Victor van Berkel

Identification of a Gammaherpesvirus Selective Chemokine Binding Protein That Inhibits Chemokine Action

ABSTRACT Chemokines are involved in recruitment and activation of hematopoietic cells at sites of infection and inflammation. The M3 gene of γHV68, a gamma-2 herpesvirus that infects and establishes a lifelong latent infection and chronic vasculitis in mice, encodes an abundant secreted protein during productive infection. The M3 gene is located in a region of the genome that is transcribed during latency. We report here that the M3 protein is a high-affinity broad-spectrum chemokine scavenger. The M3 protein bound the CC chemokines human regulated upon activation of normal T-cell expressed and secreted (RANTES), murine macrophage inflammatory protein 1α (MIP-1α), and murine monocyte chemoattractant protein 1 (MCP-1), as well as the human CXC chemokine interleukin-8, the murine C chemokine lymphotactin, and the murine CX3C chemokine fractalkine with high affinity (Kd = 1.6 to 18.7 nM). M3 protein chemokine binding was selective, since the protein did not bind seven other CXC chemokines (Kd > 1 μM). Furthermore, the M3 protein abolished calcium signaling in response to murine MIP-1α and murine MCP-1 and not to murine KC or human stromal cell-derived factor 1 (SDF-1), consistent with the binding data. The M3 protein was also capable of blocking the function of human CC and CXC chemokines, indicating the potential for therapeutic applications. Since the M3 protein lacks homology to known chemokines, chemokine receptors, or chemokine binding proteins, these studies suggest a novel herpesvirus mechanism of immune evasion.

Structural Basis of Chemokine Sequestration by a Herpesvirus Decoy Receptor

The M3 protein encoded by murine gamma herpesvirus68 (gamma HV68) functions as an immune system saboteur by the engagement of chemoattractant cytokines, thereby altering host antiviral inflammatory responses. Here we report the crystal structures of M3 both alone and in complex with the CC chemokine MCP-1. M3 is a two-domain beta sandwich protein with a unique sequence and topology, forming a tightly packed anti-parallel dimer. The stoichiometry of the MCP-1:M3 complex is 2:2, with two monomeric chemokines embedded at distal ends of the preassociated M3 dimer. Conformational flexibility and electrostatic complementation are both used by M3 to achieve high-affinity and broad-spectrum chemokine engagement. M3 also employs structural mimicry to promiscuously sequester chemokines, engaging conservative structural elements associated with both chemokine homodimerization and binding to G protein-coupled receptors.

Establishment and Maintenance of Gammaherpesvirus Latency Are Independent of Infective Dose and Route of Infection

ABSTRACT Gammaherpesviruses such as Epstein-Barr virus and Kaposis sarcoma-associated herpesvirus are important human pathogens that establish long-term latent infections. Understanding of the initiation and maintenance of latent infections has important implications for the prevention and treatment of gammaherpesvirus-related diseases. Although much is known about gammaherpesvirus pathogenesis, it is unclear how the infectious dose of a virus influences its ability to establish latent infection. To examine the relationship between the infectious dose and gammaherpesvirus latency, we inoculated wild-type mice with 0.01 to 106 PFU of murine gammaherpesvirus 68 (γHV68) and quantitatively measured latency and acute-phase replication. Surprisingly, during latency, the frequencies of ex vivo reactivation were similar over a 107-fold range of doses for i.p. infection and over a 104-fold range of doses for intranasal infection. Further, the frequencies of cells harboring viral genome during latency did not differ substantially over similar dose ranges. Although the kinetics of acute-phase replication were delayed at small doses of virus, the peak titer did not differ significantly between mice infected with a large dose of virus and those infected with a small dose of virus. The results presented here indicate that any initiation of infection leads to substantial acute-phase replication and subsequent establishment of a maximal level of latency. Thus, infections with doses as small as 0.1 PFU of γHV68 result in stable levels of acute-phase replication and latent infection. These results demonstrate that the equilibrium level of establishment of gammaherpesvirus latency is independent of the infectious dose and route of infection.

Critical role for a high-affinity chemokine-binding protein in γ-herpesvirus-induced lethal meningitis

Chemokines are involved in recruitment and activation of hematopoietic cells in sites of infection and inflammation. The M3 gene of the gamma-herpesvirus gammaHV68 encodes an abundant secreted protein that binds CC chemokines with high affinity. We report here that this gene is essential for efficient induction of lethal meningitis by gammaHV68. An M3 mutant gammaHV68 (gammaHV68-M3.stop) was 100-fold less virulent than wild-type or marker rescue control (gammaHV68-M3.MR) viruses after intracerebral inoculation. After intracerebral inoculation, gammaHV68-M3.stop grew to lower titers than gammaHV68 or gammaHV68-M3.MR in the brain but spread to and grew normally in the spleen and lung. Expression of several CC chemokines was significantly induced in the CNS by gammaHV68 infection. Consistent with M3 acting by blockade of CC chemokine action, gammaHV68 induced a neutrophilic meningeal inflammatory infiltrate, while gammaHV68-M3.stop induced an infiltrate in which lymphocytes and macrophages predominated. In contrast to the important role of M3 in lethal meningitis, M3 was not required for establishment or reactivation from latent infection or induction of chronic arteritis. These data suggest a role for chemokines in the protection of the nervous system from viral infection and that the M3 protein acts in a tissue-specific fashion during acute but not chronic gammaHV68 infection to limit CC chemokine-induced inflammatory responses.

