Biomedical subjects

Lawrence K Silbart

Publications and source records attributed to Lawrence K Silbart.

5 recordsLinked to original sources

PubMed2006

Labeling and intracellular tracking of functionally active plasmid DNA with semiconductor quantum dots.

Semiconductor nanocrystal quantum dots (QDs) allow long-term imaging in the cellular environment with high photostability. QD biolabeling techniques have previously been developed for tagging proteins and peptides as well as oligonucleotides. In this contribution, QD-decorated plasmid DNA was utilized for the first time for long-term intracellular and intranuclear tracking studies. Conjugation of plasmid DNA with phospholipid-coated QDs was accomplished using a peptide nucleic acid (PNA)-N-succinimidyl-3-(2-pyridylthio) propionate linker. Gel electrophoresis and confocal and atomic force microscopy (AFM) were used to confirm the structure of QD-DNA conjugates. AFM imaging also revealed that multiple QDs were attached in a cluster at the PNA-reactive site of the plasmid DNA. These QD-DNA conjugates were capable of expressing the reporter protein, enhanced green fluorescent protein, following transfection in Chinese hamster ovary (CHO-K1) cells with an efficiency of ca. 62%, which was comparable to the control (unconjugated) plasmid DNA.

Animals↗

PubMed2006

Quantifying specific antibody concentrations by enzyme-linked immunosorbent assay using slope correction.

Assessing the magnitude of an antibody response is important to many research and clinical endeavors; however, there are considerable differences in the experimental approaches used to achieve this end. Although the time-honored approach of end point titration has merit, the titer can often be misleading due to differences in how it is calculated or when samples contain high concentrations of low-avidity antibodies. One frequently employed alternative is to adapt commercially available enzyme-linked immunosorbent assay kits, designed to measure total antibody concentrations, to estimate antigen-specific antibody concentrations. This is accomplished by coating the specific antigen of interest in place of the capture antibody provided with the kit and then using the kit's standard curve to quantify the specific antibody concentration. This approach introduces considerable imprecision, due primarily to its reliance on a single sample dilution. This "single-point" approach fails to address differences in the slope of the sample titration curve compared to that of the standard curve. Here, we describe a general approach for estimating the effective concentration of specific antibodies, using antisera against foot-and-mouth disease virus VP1 peptide. This was accomplished by initially calculating the slope of the sample titration curve and then mathematically correcting the slope to that of a corresponding standard curve. A significantly higher degree of precision was attained using this approach rather than the single-point method.

Animals↗

PubMed2006

Incorporating problem-based learning exercises into an environmental health curriculum.

Environmental health professionals are often confronted with difficult conflicts involving multiple stakeholders. A problem-based-learning approach was used to engage students in a mock conflict resolution exercise involving the heavily publicized dispute over industrial contamination of an aquifer supplying drinking water to neighborhoods in Woburn, Massachusetts. This dispute provided the basis for the bestselling novel A Civil Action, by Jonathan Harr, as well as a major motion picture bearing the same name. Students were assigned to one of three stakeholder groups: the affected families, an industry consortium, or state and federal regulatory authorities. These stakeholder groups were then directed to participate in alternative-dispute-resolution mock negotiations, with the course instructor serving as a neutral facilitator. The "problem" the students were assigned was to reach a consent agreement that was acceptable to all three stakeholder groups. Two groups of undergraduate students (from different semesters) successfully completed all five phases of this endeavor, with nearly unanimous agreement that the approach was more rapid and potentially less expensive than the civil-litigation process portrayed in the book and movie. The alternative-dispute-resolution approach also provided a less adversarial environment, thereby allowing the industry and community groups to work together to forge a more creative, long-term agreement than that which resulted from the actual civil litigation.

Curriculum↗

PubMed2005

Correlates of immune protection in chickens vaccinated with Mycoplasma gallisepticum strain GT5 following challenge with pathogenic M. gallisepticum strain R(low).

Colonization of the avian respiratory tract with Mycoplasma gallisepticum results in a profound inflammatory response in the trachea, air sacs, conjunctiva, and lungs. A live attenuated M. gallisepticum vaccine strain, GT5, was previously shown to be protective in chickens upon challenge; however, the mechanisms by which this vaccine and others confer protection remain largely unknown. The current study evaluated several potential correlates of GT5 vaccine-mediated immune protection following challenge with the pathogenic M. gallisepticum strain R(low). GT5-vaccinated chickens developed mild tracheal lesions, consisting of few and scattered, discrete, lymphofollicular aggregates in the lamina propria. In addition, low numbers of aggregated B, CD4(+), and CD8(+) cells were observed to infiltrate the trachea, in stark contrast to the large numbers infiltrating the tracheas of sham-vaccinated chickens challenged with R(low). Lymphofollicular aggregates were rarely observed prior to day 12 postchallenge in sham-vaccinated chickens. Instead, they contained an increasingly more cellular inflammatory response characterized by expansion of the lamina propria by lymphoplasmacytic and histiocytic infiltrates. This was due in part to expansion of interfollicular zones by large numbers of infiltrating CD4(+) and CD8(+) cells and a sizeable population of immunoglobulin A (IgA)- and IgG-secreting plasma cells. GT5-vaccinated chickens also had higher serum IgG concentrations, and significantly higher numbers of M. gallisepticum-specific IgG- and IgA-secreting plasma/B cells within the trachea, than did sham-vaccinated chickens. These responses were observed as early as day 4 postchallenge, indicating the importance of antibody-mediated clearance of mycoplasma in GT5-vaccinated chickens.

Animals↗

PubMed2003

Modulating gene expression using DNA vaccines with different 3'-UTRs influences antibody titer, seroconversion and cytokine profiles.

To determine if modulating the amount of foreign antigen produced by a DNA vaccine can influence the overall intensity and cytokine polarization of the ensuing immune response, three different plasmids, each encoding the hepatitis B (HB) surface antigen, were constructed. In each construct, HBs gene expression was driven by the cytomegalovirus immediate early promoter, but differed in the 3'-untranslated regions (3'-UTR) containing the polyadenylation sequence. These 3'-UTR sequences were derived from either the hepatitis B virus (HBVpA), bovine growth hormone (BGHpA), or rabbit beta-globin (betapA). BALB/c mice were immunized intramuscularly with equimolar amounts of each plasmid and blood was collected bi-weekly. Following immunization, total IgG titers correlated with in vitro antigen production levels (from transfected CHO cells), as evidenced by the following response pattern: HBVpA>BGHpA>>betapA. All groups demonstrated a heavy bias toward a Th1 immune response, as evidenced by high serum IgG2a/IgG1 ratios and the predominance of IFN-gamma over IL-4 secretion from cultured splenocytes. In addition, the HBVpA construct resulted in a seroconversion rate of 100%, in comparison to 40-50% in the BGHpA, and 0% in the betapA group. Surprisingly, splenocytes isolated from mice immunized with the betapA construct secreted the highest levels of IFN-gamma. Taken together, these findings suggest that altering the level of gene expression not only affects the overall titer and seroconversion rates of vaccinated animals, but also may play a role in modulating cytokine profiles.

3' Untranslated Regions↗