Correction to: Intraoperative Imaging with a Portable Gamma Camera May Reduce the False-Negative Rate for Melanoma Sentinel Lymph Node Surgery.

The article "Intraoperative Imaging with a Portable Gamma Camera May Reduce the False-Negative Rate for Melanoma Sentinel Lymph Node Surgery," written by Stanley P. Leong et al., was originally published electronically on the publisher's internet portal (currently SpringerLink) on August 13, 2018, without open access.

Intraoperative Imaging with a Portable Gamma Camera May Reduce the False-Negative Rate for Melanoma Sentinel Lymph Node Surgery.

BACKGROUND: Preoperative imaging and intraoperative gamma probe (GP) localization is standard for identifying sentinel lymph nodes (SLNs) in melanoma patients. The aim of this prospective Institutional Review Board-approved study was to investigate whether an intraoperative portable gamma camera (PGC) improves SLN detection over the GP. METHODS: Lymphoscintigraphy and single photon emission computed tomography/computed tomography were performed after injection of 99mTc-Tilmanocept in melanoma patients (≥ 18 years, Breslow thickness ≥ 1.0 mm). A GP was used to localize the SLNs in each basin, which was explored by the GP to ensure that the operative field was < 10% counts of the hottest SLN. The PGC was then used after a negative GP screening. Any residual hotspots identified by the PGC were considered as additional SLNs and were removed following the 10% rule. RESULTS: Preoperative imaging of 100 patients identified 138 SLN basins, with 306 SLNs being identified by conventional surgery. The PGC localized 89 additional SLNs in 54 patients. Thus, the PGC identified an additional 23% of SLNs [95% confidence interval (CI) 18-27%]. Four of these 89 SLNs showed micrometastasis in four patients, in two of whom the only tumor-positive SLN was identified by the PGC, preventing two false-negative cases. Thus, the null hypothesis that the PGC did not detect additional positive SLNs was rejected (p = 0.000). The overall SLN positive rate was 9.9% (39/395, 95% CI 6-12), and the overall patient positive rate was increased using the PGC, from 25 to 27% (27/100). CONCLUSIONS: Intraoperative PGC imaging yielded additional SLNs in a significant number of patients over GP alone. Identification of these additional SLNs resulted in upstaging of four patients with two patients being converted from a negative to a positive status, thus, preventing two false-negative cases.

Strategies for the rapid construction of conditionally-replicating HSV-1 vectors expressing foreign genes as anticancer therapeutic agents.

Conditionally replication-competent Herpes Simplex Virus Type 1 (HSV-1) vectors expressing foreign genes have been developed as experimental therapeutic agents. Traditional methods of virus construction, including growth selection based on thymidine kinase gene expression, and color selection based on a reporter gene expression are often time-consuming and relatively inefficient. This review summarizes the various strategies developed in recent years for the rapid and efficient construction of novel conditionally replication-competent mutant HSV expressing multiple foreign genes. Additionally, two new modifications of existing strategies, which have not been previously reported, are discussed.

Impact of preexisting vector-specific immunity on vaccine potency: characterization of listeria monocytogenes-specific humoral and cellular immunity in humans and modeling studies using recombinant vaccines in mice.

Recombinant live-attenuated Listeria monocytogenes is currently being developed as a vaccine platform for treatment or prevention of malignant and infectious diseases. The effectiveness of complex biologic vaccines, such as recombinant viral and bacterial vectors, can be limited by either preexisting or vaccine-induced vector-specific immunity. We characterized the level of L. monocytogenes-specific cellular and humoral immunity present in more than 70 healthy adult subjects as a first step to understanding its possible impact on the efficacy of L. monocytogenes-based vaccines being evaluated in early-phase clinical trials. Significant L. monocytogenes-specific humoral immunity was not measured in humans, consistent with a lack of antibodies in mice immunized with wild-type L. monocytogenes. Cellular immune responses specific for listeriolysin O, a secreted bacterial protein required for potency of L. monocytogenes-derived vaccines, were detected in approximately 60% of human donors tested. In mice, while wild-type L. monocytogenes did not induce significant humoral immunity, attenuated L. monocytogenes vaccine strains induced high-titer L. monocytogenes-specific antibodies when given at high doses used for immunization. Passive transfer of L. monocytogenes-specific antiserum to naïve mice had no impact on priming antigen-specific immunity in mice immunized with a recombinant L. monocytogenes vaccine. In mice with preexisting L. monocytogenes-specific immunity, priming of naïve T cells was not prevented, and antigen-specific responses could be boosted by additional vaccinations. For the first time, our findings establish the level of L. monocytogenes-specific cellular immunity in healthy adults, and, together with modeling studies performed with mice, they support the scientific rationale for repeated L. monocytogenes vaccine immunization regimens to elicit a desired therapeutic effect.

