Phyllis Flomenberg

Risk factors for surgical site infections following spinal fusion procedures: a case-control study.

BACKGROUND Spinal fusion procedures are associated with a significant rate of surgical site infection (SSI) (1%-12%). The goal of this study was to identify modifiable risk factors for spinal fusion SSIs at a large tertiary care center. METHODS A retrospective, case-control (1:3 ratio) analysis of SSIs following posterior spine fusion procedures was performed over a 1-year period. Clinical and surgical data were collected through electronic database and chart review. Variables were evaluated by univariate analysis and multivariable logistic regression. RESULTS In total, 57 deep SSIs were identified out of 1587 procedures (3.6%). Infections were diagnosed a mean of 13.5 ± 8 days postprocedure. Staphylococcus aureus was the predominant pathogen (63%); 1/3 of these isolates were methicillin resistant. Significant patient risk factors for infection by univariate analysis included ASA score >2 and male gender. Among surgical variables, infected cases had significantly higher proportions of staged procedures and thoracic level surgeries and had a greater number of vertebrae fused. Notably, infected fusion procedures had a longer duration of closed suction drains than controls (5.1 ± 2 days vs 3.4 ± 1 day, respectively; P < .001). Drain duration (unit odds ratio [OR], 1.6 per day drain present; 95% confidence interval [CI], 1.3-1.9), body mass index (OR, 1.1; 95% CI, 1.0-1.1), and male gender (OR, 2.7; 95% CI, 1.4-5.6) were significant risk factors in the multivariate analysis. CONCLUSIONS Prolonged duration of closed suction drains is a strong independent risk factor for SSI following instrumented spinal fusion procedures. Therefore, removing drains as early as possible may lower infection rates.

Adenovirus hexon T-cell epitope is recognized by most adults and is restricted by HLA DP4, the most common class II allele

The immunogenicity of adenovirus (Ad) vectors is enhanced by virus-specific memory immune responses present in most individuals as a result of past exposure to these ubiquitous pathogens. We previously identified the first human T-cell epitope from the major capsid protein hexon, H910-924, and found that it is highly conserved among different Ad serotypes. Memory/effector T-cell responses to H910-924 were detected in 14 of 18 (78%) healthy adults by an interferon-γ ELISPOT assay. Hexon peptide-specific CD4 T-cell lines were generated from three HLA-typed donors and analyzed using a panel of HLA homozygous B-cell lines and monoclonal antibodies to HLA class II loci. These studies reveal that the hexon epitope is restricted by HLA DP4, a class II allele present in 75% of the population. Analysis of overlapping peptides and peptides with single residue mutations identified a HLA DP4-binding motif. Additionally, antibodies to the hexon peptide were detected in all donor sera by dot blot assay and ELISA. Therefore, most individuals exhibit both memory B- and T-cell responses to this highly conserved epitope on hexon, an obligate component of all Ad vectors, including ‘gutted’ vectors. These data suggest that current strategies for the use of Ad gene therapy vectors will not evade memory immune responses to Ad.

A 2-step approach to myeloablative haploidentical stem cell transplantation: a phase 1/2 trial performed with optimized T-cell dosing.

Studies of haploidentical hematopoietic stem cell transplantation (HSCT) have identified threshold doses of T cells below which severe GVHD is usually absent. However, little is known regarding optimal T-cell dosing as it relates to engraftment, immune reconstitution, and relapse. To begin to address this question, we developed a 2-step myeloablative approach to haploidentical HSCT in which 27 patients conditioned with total body irradiation (TBI) were given a fixed dose of donor T cells (HSCT step 1), followed by cyclophosphamide (CY) for T-cell tolerization. A CD34-selected HSC product (HSCT step 2) was infused after CY. A dose of 2 × 10(8)/kg of T cells resulted in consistent engraftment, immune reconstitution, and acceptable rates of GVHD. Cumulative incidences of grade III-IV GVHD, nonrelapse mortality (NRM), and relapse-related mortality were 7.4%, 22.2%, and 29.6%, respectively. With a follow-up of 28-56 months, the 3-year probability of overall survival for the whole cohort is 48% and 75% in patients without disease at HSCT. In the context of CY tolerization, a high, fixed dose of haploidentical T cells was associated with encouraging outcomes, especially in good-risk patients, and can serve as the basis for further exploration and optimization of this 2-step approach. This study is registered at www.clinicaltrials.gov as NCT00429143.

