University at Buffalo
The Witebsky Center

The Witebsky Center
University at Buffalo
Bacteriology hostmicrobe immunology parasitology virology bioinformatics mycology
The Witebsky Center The Witebsky Center
Paul R. Knight., Ph.D.
Paul R. Knight, M.D., Ph.D.
Departments of Anesthesiology and Microbiology
Phone: (716) 829-3582
Fax: (716) 829 2172

Pathogenesis of Aspiration Pneumonitis (HL48889): A major focus of our laboratory is studying the mechanism(s) involved in the development of acute lung injury/acute respiratory distress syndrome (ALI/ARDS) from a variety of pulmonary insults. The lung injury resulting from the aspiration of gastric contents, containing both acidic and small food particle components, and how it interacts with other common concomitant injuries (i.e., lung contusion and bacterial pneumonia) is of particular interest. The combination of these insults results in a synergistic and sustained ALI/ARDS. Our investigations center on the cellular mechanisms (parenchymal, as well inflammatory cells) involved in the evolution of the injury, and the cytokine/chemokine networks that direct its course. We are also investigating the role that gender and pregnancy play in the pathogenesis of gastric aspiration. Pregnancy carries an elevated risk of gastric aspiration and subsequent development of ALI/ARDS. We have found that non-pregnant females are more resistant to gastric aspiration injury than males, while pregnant females are more susceptible. The estrus cycle phase does not explain this observed resistance. We plan to continue to explore the pathogenic mechanisms that are responsible for why females are more resistant and pregnant females are more susceptible than males to gastric aspiration. In addition, the interaction of gastric aspiration and lung contusion, a combination common in blunt chest trauma, is the subject of a translational patient study that we are currently conducting (see below).

            Our laboratory is also examining the interactive role of the resident alveolar macrophage (aMØ) and alveolar epithelial cell in the inflammatory response to gastric aspiration. Based on our initial published results, we are focused on ex vivo and in vitro responses of aMØ and alveolar type II epithelial cells (AECs) following: 1) a transient, non-lethal low pH shock, 2) exposure to non-acidified small food particles (SNAP), or 3) a combined acid and small gastric particles (CASP) insult. In vitro experiments are critical because low pH aspiration produces a heterogeneous ALI, and two populations of MØ can clearly be identified post aspiration (cells that are directly exposed to a transient H+ stress and those MØ that are not). The relative size of these two populations greatly affects the overall responsiveness to a secondary insult. We have completed the in vitro experiments with the individual cell types and have begun comparing the interactions of aMØ and AECs in co-cultures following exposure to different insults (e.g., uninjured AECs and low pH injured aMØ, or low pH injured AECs and SNAP exposed aMØ). The experiments employ the strategy of assaying for the release of cytokines (i.e., TNFα & IL-1) and chemokines (i.e., MIP-2, KC, Lix, MCP-1, MIP-1α) from the homogenous cells and co-cultures in order to assess alveolar capillary wall cell crosstalk. Additionally, we have compared the degree of apoptosis/necrosis in the different cell cultures or co-cultures. These in vitro studies are providing us with a well-defined picture of the role and interaction of these primary cells of the alveolus during the pathogenesis of gastric aspiration and how the different components of the aspirate stimulate the individual alveolar cells. The findings, thus far, clearly support our hypothesis that, AECs are the primary cells involved in the pathogenesis of the acute neutrophil inflammatory response following low pH aspiration, while the aMØ-directed inflammatory response is the principal mechanism responsible for SNAP acute lung injury. As in the in vivo model, CASP aspiration results in a synergistic interaction between both. However, the low pH stress also clearly modifies the responsiveness of the aMØ population that is directly exposed to the insult. Following these studies we will proceed to ex vivo isolation of different cell types from animals with different injuries and homogenous culturing and co-culturing to compare with the in vitro findings. The results from these experiments will be compared with aMØ and AECs isolated from the lungs of animals with different in vivo aspiration injuries. We also plan to assess H+-induced perturbation of intracellular recognition signaling.

