases, cathepsin G, and related enzymes including duodenase collectively represent a special group within the chymotrypsin family which we refer to here as "granases". Enzymes of this group have lost the ancient active-site disulfide bond Cys191-Cys220 (bovine chymotrypsinogen A numbering) which is strongly conserved in classic serine proteases such as pancreatic, blood coagulation, and fibrinolysis proteases and others (granzymes A, M, K and leukocyte elastases). We sequenced the cDNA encoding bovine (Bos taurus) duodenase, a granase with unusual dual trypsin-like and chymotrypsin-like specificity. The sequence revealed a 17-residue signal peptide and two-residue (GlyLys) activation peptide typical for granases. Production of the mature enzyme is apparently accompanied by further proteolytic processing of the C-terminal pentapeptide extension of duodenase. Similar C-terminal processing is known for another dual-specific granase, human cathepsin G. Using phylogenetic analysis based on 39 granases we retraced the evolution of residues 189 and 226 crucial for serine protease primary specificity. The analysis revealed that while there is no obvious link between mutability of residue 189 and the appearance of novel catalytic properties in granases, the mutability of residue 226 evidently gives rise to different specificity subgroups within this enzyme group. The architecture of the extended substrate-binding site of granases and structural basis of duodenase dual specificity based on molecular dynamic method are discussed. We conclude that the marked selectivity of granases that is crucial to their role as regulatory proteases has evolved through the fine-tuning of specificity at three levels--primary, secondary, and conformational.

Amino Acid Sequence↗
PubMed2006

Prevention of catheter lumen occlusion with rT-PA versus heparin (Pre-CLOT): study protocol of a randomized trial [ISRCTN35253449].

BACKGROUND: Many patients with end-stage renal disease use a central venous catheter for hemodialysis access. A large majority of these catheters malfunction within one year of insertion, with up to two-thirds due to thrombosis. The optimal solution for locking the catheter between hemodialysis sessions, to decrease the risk of thrombosis and catheter malfunction, is unknown. The Prevention of Catheter Lumen Occlusion with rt-PA versus Heparin (PreCLOT) study will determine if use of weekly rt-PA, compared to regular heparin, as a catheter locking solution, will decrease the risk of catheter malfunction. METHODS/DESIGN: The study population will consist of patients requiring chronic hemodialysis thrice weekly who are dialyzed with a newly inserted permanent dual-lumen central venous catheter. Patients randomized to the treatment arm will receive rt-PA 1 mg per lumen once per week, with heparin 5,000 units per ml as a catheter locking solution for the remaining two sessions. Patients randomized to the control arm will receive heparin 5,000 units per ml as a catheter locking solution after each dialysis session. The study treatment period will be six months, with 340 patients to be recruited from 14 sites across Canada. The primary outcome will be catheter malfunction, based on mean blood flow parameters while on hemodialysis, with a secondary outcome of catheter-related bacteremia. A cost-effectiveness analysis will be undertaken to assess the cost of maintaining a catheter using rt-PA as a locking solution, compared to the use of heparin. DISCUSSION: Results from this study will determine if use of weekly rt-PA, compared to heparin, will decrease catheter malfunction, as well as assess the cost-effectiveness of these locking solutions.

Bacteremia↗
PubMed1996

Evidence for dual signaling pathways for V2 vasopressin receptor in rat inner medullary collecting duct.

