Purpose: In the first-in-human trial of ultrasonic propulsion, subjects passed collections of res... more Purpose: In the first-in-human trial of ultrasonic propulsion, subjects passed collections of residual stone fragments repositioned with a C5-2 probe. Here, effectiveness and safety in moving multiple fragments are compared between the C5-2 and a custom (SC-50) probe that produces a longer focal beam and burst duration. Materials and Methods: Effectiveness was quantified by the number of stones expelled from a calyx phantom consisting of a 30-mm deep, water-filled well in a block of tissue mimicking material. Each probe was positioned below the phantom to move stones against gravity. Single propulsion bursts of 50 ms or 3 s duration were applied to three separate targets: 10 fragments of 2 different sizes (1-2 and 2-3 mm) and a single 4 • 7 mm human stone. Safety studies consisted of porcine kidneys exposed to an extreme dose of 10-minute burst duration, including a 7-day survival study and acute studies with surgically implanted stones. Results: Although successful in the clinical trial, the shorter focal beam and maximum 50 ms burst duration of the C5-2 probe moved stones, but did not expel any stones from the phantom's 30-mm deep calyx. The results were similar with the SC-50 probe under the same 50 ms burst duration. Longer (3 s) bursts available with the SC-50 probe expelled all stones at both 4.5 and 9.5 cm ''skin-to-stone'' depths with lower probe heating compared to the C5-2. No abnormal behavior, urine chemistry, serum chemistry, or histological findings were observed within the kidney or surrounding tissues for the 10 min burst duration used in the animal studies. Conclusions: A longer focal beam and burst duration improved expulsion of a stone and multiple stone fragments from a phantom over a broad range of clinically relevant penetration depths and did not cause kidney injury in animal studies.
Small cell lung cancer (SCLC) is a recalcitrant, aggressive neuroendocrine-type cancer for which ... more Small cell lung cancer (SCLC) is a recalcitrant, aggressive neuroendocrine-type cancer for which little change to first-line, standard of care treatment has occurred within the last few decades. Unlike nonsmall cell lung cancer (NSCLC), SCLC harbors few actionable mutations for therapeutic intervention. Lysine-specific demethylase 1A (LSD1, also known as KDM1A) inhibitors were previously shown to have selective activity in SCLC models, but the underlying mechanism was elusive. Here, we found that exposure to the selective LSD1 inhibitor ORY-1001 activated the NOTCH pathway, resulting in the suppression of the transcription factor ASCL1 and *
Non-small cell lung carcinoma (NSCLC) is among the deadliest of human cancers. The CDKN2A locus, ... more Non-small cell lung carcinoma (NSCLC) is among the deadliest of human cancers. The CDKN2A locus, which houses the INK4a and ARF tumor suppressor genes, is frequently altered in NSCLC. However, the specific role of ARF in pulmonary tumorigenesis remains unclear. KRAS and other oncogenes induce the expression of ARF, thus stabilizing p53 activity and arresting cell proliferation. To address the role of ARF in Kras-driven NSCLC, we compared the susceptibility of NIH/Ola strain wild-type and Arf knockout mice to urethane-induced lung carcinogenesis. Lung tumor size, malignancy, and associated morbidity were significantly increased in Arf -/-compared to Arf +/+ animals at 25 weeks post-induction. Pulmonary tumors from Arf knockout mice exhibited increased cell proliferation and DNA damage compared to wild-type. A subgroup of tumors in Arf -/-animals presented as dedifferentiated and metastatic, with many characteristics of pulmonary sarcomatoid carcinoma, a neoplasm previously undocumented in mouse models. Our finding of a role for ARF in NSCLC is consistent with the observation that benign adenomas from Arf +/+ mice robustly expressed ARF, while ARF expression was markedly reduced in malignant adenocarcinomas. ARF expression also frequently co-localized with expression of p21 CIP1 , a transcriptional target of p53, arguing that ARF induces the p53 checkpoint to arrest cell proliferation in vivo. Together, these findings demonstrate that induction of ARF is an early response in lung tumorigenesis that mounts a strong barrier against tumor growth and malignant progression.