Killed but metabolically active Bacillus anthracis vaccines induce broad and protective immunity against anthrax.

Bacillus anthracis is the causative agent of anthrax. We have developed a novel whole-bacterial-cell anthrax vaccine utilizing B. anthracis that is killed but metabolically active (KBMA). Vaccine strains that are asporogenic and nucleotide excision repair deficient were engineered by deleting the spoIIE and uvrAB genes, rendering B. anthracis extremely sensitive to photochemical inactivation with S-59 psoralen and UV light. We also introduced point mutations into the lef and cya genes, which allowed inactive but immunogenic toxins to be produced. Photochemically inactivated vaccine strains maintained a high degree of metabolic activity and secreted protective antigen (PA), lethal factor, and edema factor. KBMA B. anthracis vaccines were avirulent in mice and induced less injection site inflammation than recombinant PA adsorbed to aluminum hydroxide gel. KBMA B. anthracis-vaccinated animals produced antibodies against numerous anthrax antigens, including high levels of anti-PA and toxin-neutralizing antibodies. Vaccination with KBMA B. anthracis fully protected mice against challenge with lethal doses of toxinogenic unencapsulated Sterne 7702 spores and rabbits against challenge with lethal pneumonic doses of fully virulent Ames strain spores. Guinea pigs vaccinated with KBMA B. anthracis were partially protected against lethal Ames spore challenge, which was comparable to vaccination with the licensed vaccine anthrax vaccine adsorbed. These data demonstrate that KBMA anthrax vaccines are well tolerated and elicit potent protective immune responses. The use of KBMA vaccines may be broadly applicable to bacterial pathogens, especially those for which the correlates of protective immunity are unknown.

Constitutive Activation of the PrfA regulon enhances the potency of vaccines based on live-attenuated and killed but metabolically active Listeria monocytogenes strains.

Recombinant vaccines derived from the facultative intracellular bacterium Listeria monocytogenes are presently undergoing early-stage clinical evaluation in oncology treatment settings. This effort has been stimulated in part due to preclinical results that illustrate potent activation of innate and adaptive immune effectors by L. monocytogenes vaccines, combined with efficacy in rigorous animal models of malignant and infectious disease. Here, we evaluated the immunologic potency of a panel of isogenic vaccine strains that varied only in prfA. PrfA is an intracellularly activated transcription factor that induces expression of virulence genes and encoded heterologous antigens (Ags) in appropriately engineered vaccine strains. Mutant strains with PrfA locked into a constitutively active state are known as PrfA* mutants. We assessed the impacts of three PrfA* mutants, G145S, G155S, and Y63C, on the immunologic potencies of live-attenuated and photochemically inactivated nucleotide excision repair mutant (killed but metabolically active [KBMA]) vaccines. While PrfA* substantially increased Ag expression in strains grown in broth culture, Ag expression levels were equivalent in infected macrophage and dendritic cell lines, conditions that more closely parallel those in the immunized host. However, only the prfA(G155S) allele conferred significantly enhanced vaccine potency to KBMA vaccines. In the KBMA vaccine background, we show that PrfA*(G155S) enhanced functional cellular immunity following an intravenous or intramuscular prime-boost immunization regimen. These results form the basis of a rationale for including the prfA(G155S) allele in future live-attenuated or KBMA L. monocytogenes vaccines advanced to the clinical setting.

Adhesion to the host cell surface is sufficient to mediate Listeria monocytogenes entry into epithelial cells.

The intestinal epithelium is the first physiological barrier breached by the Gram-positive facultative pathogen Listeria monocytogenes during an in vivo infection. Listeria monocytogenes binds to the epithelial host cell receptor E-cadherin, which mediates a physical link between the bacterium and filamentous actin (F-actin). However, the importance of anchoring the bacterium to F-actin through E-cadherin for bacterial invasion has not been tested directly in epithelial cells. Here we demonstrate that depleting αE-catenin, which indirectly links E-cadherin to F-actin, did not decrease L. monocytogenes invasion of epithelial cells in tissue culture. Instead, invasion increased due to increased bacterial adhesion to epithelial monolayers with compromised cell-cell junctions. Furthermore, expression of a mutant E-cadherin lacking the intracellular domain was sufficient for efficient L. monocytogenes invasion of epithelial cells. Importantly, direct biotin-mediated binding of bacteria to surface lipids in the plasma membrane of host epithelial cells was sufficient for uptake. Our results indicate that the only requirement for L. monocytogenes invasion of epithelial cells is adhesion to the host cell surface, and that E-cadherin-mediated coupling of the bacterium to F-actin is not required.

Effects of Supplemental Citrulline-Malate Ingestion on Blood Lactate, Cardiovascular Dynamics, and Resistance Exercise Performance in Trained Males.