Quantitative Analysis of Adenovirus-Specific CD4+ T-Cell Responses from Healthy Adults

Although nearly all adults are seropositive for adenoviruses, little is known about the cellular immune responses to these ubiquitous pathogens. We have previously identified adenovirus-specific proliferative T-cell responses in peripheral blood mononuclear cells (PBMC) from healthy adults. In this study, memory T-cell responses to adenovirus were further evaluated in healthy adult donors using a short term, quantitative enzyme-linked immunospot assay (ELISPOT) assay. Adenovirus antigen induced specific secretion of interferon-gamma (IFN-gamma) from PBMC within 12 hours of incubation. PBMC from 20 of 22 healthy donors (90.9%) expressed IFN-y in response to adenovirus. Responder cells were identified as CD4+ T cells by immunomagnetic depletion methods. Interleukin-4 (IL-4) secretion was not detected, consistent with a TH1 response. There was a 10-fold variation in the frequencies of adenovirus-specific CD4+ T cells between donors (range, 34 to 294; median, 122 per million PBMC). Adenovirus-specific T cell frequencies remained stable over periods up to 2 years among individual donors, but there was an inverse correlation between frequency and donor age. These quantitative data suggest that most adults retain adenovirus-specific cellular memory after childhood exposure. This assay may be useful for the evaluation of adenovirus-specific CD4+ T-cell responses in patients treated with adenovirus gene therapy vectors and the identification of major T-cell epitopes.

Glioblastoma patients exhibit circulating tumor-specific CD8+ T cells.

Purpose: There is growing interest in developing cellular immune therapies for glioblastoma multiforme, but little is known about tumor-specific T-cell responses. A glioblastoma multiforme–specific T-cell assay was developed using monocyte-derived dendritic cells to present tumor antigens from the established glioblastoma multiforme cell line U118. Experimental Design: Peripheral blood mononuclear cells (PBMC) and tumor cells were obtained from nine patients with newly diagnosed brain tumors: five glioblastoma multiforme, two oligodendroglioma, one ependymoma, and one astrocytoma. PBMCs were incubated overnight with autologous tumor cells or autologous dendritic cells loaded with a U118 cell lysate, and responses were detected by IFN-γ ELISPOT and cytokine flow cytometry assays. Results: PBMCs from all glioblastoma multiforme patients exhibited IFN-γ responses to autologous tumor but not to HLA-mismatched U118 cells. Glioblastoma multiforme–specific IFN-γ responses were primarily mediated by CD8+ T cells and represented ∼2% of total CD8+ T cells. Additionally, all glioblastoma multiforme patients responded to autologous dendritic cells loaded with U118 lysate but not with low-grade astrocytoma cell lysates. PBMCs from four patients with other brain tumor types and one normal donor failed to respond to U118 lysate–loaded autologous dendritic cells. These data indicate that the IFN-γ responses to U118 lysate–loaded autologous dendritic cells are glioblastoma multiforme specific. Moreover, PBMCs stimulated 1 to 2 weeks with U118 lysate–loaded dendritic cells exhibited MHC class I–restricted cytotoxicity against autologous tumor cells. Conclusions: Glioblastoma multiforme patients exhibit circulating tumor-specific CD8+ T cells that recognize shared tumor antigens from the glioblastoma multiforme cell line U118. These data show that glioblastoma multiformes are immunogenic and support the development of immunotherapy trials.

Detection of adenovirus DNA in peripheral blood mononuclear cells by polymerase chain reaction assay

Adenovirus can establish persistent infections which may reactivate and cause disease in immunocompromised hosts. Lymphocytes have been postulated to serve as a site of adenoviral persistence based upon the ability to isolate adenovirus from tonsils and to detect adenovirus DNA by Southern blot hybridization in peripheral blood mononuclear cells (PBMC). To test this hypothesis, a more sensitive and specific polymerase chain reaction (PCR) assay was developed to detect adenovirus DNA. Two sets of nested primers were designed to conserved sequences in the adenovirus E1A and hexon genes. The E1A and hexon primers amplified DNA from representative adenoviral serotypes in all six adenoviral groups (A‐F). Both primers detected a single copy of the adenovirus type 2 genome but were less sensitive for the group B type 35. None of 33 PBMC specimens from healthy adults and only one of 40 pediatric samples was positive (at a low level) for adenovirus DNA by nested PCR assay. In comparison, PBMC from two children with fatal adenoviral infection were both strongly positive for adenovirus DNA. It is concluded that, in contrast to a previous study, PBMC are not a common site of persistent group C adenoviral infection. In addition, assay of PBMC by the adenovirus‐specific PCR may help detect early invasive disease and warrants further evaluation. J. Med. Virol. 51:182–188, 1997.

Impact of a standardized protocol and antibiotic-impregnated catheters on ventriculostomy infection rates in cerebrovascular patients.

BACKGROUNDVentriculostomy infections create significant morbidity. To reduce infection rates, a standardized evidence-based catheter insertion protocol was implemented. A prospective observational study analyzed the effects of this protocol alone and with antibiotic-impregnated ventriculostomy catheters. OBJECTIVETo compare infection rates after implementing a standardized protocol for ventriculostomy catheter insertion with and without the use of antibiotic-impregnated catheters. METHODSBetween 2003 and 2008, 1961 ventriculostomies and infections were documented. A ventriculostomy infection was defined as 2 positive CSF cultures from ventriculostomy catheters with a concurrent increase in cerebrospinal fluid white blood cell count. A baseline (preprotocol) infection rate was established (period 1). Infection rates were monitored after adoption of the standardized protocol (period 2), institution of antibiotic-impregnated catheter A (period 3), discontinuation of antibiotic-impregnated catheter A (period 4), and institution of antibiotic-impregnated catheter B (period 5). RESULTSThe baseline infection rate (period 1) was 6.7% (22/327 devices). Standardized protocol (period 2) implementation did not change the infection rate (8.2%; 23/281 devices). Introduction of catheter A (period 3) reduced infections to 1.0% (2/195 devices, P = .0005). Because of technical difficulties, this catheter was discontinued (period 4), resulting in an increase in infection rate (7.6%; 12/157 devices). Catheter B (period 5) significantly decreased infections to 0.9% (9 of 1001 devices, P = .0001). The Staphylococcus infection rate for periods 1, 2, and 4 was 6.1% (47/765) compared with 0.2% (1/577) during use of antibiotic-impregnated catheters (periods 3 and 5). CONCLUSIONThe use of antibiotic-impregnated catheters resulted in a significant reduction of ventriculostomy infections and is recommended in the adult neurosurgical population.