Enhanced penetration into 3D cell culture using two and three layered gold nanoparticles

Nano-scale particles sized 10–400 nm administered systemically preferentially extravasate from tumor vasculature due to the enhanced permeability and retention effect. Therapeutic success remains elusive, however, because of inhomogeneous particle distribution within tumor tissue. Insufficient tumor vascularization limits particle transport and also results in avascular hypoxic regions with non-proliferating cells, which can regenerate tissue after nanoparticle-delivered cytotoxicity or thermal ablation. Nanoparticle surface modifications provide for increasing tumor targeting and uptake while decreasing immunogenicity and toxicity. Herein, we created novel two layer gold-nanoshell particles coated with alkanethiol and phosphatidylcholine, and three layer nanoshells additionally coated with high-density-lipoprotein. We hypothesize that these particles have enhanced penetration into 3-dimensional cell cultures modeling avascular tissue when compared to standard poly(ethylene glycol) (PEG)-coated nanoshells. Particle uptake and distribution in liver, lung, and pancreatic tumor cell cultures were evaluated using silver-enhancement staining and hyperspectral imaging with dark field microscopy. Two layer nanoshells exhibited significantly higher uptake compared to PEGylated nanoshells. This multilayer formulation may help overcome transport barriers presented by tumor vasculature, and could be further investigated in vivo as a platform for targeted cancer therapies.

Surgical management of pulmonary carcinoid tumors: sublobar resection versus lobectomy.

BACKGROUND Surgical resection of bronchopulmonary carcinoid tumors can be curative and remains the primary treatment modality. There are limited data to delineate the optimal extent of resection for this disease. METHODS A retrospective review of the 3,270 patients diagnosed with typical and atypical carcinoid tumors between 2000 and 2007 in the Surveillance Epidemiology and End Results registry was performed. RESULTS The mean follow-up period was 46 months (range, 1-95 mo). Overall survival (OS) and disease-specific survival at 5 years was 80% and 90%, respectively. The mean OS was slightly better in the lobectomy group compared with those undergoing sublobar resection (86 vs 83 mo; P = .008). After adjusting for age, this finding was no longer present (P = .513). By using multivariate analysis, sublobar resection was noninferior to lobectomy with regard to disease-specific survival and OS (P < .05). CONCLUSIONS Compared with lobectomy, sublobar resection is associated with noninferior survival in patients with typical carcinoid of the lung.

Impact of Anastomotic Techniques on Airway Complications After Lung Transplant

BACKGROUND Airway complications are a source of morbidity and expense after lung transplant. Posttransplant stenosis can occur when the donor bronchus is rendered ischemic and is dependent upon collateral flow from the pulmonary capillary system. By shortening the donor bronchus, the tissue at risk for ischemia is reduced. In an effort to reduce airway complications, one surgeon at our institution began dividing the donor bronchus at the lobar carina. METHODS This is a retrospective analysis of all transplanted patients over the 2-year period before and after the institution of the technique change. To adjust for covariates, we performed a propensity score analysis. Outcome endpoints were postoperative airway complications, specifically, the need for therapeutic bronchoscopy, dilation, stenting, or retransplant. RESULTS After instituting the practice of dividing the bronchus at the lobar carina, the incidence of airway complication for the principle surgeon decreased from 13.2% (7 of 53) to 2.1% (1 of 48), resulting in an improved freedom from airway complication for that surgeon. Compared with all transplants performed during this period, the modified anastomosis resulted in fewer airway complications: 2.1% (1 of 48) versus 8.2% (19 of 231). The propensity analysis matched the 48 patients who received the modified anastomosis with 48 patients who received the standard two ring length anastomosis by surgical colleagues. The modified anastomosis group had fewer required interventions for airway complications and had significantly better freedom from airway complication when followed over time. CONCLUSIONS Decreasing the amount of potentially ischemic tissue implanted from the donor bronchus can reduce posttransplant airway complications.