Adoptive T cell therapy (ACT) for the treatment of established cancers is actively being pursued ... more Adoptive T cell therapy (ACT) for the treatment of established cancers is actively being pursued in clinical trials. However, poor in vivo persistence and maintenance of anti-tumor activity of transferred T cells remain major problems. Transforming growth factor beta (TGFβ) is a potent immunosuppressive cytokine that is often expressed at high levels within the tumor microenvironment, potentially limiting T cell mediated anti-tumor activity. Here, we used a model of autochthonous murine prostate cancer to evaluate the effect of cell intrinsic abrogation of TGFβ signaling in self/tumor specific CD8 T cells used in ACT to target the tumor in situ. We found that persistence and anti-tumor activity of adoptively transferred effector T cells deficient in TGFβ signaling was significantly improved in the cancerous prostate. However, over time, despite persistence in peripheral lymphoid organs, the numbers of transferred cells in the prostate decreased and the residual prostate infiltrating T cells were no longer functional. These findings reveal that TGFβ negatively regulates the accumulation and effector function of transferred self/ tumor specific CD8 T cells and highlight that, when targeting a tumor antigen that is also expressed as a self-protein, additional substantive obstacles are operative within the tumor microenvironment, potentially hampering the success of ACT for solid tumors.
International Journal of Experimental Pathology, Nov 8, 2012
Melioidosis is a tropical disease caused by ingestion, percutaneous inoculation or inhalation of ... more Melioidosis is a tropical disease caused by ingestion, percutaneous inoculation or inhalation of the Gram-negative soil saprophyte Burkholderia pseudomallei. We developed a reproducible experimental murine model of pneumonic melioidosis induced by inhalation of aerosolized B. pseudomallei 1026b. In a series of experiments performed to bracket the lethal dose, we found that C57BL/6 mice were modestly more resistant than BALB/c mice (median lethal dose 334 CFU/lung vs 204 CFU/lung). We further characterized infection and pulmonary inflammation in C57BL/6 mice infected with a sublethal dose. We observed pulmonary replication and dissemination of bacteria to distant organs in the first days after infection, followed by bacterial containment by day 4 and no evidence of recrudescent infection for up to 2 months. We measured a robust host inflammatory response notable for a neutrophilic bronchoalveolar lavage fluid profile, elevated cytokines and chemokines in the lung and serum and scattered foci of neutrophilic infiltrates in the alveoli and in a perivascular distribution on histological analysis. We previously noted a similar pattern of inflammation in mice infected with aerosolized B. thailandensis. This report builds on the limited literature describing experimental murine pneumonic melioidosis induced by aerosol and characterizes pulmonary infection and resultant inflammation in C57BL/6 mice infected with aerosolized B. pseudomallei. This model has utility for the study of bacterial and host factors that contribute to the virulence of melioidosis.
Multiple sclerosis (MS) is an inflammatory disease of the CNS that causes the demyelination of ne... more Multiple sclerosis (MS) is an inflammatory disease of the CNS that causes the demyelination of nerve cells and destroys oligodendrocytes, neurons, and axons. Historically, MS has been thought to be a CD4 T cell-mediated autoimmune disease of CNS white matter. However, recent studies identified CD8 T cell infiltrates and gray matter lesions in MS patients. These findings suggest that CD8 T cells and CNS Ags other than myelin proteins may be involved during the MS disease process. In this article, we show that CD8 T cells reactive to glial fibrillary acidic protein (GFAP), a protein expressed in astrocytes, can avoid tolerance mechanisms and, depending upon the T cell-triggering event, drive unique aspects of inflammatory CNS autoimmunity. In GFAP-specific CD8 TCR-transgenic (BG1) mice, tissue resident memory-like CD8 T cells spontaneously infiltrate the gray matter and white matter of the CNS, resulting in a relapsing-remitting CNS autoimmunity. The frequency, severity, and remissions from spontaneous disease are controlled by the presence of polyclonal B cells. In contrast, a viral trigger induces GFAP-specific CD8 T effector cells to exclusively target the meninges and vascular/perivascular space of the gray and white matter of the brain, causing a rapid, acute CNS disease. These findings demonstrate that the type of CD8 T cell-triggering event can determine the presentation of distinct CNS autoimmune disease pathologies.
