Eric G. Sheu

Induction of CD8+ T cells using heterologous prime-boost immunisation strategies

Summary: One of the current challenges in vaccine design is the development of antigen delivery systems or vaccination strategies that induce higb protective levels of CD8+ T cells. These cells are crucial for protection against certain tumours and intracellular pathogens such as the liver‐stage parasite of malaria, A liver‐stage malaria vaccine should therefore include CD8+ T‐cell‐inducing components. This review provides an overview of prime‐boost immunisation strategies that result in protective CD8’ T‐cell responses against malaria with an emphasis on work from our laboratory. Possible mechanisms explaining why heterologous prime‐boost strategies, in particular boosting with replication‐impaired recombinant poxviruses, are so effective are discussed.

Enhancement of Class II-Restricted T cell Responses by Costimulatory NK Receptors for Class I MHC Proteins

An important feature of the human immune system is the ability of T cells to respond to small quantities of antigen. Class II major histocompatibility complex (MHC)-restricted T cells that expressed a costimulatory natural killer (NK) cell receptor for class I MHC proteins were cloned. In the presence of low doses of superantigen, the proliferative response of these T cell clones was three- to ninefold greater when the T cells were costimulated by way of the NK receptor. Thus, the action of costimulatory NK receptors on T cells may play a significant role in initiating and sustaining immune responses.

Enhanced CD8+ T Cell Immune Responses and Protection Elicited against Plasmodium berghei Malaria by Prime Boost Immunization Regimens Using a Novel Attenuated Fowlpox Virus

Sterile immunity can be provided against the pre-erythrocytic stages of malaria by IFN-γ-secreting CD8+ T cells that recognize parasite-infected hepatocytes. In this study, we have investigated the use of attenuated fowlpox virus (FPV) strains as recombinant vaccine vectors for eliciting CD8+ T cells against Plasmodium berghei. The gene encoding the P. berghei circumsporozoite (PbCS) protein was inserted into an FPV vaccine strain licensed for use in chickens, Webster’s FPV, and the novel FPV vaccine strain FP9 by homologous recombination. The novel FP9 strain proved more potent as a vaccine for eliciting CD8+ T cell responses against the PbCS Ag. Sequential immunization with rFP9 and recombinant modified vaccinia virus Anakara (MVA) encoding the PbCS protein, administered by clinically acceptable routes, elicited potent CD8+ T cell responses against the PbCS protein. This immunization regimen elicited substantial protection against a stringent liver-stage challenge with P. berghei and was more immunogenic and protective than DNA/MVA prime/boost immunization. However, further improvement was not achieved by sequential (triple) immunization with a DNA vaccine, FP9, and MVA.

Human NK cells: their ligands, receptors and functions.

Summary: The expression, or lack thereof, of class I MHC glycoproteins has a marked influence on natural killer cell function. Cells which do not express class I MHC molecules are susceptible to lysis by NK cells, and transfection of these targets with class I MHC genes can render these cells resistant to NK attack. This inhibition of NK‐killing is mediated by a novel family of receptors expressed mainly on NK cells, but also found on some T‐cells. The function of these class I MHC binding receptors when expressed on T‐cells is discussed also and a novel co‐stimulatory activity of NKAR described. Lastly, a novel mechanism by which human cytomegalovirus evades immune surveillance by NK cells is documented.

A Plasmodium falciparum candidate vaccine based on a six-antigen polyprotein encoded by recombinant poxviruses

To generate broadly protective T cell responses more similar to those acquired after vaccination with radiation-attenuated Plasmodium falciparum sporozoites, we have constructed candidate subunit malaria vaccines expressing six preerythrocytic antigens linked together to produce a 3,240-aa-long polyprotein (L3SEPTL). This polyprotein was expressed by a plasmid DNA vaccine vector (DNA) and by two attenuated poxvirus vectors, modified vaccinia virus Ankara (MVA) and fowlpox virus of the FP9 strain. MVAL3SEPTL boosted anti-thrombospondin-related adhesive protein (anti-TRAP) and anti-liver stage antigen 1 (anti-LSA1) CD8+ T cell responses when primed by single antigen TRAP- or LSA1-expressing DNAs, respectively, but not by DNA-L3SEPTL. However, prime boost regimes involving two heterologous viral vectors expressing L3SEPTL induced a strong cellular response directed against an LSA1 peptide located in the C-terminal region of the polyprotein. Peptide-specific T cells secreted IFN-γ and were cytotoxic. IFN-γ-secreting T cells specific for each of the six antigens were induced after vaccination with L3SEPTL, supporting the use of polyprotein inserts to induce multispecific T cells against P. falciparum. The use of polyprotein constructs in nonreplicating poxviruses should broaden the target antigen range of vaccine-induced immunity and increase the number of potential epitopes available for immunogenetically diverse human populations.