Quantitative analysis of exhaled carbonyl compounds distinguishes benign from malignant pulmonary disease

OBJECTIVES The analysis of exhaled breath is a promising noninvasive tool for the diagnosis of lung cancer, but its clinical relevance has yet to be established. We report the analysis of exhaled volatile carbonyl compounds for the identification of specific carbonyl cancer markers to differentiate benign pulmonary disease from early-stage lung cancer and to compare its diagnostic accuracy with positron emission tomography (PET) scans. METHODS Aminooxy-coated silicon microchips were used for the selective capture of exhaled carbonyls by an oximation reaction. Breath samples were collected then directed through the silicon chips by applying a vacuum. Carbonyl adducts were analyzed by Fourier transform mass spectrometry. Eighty-eight control subjects, 107 patients with lung cancer (64 stage 0, I, or II), 40 patients with benign pulmonary disease, and 7 patients with a solitary pulmonary metastasis participated. Analysis of cancer markers was performed blinded to the pathologic results. RESULTS Four carbonyls were defined as cancer markers with significantly higher concentrations in patients with lung cancer. The number of increased cancer markers distinguished benign disease from both early and stage III and IV lung cancer. For early-stage disease, defining greater than 2 increased markers as diagnostic of lung cancer resulted in 83% sensitivity and 74% specificity. PET scans for this same cohort resulted in 90% sensitivity but only 39% specificity. Markers normalized for 3 of the 4 markers after resection of the lung cancer. CONCLUSIONS Analysis of specific exhaled carbonyls can differentiate early lung cancer from benign pulmonary disease. Breath analysis was more specific than PET for a lung cancer diagnosis. Judicious use of these data may expedite the care of patients with lung cancer.

High sensitivity for lung cancer detection using analysis of exhaled carbonyl compounds.

OBJECTIVE Several volatile carbonyl compounds in exhaled breath have been identified as cancer-specific markers. The potential for these markers to serve as a screening test for lung cancer is reported. METHODS Patients with computed tomography-detected intrathoracic lesions and healthy control participants were enrolled from 2011 onward. One liter of breath was collected from a single exhalation from each participant. The contents were evacuated over a silicon microchip, captured by oximation reaction, and analyzed by mass spectrometry. Concentrations of 2-butanone, 3-hydroxy-2-butanone, 2-hydroxyacetaldehyde, and 4-hydroxyhexanal were measured. The overall population was divided into 3 groups: those with lung cancer, benign disease, and healthy controls. An elevated cancer marker was defined as ≥1.5 SDs above the mean concentration of the control population. One or more elevated cancer markers constituted a positive breath test. RESULTS In all, 156 subjects had lung cancer, 65 had benign disease, and 194 were healthy controls. A total of 103 (66.0%) lung cancer patients were early stage (stage 0, I, and II). For ≥1 elevated cancer marker, breath analysis showed a sensitivity of 93.6%, and a specificity of 85.6% for lung cancer patients. Additionally, 83.7% of stage I tumors ≤2 cm were detected; whereas only 14% of the control population tested positive. In a comparison of cancer to benign disease, specificity was proportional to the number of elevated cancer markers present. CONCLUSIONS Screening using a low-dose CT scan is associated with high cost, repeated radiation exposure, and low accrual. The high sensitivity, convenience, and low cost of breath analysis for carbonyl cancer markers suggests that it has the potential to become a primary screening modality for lung cancer.

Lung donor selection criteria

The criteria that define acceptable physiologic and social parameters for lung donation have remained constant since their empiric determination in the 1980s. These criteria include a donor age between 25-40, a arterial partial pressure of oxygen (PaO2)/FiO2 ratio greater than 350, no smoking history, a clear chest X-ray, clean bronchoscopy, and a minimal ischemic time. Due to the paucity of organ donors, and the increasing number of patients requiring lung transplant, finding a donor that meets all of these criteria is quite rare. As such, many transplants have been performed where the donor does not meet these stringent criteria. Over the last decade, numerous reports have been published examining the effects of individual acceptance criteria on lung transplant survival and graft function. These studies suggest that there is little impact of the historical criteria on either short or long term outcomes. For age, donors should be within 18 to 64 years old. Gender may relay benefit to all female recipients especially in male to female transplants, although results are mixed in these studies. Race matched donor/recipients have improved outcomes and African American donors convey worse prognosis. Smoking donors may decrease recipient survival post transplant, but provide a life saving opportunity for recipients that may otherwise remain on the transplant waiting list. No specific gram stain or bronchoscopic findings are reflected in recipient outcomes. Chest radiographs are a poor indicator of lung donor function and should not adversely affect organ usage aside for concerns over malignancy. Ischemic time greater than six hours has no documented adverse effects on recipient mortality and should not limit donor retrieval distances. Brain dead donors and deceased donors have equivalent prognosis. Initial PaO2/FiO2 ratios less than 300 should not dissuade donor organ usage, although recruitment techniques should be implemented with intent to transplant.