International Journal of Pharmaceutics, Aug 1, 1988
The absorption of recombinant methionyl human growth hormone (Met-hGH) from the nasal mucosa into... more The absorption of recombinant methionyl human growth hormone (Met-hGH) from the nasal mucosa into the systemic circulation was studied in anesthetized rats. Met-hGH was administered intranasally (i.n.), intramuscularly (i.m.) and intravenously (i.v.) to determine the relative and absolute bioavailability of an intranasal Met-hGH formulation. In the absence of detergent enhancers, the absolute bioavailability of met hGH was < 1%. Hypotonicity enhanced absorption slightly. The absolute bioavailability of Met-hGH increased markedly in the presence of the non-ionic surfactant, polyoxyethylene 9-1auryl ether (lauretb-9). The histological changes in the nasal mucosa of rats treated with 1% laureth-9 were severe, however, and included complete removal of the nasal epithelium in places. The bile salt sodium glycocholate was also evaluated for its permeation enhancing abilities. At 0.5% glycocholate there were few noticeable histological changes relative to controls and the absolute bioavailability of Met-hGH was approximately 7-8%. While absorption of Met-hGH from the nasal epithelium is demonstrated, the effects of permeation enhancing detergents on the delicate nasal mucosa must be better understood before the intranasal route of administration may be considered suitable for delivering Met-hGH to the systemic circulation.
The combination of genetic and environmental factors that contribute to human autoimmune response... more The combination of genetic and environmental factors that contribute to human autoimmune responses has made potential triggers of these diseases difficult to identify. We examined how experimental allergic encephalomyelitis (EAE), an animal model of multiple sclerosis, is triggered using TCR-transgenic mice specific for myelin basic protein (MBP). In these TCR-transgenic mice, EAE can be actively induced and also occurs spontaneously. The incidence of spontaneous EAE in this model is largely confined to adolescence and early adulthood and is more prevalent among males than females, indicating that hormonal influences may contribute to triggering central nervous system autoimmune disease. Disease induction studies show that not all stimuli that activate MBP-specific T cells in vivo also induce EAE. Immunization with MBP peptide stimulates the transgenic T cells to produce Thl cytokines; however, the activated T cells do not accumulate in the central nervous system and induce EAE unless pertussis toxin is also administered. EAE can be induced by intrathecal injection of either stimulated or nonstimulated transgenic T cells into nontransgenic or transgenic recipients. Therefore, gaining access to the central nervous system appears to be the critical step in this model for the induction of EAE, regardless of the activation state of the T cells. The lournal of Immunology, 1997, 159: 497-507. T cell tolerance is induced by clonal deletion or induction of anergy following exposure to self Ags in the thymus and periphery (1-4). Peripheral tolerance is also dependent on maintaining a state of ignorance in T cells, in which Ags are ignored unless they are presented by cells with specific costimulatory activation signals ( 5 , 6). A breakdown in tolerance and the mechanisms that control activated T cell responses can result in autoimmune disease. One theory suggests that autoimmune disease is initiated when ignorant, self-reactive T cells are activated by pathogens whose structures mimic self Ags (7-9). Activated, selfreactive T cells may be difficult to regulate because of the chronic presence of self Ag. In addition, inflammatory conditions may exacerbate autoimmune responses in a target tissue through nonspecific recruitment of T cells that may fortuitously have specificities for other self Ags found at this site 11). Autoimmune responses to central nervous system (CNS)3 Ags have been studied using EAE, an animal model of the human disease multiple sclerosis. Like MS, EAE is an inflammatory, demyelinating disease manifested by acute, chronic, or relapsing paralysis (12). EAE is commonly induced either by
Figure S5 from Chimeric Antigen Receptor T Cell–Mediated Neurotoxicity in Nonhuman Primates
CSF and serum levels of 21 cytokines and chemokines measured before and at indicated days after a... more CSF and serum levels of 21 cytokines and chemokines measured before and at indicated days after adoptive CD20 CAR T cell transfer.