Previous studies have demonstrated that both the V2-receptor agonist, 1-desamino-8-D-arginine vasopressin (dDAVP), and the V1a-receptor agonist, [Phe2, Orn8]vasotocin (PO-VT), increase intracellular calcium concentration ([Ca2+]i) in the rat inner medullary collecting duct (IMCD). The present studies were done to clarify the receptor subtype(s) responsible for calcium mobilization. Measurements of [Ca2+]i, using fura 2 in microdissected IMCD segments, confirmed that arginine vasopressin (AVP), dDAVP, and PO-VT stimulate an increase in [Ca2+]i and that the response to all three agents could be blocked by the specific V2-receptor antagonist, [d(CH2)5(1),D-Ile2, Ile4, Arg8]vasopressin (II-VP). These results would suggest that all three agents acted through the V2 receptor. Furthermore, we showed that PO-VT increased cAMP production in IMCD suspensions and water permeability in isolated perfused tubules. These responses were also blocked by II-VP, indicating that PO-VT is also a V2 agonist in the IMCD. Finally, we utilized the quantitative reverse transcription-polymerase chain reaction technique of Wiesner (Nucleic Acids Res. 20: 5863-5864, 1992) to evaluate V1a and V2 mRNA levels in rat collecting duct. In terminal IMCD, we estimated > 30 copies/cell for V2 receptor mRNA but less than 1 copy/cell of V1a receptor mRNA, thus there is littler or no V1a mRNA expression in the terminal IMCD. These results suggest that calcium mobilization in response to vasopressin analogues is associated with the V2 receptor and that the V2 receptor is linked to both adenylyl cyclase and calcium mobilization in the rat IMCD.

Animals↗
PubMed1995

Transcriptional enhancer activity of hr5 requires dual-palindrome half sites that mediate binding of a dimeric form of the baculovirus transregulator IE1.

The hr5 enhancer element stimulates early viral transcription and may function as an origin of DNA replication for Autographa californica nuclear polyhedrosis virus (AcMNPV). The smallest functional unit of hr5 is a 28-bp repeat consisting of an imperfect palindrome (28-mer). To identify essential sequences and examine the molecular basis of hr5 activity, the effects of site-directed mutations on transcriptional enhancement by the 28-mer and binding of the AcMNPV transregulator IE1 were investigated. In transfection assays and infections with AcMNPV recombinants, activation of a basal viral promoter required sequences within both halves of the 28-mer. Basal promoter activation also required a critical spacing between these half sites. Mobility shift assays indicated that hr5 probes containing a single 28-mer were bound by in vitro-synthesized IE1. Competition assays using DNA fragments that contained mutated 28-mers demonstrated that both half sites were required for optimal binding of IE1. Similar assays using mutated 28-mer DNAs and nuclear extracts indicated that the relative affinity with which AcMNPV infection-specific proteins bound to the 28-mer was similar to that of in vitro-synthesized IE1. By using a combination of DNA binding and antibody supershift assays, it was demonstrated that IE1 binds to the 28-mer as a dimer. Collectively, these findings support a model in which symmetrical IE1 binding and simultaneous interaction with each half site are required for IE1-mediated transcriptional enhancement by hr5. Thus, sequence-specific binding may be one of the mechanisms by which IE1 directly or indirectly transregulates baculovirus gene expression.

Animals↗
PubMed2006

Disenrollment from an acute/long-term managed care program (PACE).

BACKGROUND: The Program of All-Inclusive Care for the Elderly (PACE) is an acute/long-term managed care plan designed to care for the most frail and vulnerable Medicare beneficiaries. To our knowledge, this is a first study to examine patterns and predictors of disenrollment from PACE. OBJECTIVE: PACE, with its comprehensive delivery system, dual capitation, and a focus on the most vulnerable population, may be expected to achieve low rates of exit and little selective dissenrollment. This study examines whether these goals have been accomplished. RESEARCH DESIGN: The study includes 30 PACE programs and 14,657 individuals enrolled in them. Individual-level records, obtained from an administrative database, contain information on sociodemographics, caregiver support, health status and disability, medical history, service utilization, and disenrollment. Program-level variables also were included. Cox proportional hazard models, with time-varying and time-invariant covariates, were employed to predict time to disenrollment. RESULTS: Our findings show a low level of disenrollment. We find no increase in disenrollment risk by age, functional or cognitive impairment, Medicaid eligibility, or diagnoses. Certain characteristics (eg, nursing treatments) appear to reduce the disenrollment hazard, whereas others (eg, hospital admissions, private pay status) significantly increase it. The risk of disenrollment also increases with longer nursing home stays, until 80-90 days, whereupon it begins to decline. CONCLUSIONS: Both enrollee and program-level attributes predict program disenrollment. Programmatic and quality of care improvements may be needed to further minimize disenrollment, particularly in programs experiencing rates that are substantially greater than the average.