            We are also examining the role of TNFα, IL-6, IL-10, and MCP-1 in the establishment of the synergistic lung injury phenotype following acid aspiration using over expression and gene deletion. Additionally, the risk of impaired bacterial clearance from the lungs of mice that experience acid aspiration-induced changes to the pulmonary cytokine milieu, as well as cytokine therapy are being assessed. An exciting and novel direction the experimental strategy has taken is the use of cDNA or siRNA complexed to nanoparticles. This approach has many advantages over using the defective viral vector lung delivery that we originally planned on using. We have synthesized a plasmid containing a red fluorescent protein (RFP)-TNFa fusion protein and successfully transfected cells. We are complexing this plasmid to gold nanorods (GNRs) and will transfect alveolar cells, in vivo, to supplement TNFa production in acid ALI. We have previously transfected cells in vivo with cDNA or siRNA nanoplexes. Based on delivery techniques and surface charge engineering of the nanoplexes, selective targeting of specific cells of the upper airways or alveoli has been performed.

      Regarding our studies on secondary bacterial infection model following acid aspiration, we have found that acid aspiration interferes with Toll-like receptor (TLR)-4 (as well as other TLRs) signaling in aMØ. We also plan to examine the role of the low pH induced decrease in TNFα from aMØ as an important mechanism involved in the synergistic acid and particulate aspiration lung injury, as well as the predisposition to a secondary bacterial infection. A transient or sustained low pH stress to aMØ impairs TLR-activated innate immune responses and subsequent pulmonary bacterial clearance, thereby predisposing the patient to a secondary bacterial pneumonia. Our previous work has identified changes secondary to aMØ low pH stress that are predicted to play an important role in the impairment of innate antibacterial immunity, specifically suppression of LPS-induced aMØ TLR4 signaling. We have also demonstrated low pH inhibition of additional surface membrane- (TLR2) and intracellular-expressed TLR (TLR3 and TLR9) signaling. We hypothesize that alterations of these innate immune responses are responsible for the increased susceptibility of patients to bacterial pneumonia following a low pH stress. We are currently examining the cellular mechanisms involved in low pH induced impairment of aMØ TLR innate immune signaling pathways in vivo, ex vivo and in vitro in mouse models of E. coli and S. pneumoniae pneumonia developed in our laboratory. We have hypothesized that decreased TLR4 signaling by the aMØ following a low pH stress plays an important role in the impaired clearance of bacteria from the lung. We are also examining the interactions of bacterial virulence factors (VFs) with the low pH impaired aMØ TLR4 signaling pathways. We have hypothesized that E. coli and S. pneumoniae VFs interact with H+-induced changes in aMØ TLR4 signaling to increase protection of the bacteria against early host antibacterial responses. We will specifically examine the capsules of both bacteria, the TLR4 binding VF, pneumolysin, and the newly identified E. coli secretory homologue TcpC, that impedes TLR4 signaling through MyD88. Finally we propose to employ site-specific delivery of multimodal nanoparticle-tagged TNFα cDNA (see above) into specific resident cells that constitute the alveoli in order to examine the role of suppression of TNFα levels following increased H+-induced inhibition of aMØ TLR4 activation in the impaired clearance of bacteria from the lung, as well as assess a novel therapeutic approach. We predict that reversing the inhibition of this key proximal event in innate host defense will improve bacterial clearance from the lung following a low pH stress.

      Finally, as mentioned above, a comparative mechanistic analysis of pulmonary and systemic inflammatory responses in mice and patients with aspiration ALI is being performed. We have hypothesized that mice and humans will locally and systemically display comparable inflammatory response profiles. We have continued to recruited patients with and without observed gastric aspiration to obtain samples. The inflammatory cell counts from the BAL, and total cell and differentiation counts determined on the blood samples are performed immediately. The BAL supernatant and the lipopolysaccharide-stimulated and non-stimulated blood samples are being stored at -70°C for “batch” cytokine analysis as detailed. These samples will be directly compared with the murine models of different gastric aspiration lung injuries. In a set of experiments related to this aim, we have recently examined gastric aspiration with/without LC. Without knowing the initiating event a diagnosis can be made. This has important clinical implications since that aspiration is often unwitnessed, and a diagnosis of exclusion.