Figure S4 from Chimeric Antigen Receptor T Cell–Mediated Neurotoxicity in Nonhuman Primates
A, Immunofluorescence images for CD20 (green), CD3 (red) and DAPI (blue) in R.304 before (pre, da... more A, Immunofluorescence images for CD20 (green), CD3 (red) and DAPI (blue) in R.304 before (pre, day -7) and following CD20 CAR T cell transfer (day 7 (d7)), in lymph node (LN) and brain (day 8 (d8)) and in normal RM brain. B, H&amp;E CSF cytospin (day 8, R.304) shows marked lymphocytosis following CD20 CAR T cell transfer. C, Fraction (%) of EGFRt+ or EGFRt- CD4 an CD8 T cells expressing �4 and �1 integrins, in the blood of normal RM controls (n = 5), on day 0, pre-CD20 CAR infusion (d0, n = 4) and on day 7 or 8 (peak CD20 CAR expansion, (d7/8, n = 4)), in the CSF (on day 8 (R.304); d8, n = 1) and in the brain (on day 8 (R.304); d8, n = 1 ). * P &lt; 0.05.
Exposure to fine particulate air pollution (PM 2.5 ) is strongly associated with cardiovascular m... more Exposure to fine particulate air pollution (PM 2.5 ) is strongly associated with cardiovascular morbidity and mortality. Exposure to PM 2.5 during pregnancy promotes reduced birthweight, and the associated adverse intrauterine conditions may also promote adult risk of cardiovascular disease. Here, we investigated the potential for in utero exposure to diesel exhaust (DE) air pollution, a major source of urban PM 2.5 , to promote adverse intrauterine conditions and influence adult susceptibility to disease. We exposed pregnant female C57Bl/6J mice to DE (<300 mg/m 3 PM 2.5 , 6 hrs/day, 5 days/week) from embryonic day (E) 0.5 to 17.5. At E17.5 embryos were collected for gravimetric analysis and assessed for evidence of resorption. Placental tissues underwent pathological examination to assess the extent of injury, inflammatory cell infiltration, and oxidative stress. In addition, some dams that were exposed to DE were allowed to give birth to pups and raise offspring in filtered air (FA) conditions. At 10-weeks of age, body weight and blood pressure were measured. At 12-weeks of age, cardiac function was assessed by echocardiography. Susceptibility to pressure overload-induced heart failure was then determined after transverse aortic constriction surgery. We found that in utero exposure to DE increases embryo resorption, and promotes placental hemorrhage, focal necrosis, compaction of labyrinth vascular spaces, inflammatory cell infiltration and oxidative stress. In addition, we observed that in utero DE exposure increased body weight, but counterintuitively reduced blood pressure without any changes in baseline cardiac function in adult male mice. Importantly, we observed these mice to have increased susceptibility to pressure-overload induced heart failure, suggesting this in utero exposure to DE 'reprograms' the heart to a heightened susceptibility to failure. These observations provide important data to suggest that developmental exposure to air pollution may strongly influence adult susceptibility to cardiovascular disease.