Aged↗
PubMed2006

The developmental association of relationship quality, hormonal contraceptive choice and condom non-use among adolescent women.

PURPOSE: Consistent condom use is critical to efforts to prevent sexually transmitted infections among adolescents, but condom use may decline as relationships and contraceptive needs change. The purpose of this research is to assess changes in condom non-use longitudinally in the context of changes in relationship quality, coital frequency and hormonal contraceptive choice. METHODS: Participants were women (aged 14-17 years at enrollment) recruited from three urban adolescent medicine clinics. Data were collected at three-month intervals using a face-to-face structured interview. Participants were able to contribute up to 10 interviews, but on average contributed 4.2 interviews over the 27-month period. Independent variables assessed partner-specific relationship quality (five items; scale range 5-25; alpha = .92, e.g., this partner is a very important person to me); and, number of coital events with a specific partner. Additional items assessed experience with oral contraceptive pills (OCP) use and injected depo medroxy-progesterone acetate (DMPA). The outcome variable was number of coital events without condom use during the past three months. Analyses were conducted as a three-level hierarchical linear growth curve model using HLM 6. The Level 1 predictor was time, to test the hypothesis that condom non-use increases over time. Level 2 predictors assessed relationship quality and coital frequency across all partners to assess hypotheses that participants' condom non-use increases over time as a function of relationship quality and coital frequency. Level 3 predictors assessed the participant-level influence of OCP or DMPA experience on time-related changes in condom non-use. RESULTS: A total of 176 women reported 279 sex partners and contributed 478 visits. Both average coital frequency and average condom non-use linearly increased during the 27-month follow-up. At any given follow-up, about 35% reported recent OCP use, and 65% reported DMPA use. HLM analyses showed that condom non-use increased as a function of time (beta = .12; p = .03, Level 1 analysis). Increased condom non-use over time was primarily a function of increased coital frequency (beta = .01; p = .00), although higher levels of relationship quality were associated with increased condom non-use at enrollment (beta = .44; p = .00, Level 2 analysis). The temporal rise in condom non-use significantly increased among DMPA users (beta = .06; p = .00) but not OCP users (Level 3 analysis) (beta = -.04; p = .06). CONCLUSIONS: Developmentally, relationship characteristics and coital frequency appear to have increasing weight in decisions about condom use. Hormonal contraceptive methods are not equivalently associated with the overall temporal decline in condom use. Future research associated with dual contraceptive/condom use should address differential factors associated condom use in combination with different hormonal methods.

Adolescent↗
PubMed2004

Measuring quality of pharmacotherapy for depression in a national health care system.

OBJECTIVES: This article examines the use of antidepressant medication in the treatment of major depression in the Department of Veterans Affairs (VA) during the 2001 fiscal year and considers the relationship of patient, drug, provider, and facility to adherence to medication treatment guidelines. METHODS: Prescription drug records for all VA outpatients diagnosed with major depression (International Classification of Diseases, 9th edition, code 296.2 or 296.3) were collected for October 2000 through September 2001. Indicators were constructed that noted whether patients newly treated with antidepressants (i.e., with no prescription in the previous 8 weeks) received at least 180 days (continuation phase) of antidepressant drug treatment (84- and 140-day measures were also considered). Logistic regression with and without center fixed effects and generalized estimation equations were used to identify patient, drug, and facility characteristics that were associated with these treatment quality indicators. RESULTS: Of the 27,713 patients in the final sample, 54% received at least 181 days of treatment. This is higher than recent rates reported by Health Employer Data and Information Set (HEDIS) for the general population, although our measures and those used by HEDIS are not exactly the same. Women, married patients, older patients, and whites were more likely than others to have higher-quality antidepressant drug treatment. Contrary to previous research, we find few significant differences among specific antidepressant agents prescribed in this large sample. Comorbid substance abuse was associated with fewer days of treatment, whereas other psychiatric comorbidities increased the length of treatment. We found few differences resulting from provider type. Although significant differences among facilities were found in the unadjusted rates (similar to those used by HEDIS), these diminished greatly after controlling for relevant covariates. CONCLUSIONS: In the nation's largest mental health system, quality of pharmacotherapy for depression, at least by one standard measure, is relatively good. We found the specific antidepressant drug used has little impact on quality. In considering differences among facilities, we found that it is critical to control for relevant patient characteristics.

