Recognition of Highly Diverse Type-1 and -2 Porcine Reproductive and Respiratory Syndrome Viruses (PRRSVs) by T-Lymphocytes Induced in Pigs after Experimental Infection with a Type-2 PRRSV Strain.

BACKGROUND/AIM: Live attenuated vaccines confer partial protection in pigs before the appearance of neutralizing antibodies, suggesting the contribution of cell-mediated immunity (CMI). However, PRRSV-specific T-lymphocyte responses and protective mechanisms need to be further defined. To this end, the hypothesis was tested that PRRSV-specific T-lymphocytes induced by exposure to type-2 PRRSV can recognize diverse isolates. METHODS: An IFN-gamma ELISpot assay was used to enumerate PRRSV-specific T-lymphocytes from PRRSVSD23983-infected gilts and piglets born after in utero infection against 12 serologically and genetically distinct type-1 and -2 PRRSV isolates. The IFN-gamma ELISpot assay using synthetic peptides spanning all open reading frames of PRRSVSD23983 was utilized to localize epitopes recognized by T-lymphocytes. Virus neutralization tests were carried out using the challenge strain (type-2 PRRSVSD23983) and another strain (type-2 PRRSVVR2332) with high genetic similarity to evaluate cross-reactivity of neutralizing antibodies in gilts after PRRSVSD23983 infection. RESULTS: At 72 days post infection, T-lymphocytes from one of three PRRSVSD23983-infected gilts recognized all 12 diverse PRRSV isolates, while T-lymphocytes from the other two gilts recognized all but one isolate. Furthermore, five of nine 14-day-old piglets infected in utero with PRRSVSD23983 had broadly reactive T-lymphocytes, including one piglet that recognized all 12 isolates. Overlapping peptides encompassing all open reading frames of PRRSVSD23983 were used to identify ≥28 peptides with T-lymphocyte epitopes from 10 viral proteins. This included one peptide from the M protein that was recognized by T-lymphocytes from all three gilts representing two completely mismatched MHC haplotypes. In contrast to the broadly reactive T-lymphocytes, neutralizing antibody responses were specific to the infecting PRRSVSD23983 isolate. CONCLUSION: These results demonstrated that T-lymphocytes recognizing antigenically and genetically diverse isolates were induced by infection with a type 2 PRRSV strain (SD23983). If these reponses have cytotoxic or other protective functions, they may help overcome the suboptimal heterologous protection conferred by conventional vaccines.

Tanning lamps: health effects and reclassification by the Food and Drug Administration.

Tanning lamps have long been considered a class I medical device under regulation by the Food and Drug Administration (FDA). A growing body of research has repeatedly documented the association between elective indoor tanning and several negative health consequences. These accepted findings have prompted action by the FDA to officially reclassify tanning lamps as a class II medical device. The main purpose of this review is to update practitioners on the current state of tanning lamp classification and highlight the practical implications of this recent change. This information can be used by clinicians to easily reference this important action, and empower patients with a better understanding of the risks associated with indoor tanning.

Evaluation of two mutants of Mycobacterium avium subsp. paratuberculosis as candidates for a live attenuated vaccine for Johne's disease.

Control of Johne's disease, caused by Mycobacterium avium subsp. paratuberculosis, has been difficult because of a lack of an effective vaccine. To address this problem we used targeted gene disruption to develop candidate mutants with impaired capacity to survive ex vivo and in vivo to test as a vaccine. We selected relA and pknG, genes known to be important virulence factors in Mycobacterium tuberculosis and Mycobacterium bovis, for initial studies. Deletion mutants were made in a wild type Map (K10) and its recombinant strain expressing the green fluorescent protein (K10-GFP). Comparison of survival in an ex vivo assay revealed deletion of either gene attenuated survival in monocyte-derived macrophages compared to survival of wild-type K10. In contrast, study in calves revealed survival in vivo was mainly affected by deletion of relA. Bacteria were detected in tissues from wild-type and the pknG mutant infected calves by bacterial culture and PCR at three months post infection. No bacteria were detected in tissues from calves infected with the relA mutant (P<0.05). Flow cytometric analysis of the immune response to the wild-type K10-GFP and the mutant strains showed deletion of either gene did not affect their capacity to elicit a strong proliferative response to soluble antigen extract or live Map. Quantitative RT-PCR revealed genes encoding IFN-γ, IL-17, IL-22, T-bet, RORC, and granulysin were up-regulated in PBMC stimulated with live Map three months post infection compared to the response of PBMC pre-infection. A challenge study in kid goats showed deletion of pknG did not interfere with establishment of an infection. As in calves, deletion of relA attenuated survival in vivo. The mutant also elicited an immune response that limited colonization by challenge wild type Map. The findings show the relA mutant is a good candidate for development of a live attenuated vaccine for Johne's disease.

Flow cytometric detection of prostate tumor cells using chemoaffinity labels.

BACKGROUND: The enzyme-biomarker prostate-specific membrane antigen (PSMA) is an emerging target for imaging and therapeutic applications for prostate cancer. However, the use of PSMA for detecting circulating prostate tumor cells remains under-explored. The present study focuses on the specific labeling of PSMA+ prostate cancer cells with a fluorescent PSMA inhibitor and the quantitation of PSMA+ cells in blood by flow cytometry (FC) using a gating strategy to separate labeled PSMA+ cells from peripheral blood mononuclear cells. METHODS: Suspensions of PSMA+ (LNCaP) and PSMA- (DU145) cells were incubated with the fluorescent PSMA inhibitor FAMX-CTT-54. Incubation parameters (time, temperature, and label concentration) were varied to optimize cell labeling. A gating protocol based on double fluorescent labeling of CD45 and PSMA was developed for the quantitiation of LNCaP cells in the presence of white blood cells from bovine blood. Nonfluorescent beads were added to the labeled cell mixture and served as internal standard for precise cellular quantification of LNCaP cells by flow cytometry. RESULTS: The fluorescent PSMA inhibitor FAMX-CTT-54 was specific for PSMA+ cells. The minimum time and concentration of FAMX-CTT-54 for effective labeling of PSMA+ cell suspensions at 37°C was 7.5 min and 35 nM, respectively; no labeling was observed on PSMA- cells. Co-incubation or pre-incubation of PSMA+ cells with the unlabeled PSMA inhibitor CTT-54 resulted in a concentration-dependent reduction in fluorescent labeling with FAMX-CTT-54 thereby confirming that the labeling was specific for PSMA. In blood samples in which LNCaP cells were added, an average of five cells were detected in a 115 µl sample of the most dilute sample examined (29 cells/ml); three cells were expected theoretically. The greater loss of labeling of PSMA+ cells with FAMX-CTT-54 when pre-incubated with CTT-54 is consistent with the irreversible mode of binding of CTT-54 to PSMA and subsequent internalization of the PSMA-inhibitor complex. CONCLUSIONS: The results suggest that fluorescent PSMA inhibitors can be utilized to effectively detect and quantify PSMA+ cells by FC. These results support the use of such compounds in the application of FC to detect, quantify, and characterize circulating prostate tumor cells.