American Journal of Physiology-lung Cellular and Molecular Physiology, Jul 1, 2004
To determine the role of respiratory epithelial cells in the inflammatory response to inhaled end... more To determine the role of respiratory epithelial cells in the inflammatory response to inhaled endotoxin, we selectively inhibited NF-B activation in the respiratory epithelium using a mutant IB-␣ construct that functioned as a dominant negative inhibitor of NF-B translocation (dnIB-␣). We developed two lines of transgenic mice in which expression of dnIB-␣ was targeted to the distal airway epithelium using the human surfactant apoprotein C promoter. Transgene expression was localized to the epithelium of the terminal bronchioles and alveoli. After inhalation of LPS, nuclear translocation of NF-B was evident in bronchiolar epithelium of nontransgenic but not of transgenic mice. This defect was associated with impaired neutrophilic lung inflammation 4 h after LPS challenge and diminished levels of TNF-␣, IL-1, macrophage inflammatory protein-2, and KC in lung homogenates. Expression of TNF-␣ within bronchiolar epithelial cells and of VCAM-1 within peribronchiolar endothelial cells was reduced in transgenic animals. Thus targeted inhibition of NF-B activation in distal airway epithelial cells impaired the inflammatory response to inhaled LPS. These data provide causal evidence that distal airway epithelial cells and the signals they transduce play a physiological role in lung inflammation in vivo.
Staphylococcus aureus is an important cause of acute bacterial pneumonia. Toll-like receptor 2 (T... more Staphylococcus aureus is an important cause of acute bacterial pneumonia. Toll-like receptor 2 (TLR2) recognizes multiple components of the bacterial cell wall and activates innate immune responses to gram-positive bacteria. We hypothesized that TLR2 would have an important role in pulmonary host defense against S. aureus. TLR null (TLR2 À/À ) mice and wild type (WT) C57BL/6 controls were challenged with aerosolized S. aureus at a range of inocula for kinetic studies of cytokine and antimicrobial peptide expression, lung inflammation, bacterial killing by alveolar macrophages, and bacterial clearance. Survival was measured after intranasal infection. Pulmonary induction of most pro-inflammatory cytokines was significantly blunted in TLR2 À/À mice 4 and 24 h after infection in comparison with WT controls. Bronchoalveolar concentrations of cathelicidin-related antimicrobial peptide also were reduced in TLR2 À/À mice. Lung inflammation, measured by enumeration of bronchoalveolar neutrophils and scoring of histological sections, was significantly blunted in TLR2 À/À mice. Phagocytosis of S. aureus by alveolar macrophages in vivo after low-dose infection was unimpaired, but viability of ingested bacteria was significantly greater in TLR2 À/À mice. Bacterial clearance from the lungs was slightly impaired in TLR2 À/À mice after low-dose infection only; bacterial elimination from the lungs was slightly accelerated in the TLR2 À/À mice after high-dose infection. Survival after high-dose intranasal challenge was 50-60% in both groups. TLR2 has a significant role in early innate immune responses to S. aureus in the lungs but is not required for bacterial clearance and survival from S. aureus pneumonia.
Figure S1 from Chimeric Antigen Receptor T Cell–Mediated Neurotoxicity in Nonhuman Primates
A, Schema of the CD20 CAR construct. B, Cytolytic activity of human CD19 CAR T cells (solid lines... more A, Schema of the CD20 CAR construct. B, Cytolytic activity of human CD19 CAR T cells (solid lines) and mock (dashed lines) T cells against 51Cr-labeled human K562 and CD19-K562 and RM B-LCL1 and B-LCL2.
Figure S3 from Chimeric Antigen Receptor T Cell–Mediated Neurotoxicity in Nonhuman Primates
Serum levels of 24 cytokines and chemokines measured before and at indicated days after adoptive ... more Serum levels of 24 cytokines and chemokines measured before and at indicated days after adoptive GFP or CD20 CAR T cell transfer in the blood.