Adult↗
PubMed1996

Correlation of genetic and serologic approaches to HIV-1 subtyping in Thailand.

The aim of this study was to compare the performance of differential polymerase chain reaction (PCR) typing and peptide enzyme-linked immunosorbent assay (V3-EIA) for human immunodeficiency virus type 1 (HIV-1) subtyping in Thailand using heteroduplex mobility assay (HMA) as the reference standard. Paired peripheral blood mononuclear cells (PBMC) and sera were collected from 38 HIV-1 seropositive persons in Thailand. HMA was done by standard methods; differential PCR employs primer pairs that differentially amplify either subtype E or B. V3-EIA used peptides specific for subtypes E or B. Thirty-two cases (84%) were found by HMA to be infected with subtype E: and six with (16%) subtype B. The results obtained with differential PCR were 100% concordant with those of HMA; V3 EIA correctly predicted the subtype in 95% (36 of 38). Six samples that molecularly subtyped as E were repeatedly dual reactive by screening V3-EIA, but these resolved to subtype E using an antigen-limiting EIA. Two samples were serologically nontypeable because of overall low levels of V3 antibody. Using HMA as the standard, differential PCR was shown to subtype HIV-1 reliably from patient PBMC samples. V3-EIA correctly predicted HIV-1 subtype in most (95%) of our cases. Because of the less rigorous sampling requirements, specimen processing, and logistical and technical requirements of serotyping compared with molecular techniques, it appears to be practical for screening purposes in a field environment. Samples that cannot be definitively subtyped serologically should undergo differential PCR and antigen-limiting V3 EIA. These approaches to HIV-1 subtyping should be used in complementary fashion in Thailand, where subtypes B and E are currently known to cocirculate.

Adult↗
PubMed2005

Noninfectious virus-like particle antigen for detection of swine vesicular disease virus antibodies in pigs by enzyme-linked immunosorbent assay.

An inactivated SVDV antigen is used in current enzyme-linked immunosorbent assays (ELISAs) for the detection of antibodies to swine vesicular disease virus (SVDV). To develop a noninfectious recombinant alternative, we produced SVDV-like particles (VLPs) morphologically and antigenically resembling authentic SVDV particles by using a dual baculovirus recombinant, which expresses simultaneously the P1 and 3CD protein genes of SVDV under different promoters. Antigenic differences between recombinant VLPs and SVDV particles were not statistically significant in results obtained with a 5B7-ELISA kit, indicating that the VLPs could be used in the place of SVDV antigen in ELISA kits. We developed a blocking ELISA using the VLPs and SVDV-specific neutralizing monoclonal antibody 3H10 (VLP-ELISA) for detection of SVDV serum antibodies in pigs. The VLP-ELISA showed a high specificity of 99.9% when tested with pig sera that are negative for SVDV neutralization (n=1,041). When tested using sera (n=186) collected periodically from pigs (n=19) with experimental infection with each of three different strains of SVDV, the VLP-ELISA detected SVDV serum antibodies as early as 3 days postinfection and continued to detect the antibodies from all infected pigs until termination of the experiments (up to 121 days postinfection). This test performance was similar to that of the gold standard virus neutralization test and indicates that the VLP-ELISA is a highly specific and sensitive method for the detection of SVDV serum antibodies in pigs. This is the first report of the production and diagnostic application of recombinant VLPs of SVDV. Further potential uses of the VLPs are discussed.