Invariant Valpha14 chain NKT cells promote Plasmodium berghei circumsporozoite protein-specific gamma interferon- and tumor necrosis factor alpha-producing CD8+ T cells in the liver after poxvirus vaccination of mice

ABSTRACT Understanding the protective mechanism in the liver induced by recombinant vaccines against the pre-erythrocytic stages of malaria is important for vaccine development. Most studies in mice have focused on splenic and peripheral blood T cells and identified gamma interferon (IFN-γ)-producing CD8+ T cells as correlates of protection, which can be induced by prime-boost vaccination with recombinant poxviruses. Invariant natural killer T (Vα14iNKT) cells can also protect against liver stage malaria, when activated, and are abundant in the liver. Since poxviruses have nonspecific immunomodulating effects, which are incompletely understood, we investigated whether recombinant poxviruses affect the protective properties of hepatic Vα14iNKT cells and thus vaccine efficacy. We show that intradermal vaccination with recombinant poxviruses activated Vα14iNKT cells and NK cells in the livers of BALB/c mice while inducing IFN-γ- and tumor necrosis factor alpha (TNF-α)-producing pre-erythrocytic stage antigen-specific CD8+ T cells. Greater numbers of hepatic Vα14iNKT cells secreted interleukin-4 than IFN-γ. Vaccinated Vα14iNKT-cell-deficient mice had lower, but still protective levels of hepatic and splenic IFN-γ+ and TNF-α+ CD8+ T cells and better protection rates later after challenge with Plasmodium berghei sporozoites. Therefore, vaccine-activated hepatic Vα14iNKT cells help in generating specific T cells but are not required for protection induced by recombinant poxviruses. Furthermore, double-positive INF-γ+/TNF-α+ CD8+ T cells were enriched in protected livers, suggesting that cells expressing both of these cytokines may be most relevant for protection.

Critical role of identification of the second gland during unilateral parathyroid surgery: a prospective review of 119 patients with concordant localization.

HYPOTHESIS We aimed to validate the effectiveness of a protocol for primary hyperparathyroidism in which intraoperative parathyroid hormone measurement (IOPTH) was not routinely used during minimally invasive parathyroidectomy for patients with dual localization by technetium Tc 99m sestamibi (MIBI) and ultrasonography and hypothesized that our rate of surgical failure would be less than 3% for patients with concordant localization. DESIGN Prospective cohort study. SETTING Brigham and Womens Hospital, Boston, Massachusetts. PATIENTS One hundred nineteen patients with primary hyperparathyroidism and dual localization. MAIN OUTCOME MEASURES Incidence of surgical cure following minimally invasive parathyroidectomy (MIP) without the use of IOPTH for patients with dual localization. RESULTS A total of 324 patients with primary hyperparathyroidism underwent parathyroid exploration between October 1, 2005, and September 30, 2009. In 136 patients (42.0%), imaging was concordant by MIBI and ultrasonography, and 119 patients were scheduled for MIP. Our protocol for MIP without IOPTH was successful in 115 patients (97%), with 13 cases converted to bilateral exploration based on intraoperative suspicion of multiglandular disease. Eight of 13 conversions (62%) revealed multiglandular disease that was undetected on imaging, 6 of which were apparent from examination of the ipsilateral second parathyroid gland. Four of 136 patients (3%) had persistent postoperative hypercalcemia necessitating reoperation, and all 4 had an adjacent but unseen second adenoma. There was no significant difference in the surgical cure rate following MIP without IOPTH for this prospective study vs a previously published retrospective analysis by our group (97% vs 98%, P = .47). CONCLUSIONS Focused parathyroid gland exploration without IOPTH can be successfully performed in a select group of patients with dual localization by MIBI and ultrasonography. However, identification of the second ipsilateral gland may be critical to ruling out undetected multiglandular disease.