Characterization of a Single-Cycle Rabies Virus-Based Vaccine Vector

ABSTRACT Recombinant rabies virus (RV)-based vectors have demonstrated their efficacy in generating long-term, antigen-specific immune responses in murine and monkey models. However, replication-competent viral vectors pose significant safety concerns due to vector pathogenicity. RV pathogenicity is largely attributed to its glycoprotein (RV-G), which facilitates the attachment and entry of RV into host cells. We have developed a live, single-cycle RV by deletion of the G gene from an RV vaccine vector expressing HIV-1 Gag (SPBN-ΔG-Gag). Passage of SPBN-ΔG-Gag on cells stably expressing RV-G allowed efficient propagation of the G-deleted RV. The in vivo immunogenicity data comparing single-cycle RV to a replication-competent control (BNSP-Gag) showed lower RV-specific antibodies; however, the overall isotype profiles (IgG2a/IgG1) were similar for the two vaccine vectors. Despite this difference, mice immunized with SPBN-ΔG-Gag and BNSP-Gag mounted similar levels of Gag-specific CD8+ T-cell responses as measured by major histocompatibility complex class I Gag-tetramer staining, gamma interferon-enzyme-linked immunospot assay, and cytotoxic T-cell assay. Moreover, these cellular responses were maintained equally at immunization titers as low as 103 focus-forming units for both RV vaccine vectors. CD8+ T-cell responses were significantly enhanced by a boost with a single-cycle RV complemented with a heterologous vesicular stomatitis virus glycoprotein. These findings demonstrate that single-cycle RV is an effective alternative to replication-competent RV vectors for future development of vaccines for HIV-1 and other infectious diseases.

Human T-Cell Responses to Vaccinia Virus Envelope Proteins

ABSTRACT One approach for a safer smallpox vaccine is to utilize recombinant subunits rather than live vaccinia virus (VACV). The products of the VACV envelope genes A27L, L1R, B5R, and A33R induce protective antibodies in animal models. We propose that proteins that elicit T-cell responses, as well as neutralizing antibodies, will be important to include in a molecular vaccine. To evaluate VACV-specific memory T-cell responses, peripheral blood mononuclear cells (PBMC) from four VACV vaccinees were tested against whole VACV and the individual envelope proteins A27, B5, L1, and A33, using gamma interferon enzyme-linked immunospot and cytokine flow cytometry assays. PBMC were stimulated with autologous dendritic cells infected with VACV or electroporated with individual VACV protein mRNAs. T-cell lines from all donors, vaccinated from 1 month to over 20 years ago, recognized all four VACV envelope proteins. Both CD4+ and CD8+ T-cell responses to each protein were detected. Further analysis focused on representative proteins B5 and A27. PBMC from a recent vaccinee exhibited high frequencies of CD4+ and CD8+ T-cell precursors to both B5 (19.8 and 20%, respectively) and A27 (6.8 and 3.7%). In comparison, B5- and A27-specific T-cell frequencies ranged from 0.4 to 1.3% in a donor vaccinated 3 years ago. Multiple CD4+ and CD8+ T-cell epitopes were identified from both A27 and B5, using overlapping 15-mer peptides. These data suggest that all four VACV envelope proteins may contribute to protective immunity, not only by inducing antibody responses, but also by eliciting T-cell responses.

Spontaneous, persistent infection of a B-cell lymphoma with adenovirus

An adenovirus culture‐positive lymphoblastoid cell line was derived from a bone marrow transplant recipient with fatal B‐cell lymphoproliferative disease and adenovirus pneumonia. At autopsy, focal areas of the lymphoma infiltrating the patients lung were positive for adenovirus proteins by immunohistochemical staining. The Epstein‐Barr virus‐transformed B‐cell line Mk, established from pleural fluid cells, contained adenovirus virions in both the nucleus and the cytoplasm by electron microscopy. The majority of Mk cells expressed adenovirus proteins and produced a high level of infectious adenovirus by plaque assay analysis. However, in contrast to the rapid cell death induced by adenovirus in other permissive cell lines, Mk was maintained stably in tissue culture for 6 months. These data indicate that adenoviral replication is not sufficient for cell lysis and confirm that adenovirus can cause persistent infection in human lymphoid cells in vivo.