Animals↗
PubMed2002

Molecular and biochemical characterization of two plant inositol polyphosphate 6-/3-/5-kinases.

Despite the high deposition of inositol hexakisphosphate (IP(6)), also known as phytate or phytin, in certain plant tissues little is known at the molecular level about the pathway(s) involved in its production. In budding yeast, IP(6) synthesis occurs through the sequential phosphorylation of I(1,4,5)P(3) by two gene products, Ipk2 and Ipk1, a IP(3)/IP(4) dual-specificity 6-/3-kinase and an inositol 1,3,4,5,6-pentakisphosphate 2-kinase, respectively. Here we report the identification and characterization of two inositol polyphosphate kinases from Arabidopsis thaliana, designated AtIpk2alpha and AtIpk2beta that are encoded by distinct genes on chromosome 5 and that are ubiquitously expressed in mature tissue. The primary structures of AtIpk2alpha and AtIpk2beta are 70% identical to each other and 12-18% identical to Ipk2s from yeast and mammals. Similar to yeast Ipk2, purified recombinant AtIpk2alpha and AtIpk2beta have 6-/3-kinase activities that sequentially phosphorylate I(1,4,5)P(3) to generate I(1,3,4,5,6)P(5) predominantly via an I(1,4,5,6)P(4) intermediate. While I(1,3,4,5)P(4) is a substrate for the plant Ipk2s, it does not appear to be a detectable product of the IP(3) reaction. Additionally, we report that the plant and yeast Ipk2 have a novel 5-kinase activity toward I(1,3,4,6)P(4) and I(1,2,3,4,6)P(5), which would allow these proteins to participate in at least two proposed pathways in the synthesis of IP(6). Heterologous expression of either plant isoform in an ipk2 mutant yeast strain restores IP(4) and IP(5) production in vivo and rescues its temperature-sensitive growth defects. Collectively our results provide a molecular basis for the synthesis of higher inositol polyphosphates in plants through multiple routes and indicate that the 6-/3-/5-kinase activities found in plant extracts may be encoded by the IPK2 gene class.

Amino Acid Sequence↗
PubMed2002

Postpartum anoestrus is reduced by increasing the within-day milking to suckling interval in dual purpose cows.