Inhibition of rat gut reperfusion injury with an agent developed for the mouse. Evidence that amplification of injury by innate immunity is conserved between two animal species

Murine reperfusion injury follows binding of specific IgM natural antibodies to neo-antigens exposed in ischemic tissue. Peptides that mimic the site of antibody binding in the injury prevent IgM binding when administered intravenously before reperfusion. To determine whether this pathogenic sequence is restricted to mice, we have tested the ability of the peptide to prevent reperfusion injury in a dissimilar species, the rat. Sprague-Dawley rats were subjected to 40 min of mesenteric ischemia followed by 180 min of reperfusion. The peptide mimic was administered intravenously prior to reperfusion. Gut injury was quantified using a scoring system based on the hematoxylin-and-eosin section. (125)I-labeled albumin was used to assess local (gut) and remote (lung) injury. The macroscopic appearance of bowel from peptide-treated animals was less edematous and hemorrhagic. Microscopic analysis showed a significantly reduced injury score in peptide-treated animals. Permeability data indicated a significant reduction in local and remote injury in peptide-treated animals. The data demonstrate attenuation of rat gut microvillus injury, of gut edema, and of remote injury following mesenteric ischemia-reperfusion due to administration of an intravenous peptide mimic of a murine ischemia neo-antigen, indicating a second species uses a similar ischemia neo-antigen and corresponding natural antibody specificity to amplify reperfusion injury to the point of necrosis. This mechanism of inflammation is potentially applicable to higher species.

An appraisal of the learning curve in robotic general surgery

BackgroundRobotic-assisted surgery is used with increasing frequency in general surgery for a variety of applications. In spite of this increase in usage, the learning curve is not yet defined. This study reviews the literature on the learning curve in robotic general surgery to inform adopters of the technology.MethodsPubMed and EMBASE searches yielded 3690 abstracts published between July 1986 and March 2016. The abstracts were evaluated based on the following inclusion criteria: written in English, reporting original work, focus on general surgery operations, and with explicit statistical methods.ResultsTwenty-six full-length articles were included in final analysis. The articles described the learning curves in colorectal (9 articles, 35%), foregut/bariatric (8, 31%), biliary (5, 19%), and solid organ (4, 15%) surgery. Eighteen of 26 (69%) articles report single-surgeon experiences. Time was used as a measure of the learning curve in all studies (100%); outcomes were examined in 10 (38%). In 12 studies (46%), the authors identified three phases of the learning curve. Numbers of cases needed to achieve plateau performance were wide-ranging but overlapping for different kinds of operations: 19–128 cases for colorectal, 8–95 for foregut/bariatric, 20–48 for biliary, and 10–80 for solid organ surgery.ConclusionAlthough robotic surgery is increasingly utilized in general surgery, the literature provides few guidelines on the learning curve for adoption. In this heterogeneous sample of reviewed articles, the number of cases needed to achieve plateau performance varies by case type and the learning curve may have multiple phases as surgeons add more complex cases to their case mix with growing experience. Time is the most common determinant for the learning curve. The literature lacks a uniform assessment of outcomes and complications, which would arguably reflect expertise in a more meaningful way than time to perform the operation alone.

Evaluation of the LINX antireflux procedure.

Purpose of review To evaluate the current data on the safety, efficacy, and indications for magnetic sphincter augmentation (MSA) using the LINX device to treat gastroesophageal reflux disease (GERD). Recent findings The LINX device has demonstrated excellent safety and GERD efficacy in several recent nonblinded, single arm studies with strict inclusion criteria and up to 3 years follow-up. Dysphagia has been the most common adverse effect occurring after LINX. Other gastrointestinal side-effects seen after laparoscopic fundoplication (bloating, gas, and inability to belch) may be less common after LINX. Summary The LINX device is a safe, well tolerated, and effective therapy for GERD in the short term. MSA should be considered for selected GERD patients without significant anatomic or motility defects. However, the long-term safety and efficacy of LINX – both alone and in comparison to current GERD therapies – remains to be determined.