Influenza Therapy by Au-nanorod 5'PPP-NS1-siRNA/cDNA Targeting of Bronchial Cells (AI084410): This work proposes to develop two novel prophylactic and therapeutic non-viral gene transfer strategies that target pulmonary cells in vivo employing nanotechnology. The lung is especially well suited for these treatment strategies as direct contact with the environment provides a portal for inhalation administration of cDNA and siRNA conjugated nanoplexes. The emergence of drug-resistant strains of human influenza A and B viruses (including swine flu), as well as avian H5N1 influenza viruses with pandemic potential to one or both classes of approved antiviral agents underscores the importance of developing novel antiviral strategies. The primary objective is to construct an electrostatic complex between a cationic nanoparticle (i.e., Gold Nanorods, GNR) and anionic genetic material (i.e., cDNA or siRNA). These nanoplexes will be engineered such that they can be taken-up and express bioactivity in large airway (i.e., bronchial) epithelial cells with little or no untoward cellular or pulmonary responses. The siRNA/cDNA constructs, which have just recently been synthesized, have dual actions of suppressing the translation of the influenza virulence factor, NS1, as well as independently stimulating type I interferon production through activation of the RIG-I pathway. Stimulation of this antiviral innate immune pathway occurs as a result of a triphosphate (PPP) moiety attached to the 5’ end of the siRNA. We will preferentially administer the GNR-5’PPP-NS1siRNA or its counterpart cDNA nanoplexes to the tracheal and bronchial epithelium in vivo, thereby increasing the safety of the treatment. Extension of these nanotechnological approaches can also be applied to treat other infectious, as well as non-infectious acute lung injuries. Following these studies we propose to examine the therapeutic efficacy of using 5’PPP NS1siRNA and cDNA-nanoplex targeting of large airway epithelial cells in vivo before and during influenza. In addition to assessing the clearance of influenza virus from the respiratory tract, we will examine the ability of 5’PPP-NS1siRNA or cDNA-nanoplexes to stimulate innate antiviral immunity, resulting in alteration of the inflammatory cytokine milieu, adaptive immune response, and antibacterial host defense, as well as prevent or reduce the degree of viral induced respiratory injury and impairment of bacterial clearance. We have predicted that these large airway epithelial-targeted nanoplexes will lead to prophylactic and therapeutic options that can prevent or significantly reduce the morbidity and severity of symptoms of influenza including the “swine flu” and the highly pathogenic H5N1 “bird flu”, and the risk of secondary bacterial pneumonia, which is the major cause of death secondary to influenza. It is our goal to have a nanoparticle mediated novel antiviral prophylactic and therapeutic strategy available for Investigational New Drug filing with the FDA to go for Phase 1 clinical trials as a result of these experiments.

Brain-derived Tumor Necrosis Factor (TNF) and Adrenergic Responses in Neuropathic Pain: This project is being carried out in a collaborative effort with Dr. Tracey Ignatowski’s (Department of Pathology) laboratory. The goal of this project is to examine the role of cytokine-induced adrenergic neuroplastic changes in the central nervous sytem (CNS) to the pathogenesis of hyperalgesia. Early experiments demonstrate that in the Bennett model of neuropathic pain, expression of TNFa increases in nuclei of the CNS associated with adrenergic nerve cell bodies. These changes are associated with alterations in the a2-adrenergic auto-feedback response. Local administration of anti-TNFa antibody inhibits both the development of hyperalgesia and adrenergic plasticity. Recently we have been successful in transfecting neurons in the hippocampus in vivo with the GNR RFP-TNFa fusion protein nanoplex to further explore the role of this cytokine as a mechanism involved in neuropathic pain. Development of effective treatment regimes for this often-time crippling condition of persistent pain is the ultimate goal of this project.


Recent Publications:

Knight PR, Tait AR:  Operating Room Theatre Transmission of Infection.  In Wylie and Churchill-Davidson’s A Practice of Anesthesia 7th Edition.  Edited by TEJ Healy and PR Knight.  Hodder & Stoughton Ltd. Sevenoaks, UL  (In Press).

Shanley T., Davidson, BA, Nader, ND, Bless N., Vasti N., Ward PA., Johnson, KJ., Knight PR., et al:  Role of Macrophage Inflammatory Protein-2 in Aspiration-induced Lung Injury.  Crit Care Med 28:2437-2444, 2000.

Nader, ND, Ignatowski. TA, Kurek, CJ, Knight, PR, Spengler, RN.  Clonidine Suppresses Plasma and Cerebrospinal Fluid Concentrations of TNFa during the Perioperative Period.  Anesth Analg. 93:363-369, 2001.

Knight, PR, Bacon DR: An Unexplained Death:  Hannah Greener and Chloroform.  In Press Anesthesiology, 2002.

Patel, AB, Sokolowski, JJ, Davidson, BA, Knight, PR, Holm, BA:  Halothane Potentiation of Hydrogen Peroxide-induced Inhibition of Surfactant Synthesis:  The Role of Type II cell Energy Status.  Anesth Analg 94:943-947, 2002.