Citrulline-malate (CM) has been proposed to provide an ergogenic effect during resistance exercise; however, there is a paucity of research investigating these claims. Therefore, we investigated the impact that CM supplementation would have on repeated bouts of resistance exercise. Fourteen resistance-trained males participated in a randomized, counterbalanced, double-blind study. Subjects were randomly assigned to placebo (PL) or CM (8 g) and performed three sets each of chin-ups, reverse chin-ups, and push-ups to failure. One week later, subjects ingested the other supplement and performed the same protocol. Blood lactate (BLa), heart rate (HR), and blood pressure (BP) were measured preexercise, with BLa measured a second time immediately following the last set, while HR and BP were measured 5 and 10 min postexercise. Citrulline-malate ingestion significantly increased the amount of repetitions performed for each exercise (chin-ups: PL = 28.4 ± 7.1, CM = 32.2 ± 5.6, p = .003; reverse chin-ups: PL = 26.6 ± 5.6, CM = 32.1 ± 7.1, p = .017; push-ups: PL = 89.1 ± 37.4, CM = 97.7 ± 36.1, p < .001). Blood lactate data indicated a time effect (p < .001), but no treatment differences (p = .935). Systolic BP data did not show differences for time (p = .078) or treatment (p = .119). Diastolic BP data did not show differences for time (p = .069), but indicated treatment differences (p = .014) for subjects ingesting CM. Collectively, these findings suggests that CM increased upper-body resistance performance in trained college-age males.

Inactivation of parvovirus B19 in human platelet concentrates by treatment with amotosalen and ultraviolet A illumination.

BACKGROUND: The human erythrovirus B19 (B19) is a small (18- to 26-nm) nonenveloped virus with a single-stranded DNA genome of 5.6 kb. B19 is clinically significant and is also generally resistant to pathogen inactivation methods. Photochemical treatment (PCT) with amotosalen and ultraviolet A (UVA) inactivates viruses, bacteria, and protozoa in platelets (PLTs) and plasma prepared for transfusion. In this study, the capacity of PCT to inactivate B19 in human PLT concentrates was evaluated. STUDY DESIGN AND METHODS: B19 inactivation was measured by a novel enzyme-linked immunosorbent spot (ELISPOT) erythroid progenitor cell infectivity assay and by inhibition of long-range (up to 4.3 kb) polymerase chain reaction (PCR), under conditions where the whole coding region of the viral genome was amplified. B19-infected plasma was used to test whether incubation of amotosalen with virus before PCT enhanced inactivation compared to immediate PCT. RESULTS: Inactivation of up to 5.8 log of B19 as measured by the infectivity assay, or up to 6 logs as measured by PCR inhibition can be achieved under non-limiting conditions. Inactivation efficacy was found to increase with incubation prior to UVA illumination. Without incubation prior to illumination 2.1 +0.4 log was inactivated as determined by infectivity assay. When measured by PCR inhibition, inactivation varied inversely with amplicon size. When primers that spanned the entire coding region of the B19 genome were used, maximum inhibition of PCR amplification was demonstrated. CONCLUSION: Under defined conditions, PCT with amotosalen combined with UVA light can be used to inactivate B19, a clinically significant virus that can be transmitted through blood transfusion, and heretofore has been demonstrated to be refractory to inactivation.

Listeria-based cancer vaccines that segregate immunogenicity from toxicity.

The facultative intracellular bacterium Listeria monocytogenes is being developed as a cancer vaccine platform because of its ability to induce potent innate and adaptive immunity. For successful clinical application, it is essential to develop a Listeria platform strain that is safe yet retains the potency of vaccines based on wild-type bacteria. Here, we report the development of a recombinant live-attenuated vaccine platform strain that retains the potency of the fully virulent pathogen, combined with a >1,000-fold reduction in toxicity, as compared with wild-type Listeria. By selectively deleting two virulence factors, ActA (DeltaactA) and Internalin B (DeltainlB), the immunopotency of Listeria was maintained and its toxicity was diminished in vivo, largely by blocking the direct internalin B-mediated infection of nonphagocytic cells, such as hepatocytes, and the indirect ActA-mediated infection by cell-to-cell spread from adjacent phagocytic cells. In contrast, infection of phagocytic cells was not affected, leaving intact the ability of Listeria to stimulate innate immunity and to induce antigenspecific cellular responses. Listeria DeltaactA/DeltainlB-based vaccines were rapidly cleared from mice after immunization and induced potent and durable effector and memory T-cell responses with no measurable liver toxicity. Therapeutic vaccination of BALB/c mice bearing murine CT26 colon tumor lung metastases or palpable s.c. tumors (>100 mm(3)) with recombinant Listeria DeltaactA/DeltainlB expressing an endogenous tumor antigen resulted in breaking of self-tolerance and long-term survival. We propose that recombinant Listeria DeltaactA/DeltainlB expressing human tumor-associated antigens represents an attractive therapeutic strategy for further development and testing in human clinical trials.