In the dual purpose milk/beef production system practised in Mexico, the cows are milked once a day in the presence of the calf and then the calf suckles for the next 1-8 h. It was hypothesised that the imposition of milking and suckling in close succession contributes to the long postpartum anoestrous periods experienced by cows in this system. Therefore, separating these two inhibiting factors, by delaying the start of suckling after milking would lower the impact on the hypothalamus-hypophysial axis and allow it to recover more readily. This hypothesis was tested in two experiments using Bostaurus x Bosindicus cows which weighed 541.3 +/- 12.7 kg (mean +/- S.E.M.; experiment 1) and 537.8 +/- 10.6 kg (experiment 2) and had a body condition score of 5-6 (scale 1-9) at calving. The cows and calves were maintained on separate pastures and the cows were offered concentrate supplements. Experiment 1 was conducted in the dry season and cows were offered a daily supplement of 3 kg of 17% crude protein (CP) concentrates plus 1 kg molasses per cow. Experiment 2 was conducted during the wet season and the nutritional supplement consisted of only 1 kg of 17% CP plus 1 kg molasses per cow per day. Each experiment lasted for the first 100 days postpartum and the cows were not mated during this time. Cows were hand-milked once a day in the presence of the calf to stimulate milk let-down. In experiment 1, 30 multiparous cows were assigned at random by date of calving, to one of three treatments: controlled suckling (CS; n = 10), delayed suckling (DS; n = 10) and prolonged delayed suckling (PDS; n = 10). The suckling period on all treatments lasted for 2 h and started at 0 h (CS), 4 h (DS) or 8 h (PDS) after milking. In experiment, 34 multiparous cows were assigned at random to three treatments: CS (n = 11), PDS (n = 11) or prolonged delayed short suckling (PDSS; n = 12), in this last treatment the suckling period was reduced to only 30 min. In both experiments, one quarter of the udder of cows on treatment CS was not milked but all the udder was milked on the other treatments. Body weights of cows and calves and cow milk yields were recorded. Weekly blood samples were collected for plasma progesterone assay to detect the first postpartum corpus luteum. Data were analysed by ANOVA and chi-square test. Cows in experiment 1 had similar weight changes during the first 100 days postpartum. Milk production was higher for DS (8.0 +/- 0.7 kg per day) and PDS (7.0 +/- 0.5 kg per day) cows than for CS (5.2 +/- 0.7 kg per day) cows (P < 0.01). Calf daily gain was greater (P < 0.05) for PDS (827 +/- 35 g per day), than CS (702 +/- 26 g per day) or DS (715 +/- 29 g per day). The proportion of cows ovulating during the first 100 days postpartum were 50.0, 70.0 and 100.0%, for CS, DS and PDS, respectively (P < 0.05). The intervals from calving to first significant rise in progesterone were 70.4 +/- 1.6, 72.1 +/- 1.3 and 64.6 +/- 1.5 days for CS, DS and PDS (P > 0.05), respectively. In experiment 2, cow body weight during the first 100 days postpartum did not differ between treatments. Milk production was not significantly different between CS (7.3 +/- 1.0 kg per day) and PDSS (6.8 +/- 1.0 kg per day), but was higher (P < 0.05) for CS than PDS (5.2 +/- 1.0 kg per day). Calf daily gain was lower for CS (576 +/- 31 g per day; P < 0.05) than PDS (779 +/- 38 g per day) and PDSS (780 +/- 20 g per day). The proportion of cows ovulating was higher (P < 0.05) for treatments PDS (100%) and CS (81.8%) than PDSS (50%). Calving to first ovulation interval was shorter (P < 0.05) for PDS (57.3 +/- 4.5 days) and PDSS (49.7 +/- 3.0 days) cows than CS (72.1 +/- 3.5 days) cows. It is concluded that delaying suckling for 8 h after milking, increases the proportion of cows ovulating within 100 days of calving, shortened the calving to first ovulation interval and improved calf performance without adversely affecting cow milk yield or body weight change.

Anestrus↗
PubMed2005

Stochastic modeling of diffuse pesticide losses from a small agricultural catchment.

The objective of this study was to identify the main sources of variation in pesticide losses at field and catchment scales using the dual permeability model MACRO. Stochastic simulations of the leaching of the herbicide MCPA (4-chloro-2-methylphenoxyacetic acid) were compared with seven years of measured concentrations in a stream draining a small agricultural catchment and one year of measured concentrations at the outlet of a field located within the catchment. MACRO was parameterized from measured probability distributions accounting for spatial variability of soil properties and local pedotransfer functions derived from information gathered in field- and catchment-scale soil surveys. At the field scale, a single deterministic simulation using the means of the input distributions was also performed. The deterministic run failed to reproduce the summer outflows when most leaching occurred, and greatly underestimated pesticide leaching. In contrast, the stochastic simulations successfully predicted the hydrologic response of the field and catchment and there was a good resemblance between the simulations and measured MCPA concentrations at the field outlet. At the catchment scale, the stochastic approach underestimated the concentrations of MCPA in the stream, probably mostly due to point sources, but perhaps also because the distributions used for the input variables did not accurately reflect conditions in the catchment. Sensitivity analyses showed that the most important factors affecting MACRO modeled diffuse MCPA losses from this catchment were soil properties controlling macropore flow, precipitation following application, and organic carbon content.

Agriculture↗