Covey WC, Ignatowski TA, Knight PR, Nader ND, Spenger RN:  Expression of neuron-associated TNFa in the brain is increased during persistent pain.  Regional Anes. 27:357-366, 2002.

Russo TA, Bartholomew JA, Davidson BA, Helinska JD, Carlino CB, Knight PR, Beers MF, Atochina EN, Notter, RH and Holm: Total Extracellular Surfactant is Increased but Abnormal in a rat Model of gram-negative Bacterial Pneumonia.  In press. Am. J. Physiology-lung.

Fernandez SF, Ming M-H, Davidson BA, Knight PR, Izzo JL: Cytosolic Calcium-dependency of Sympathetic Neuronal Responses to Angiotensin II:  The Calcium Switch.  Accepted pending revision.

Russo TA, Davidson BA, Prior R, Carlino CB, Helinska JD, Knight PR:  The roles of capsule and O-specific antigen from an extaintestinal  isolate in the pathogenesis of E. coli in gram-negative pneumonitis.  Submitted.  Am. Journal of Physiology.

Russo TA. Wang Z. Davidson BA. Genagon SA. Beanan JM. Olson R. Holm BA. Knight PR 3rd. Chess PR. Notter RH. Surfactant dysfunction and lung injury due to the E. coli virulence fctor hemolysin in a rat pneumonia model. American Journal of Physiology - Lung Cellular & Molecular Physiology. 292(3):L632-43, 2007 Mar.

Segal TA. Wang Z. Davidson BA.Hutson AD. Russo TA. Holm BA. Mullan B. Habitzruther M. Holland SM. Knight PR 3rd. Acid aspiration-induced lung inflammation and injury are exacerbated in NADPH oxidase-deficient mice. American Journal of Physiology - Lung Cellular & Molecular Physiology. 292(3):L760-8, 2007 Mar.

Spengler RN. Sud R. Knight PR. Ignatowski TA. Antinociception mediated by alpha (2)-adrenergic activiation involves increasing tumor necrosis factor alpha (TNFalpha) expression and restoring TNFalpha and alpha(2)-adrenergic inhibition of norepinephrine release.Neuropharmacology. 52(2):576-89, 2007 Feb.

Not Peer Reviewed:

Knight PR, Curtis L. Mendelson, M.D.: His Role in the Development of the Specialty of Anesthesiology, ASA Newsletter 63:14-16, (Sept) 1999.

Knight PR:  Developing Investigators in Anesthesiology.  ASA Newsletter 64:36, (March) 2000.

Knight PR and Holm BA:  The three components of hyperoxia (Editorial).  Anesthesiology, 93:3-5, 2000.

Chapters in Books:

Tait AR, Knight PR: Upper Respiratory Infection.  In Decision Making in Anesthesiology, 3rd Edition. Edited by LL Bready, RM Mullins, SH Noorily, RB Smith.  Mosby, Inc. St. Louis, Missouri pgs 96-97, 2000.

Bui D, Knight PR:  IgA Deficiency; Knight PR:  Immune Suppression; Knight PR, Russo TA: Rickettsial Disease/Q Fever; Knight PR, Russo TA: Rocky Mountain Spotted Fever.  In Essence of Anesthesia Practice  2nd Edition, Edited by M.F. Roizen and L.A. Fleisher, W.B. Saunders Company, Philadelphia, Pennslyvania. Pgs 192, 193, 280, 289.  2002.

Knight PR, Tait AR:  Operating Room Theatre Transmission of Infection.  In Sylie and Churchill-Davidson's A Practice of Anesthesia 7th Edition.  Edited by TEJ Healy and PR Knight.  Hodder & Stoughton LTD., Sevenoaks, UK. (In Press).

Abstracts, Preliminary Communications, Panel Discussions:

Nader ND, Li CM, Khadra WZ, Panos AL, Knight PR:  Sevoflurane-vaporized cardioplegia improves myocardial wall motion after coronary revascularization.  Anesthesiology 93:A101, 2002.

Nader ND, Li CM, Khadra WZ, Panos AL, Knight PR:  Sevoflurane-vaporized cardioplegia decreases neutrophil activation following cardiopulmonary bypass.  Anesthesiology 93A169, 2000.

Plata ET, Helinski JD, Davidson BA, Soloway P, Knight PR:  The protective role of TIMP-1 in an acid aspiration ouse model.  Anesthesiology 93:A1333, 2000.