Purpose: In the first-in-human trial of ultrasonic propulsion, subjects passed collections of res... more Purpose: In the first-in-human trial of ultrasonic propulsion, subjects passed collections of residual stone fragments repositioned with a C5-2 probe. Here, effectiveness and safety in moving multiple fragments are compared between the C5-2 and a custom (SC-50) probe that produces a longer focal beam and burst duration. Materials and Methods: Effectiveness was quantified by the number of stones expelled from a calyx phantom consisting of a 30-mm deep, water-filled well in a block of tissue mimicking material. Each probe was positioned below the phantom to move stones against gravity. Single propulsion bursts of 50 ms or 3 s duration were applied to three separate targets: 10 fragments of 2 different sizes (1-2 and 2-3 mm) and a single 4 • 7 mm human stone. Safety studies consisted of porcine kidneys exposed to an extreme dose of 10-minute burst duration, including a 7-day survival study and acute studies with surgically implanted stones. Results: Although successful in the clinical trial, the shorter focal beam and maximum 50 ms burst duration of the C5-2 probe moved stones, but did not expel any stones from the phantom's 30-mm deep calyx. The results were similar with the SC-50 probe under the same 50 ms burst duration. Longer (3 s) bursts available with the SC-50 probe expelled all stones at both 4.5 and 9.5 cm ''skin-to-stone'' depths with lower probe heating compared to the C5-2. No abnormal behavior, urine chemistry, serum chemistry, or histological findings were observed within the kidney or surrounding tissues for the 10 min burst duration used in the animal studies. Conclusions: A longer focal beam and burst duration improved expulsion of a stone and multiple stone fragments from a phantom over a broad range of clinically relevant penetration depths and did not cause kidney injury in animal studies.
Small cell lung cancer (SCLC) is a recalcitrant, aggressive neuroendocrine-type cancer for which ... more Small cell lung cancer (SCLC) is a recalcitrant, aggressive neuroendocrine-type cancer for which little change to first-line, standard of care treatment has occurred within the last few decades. Unlike nonsmall cell lung cancer (NSCLC), SCLC harbors few actionable mutations for therapeutic intervention. Lysine-specific demethylase 1A (LSD1, also known as KDM1A) inhibitors were previously shown to have selective activity in SCLC models, but the underlying mechanism was elusive. Here, we found that exposure to the selective LSD1 inhibitor ORY-1001 activated the NOTCH pathway, resulting in the suppression of the transcription factor ASCL1 and *
Non-small cell lung carcinoma (NSCLC) is among the deadliest of human cancers. The CDKN2A locus, ... more Non-small cell lung carcinoma (NSCLC) is among the deadliest of human cancers. The CDKN2A locus, which houses the INK4a and ARF tumor suppressor genes, is frequently altered in NSCLC. However, the specific role of ARF in pulmonary tumorigenesis remains unclear. KRAS and other oncogenes induce the expression of ARF, thus stabilizing p53 activity and arresting cell proliferation. To address the role of ARF in Kras-driven NSCLC, we compared the susceptibility of NIH/Ola strain wild-type and Arf knockout mice to urethane-induced lung carcinogenesis. Lung tumor size, malignancy, and associated morbidity were significantly increased in Arf -/-compared to Arf +/+ animals at 25 weeks post-induction. Pulmonary tumors from Arf knockout mice exhibited increased cell proliferation and DNA damage compared to wild-type. A subgroup of tumors in Arf -/-animals presented as dedifferentiated and metastatic, with many characteristics of pulmonary sarcomatoid carcinoma, a neoplasm previously undocumented in mouse models. Our finding of a role for ARF in NSCLC is consistent with the observation that benign adenomas from Arf +/+ mice robustly expressed ARF, while ARF expression was markedly reduced in malignant adenocarcinomas. ARF expression also frequently co-localized with expression of p21 CIP1 , a transcriptional target of p53, arguing that ARF induces the p53 checkpoint to arrest cell proliferation in vivo. Together, these findings demonstrate that induction of ARF is an early response in lung tumorigenesis that mounts a strong barrier against tumor growth and malignant progression.
Adoptive T cell therapy (ACT) for the treatment of established cancers is actively being pursued ... more Adoptive T cell therapy (ACT) for the treatment of established cancers is actively being pursued in clinical trials. However, poor in vivo persistence and maintenance of anti-tumor activity of transferred T cells remain major problems. Transforming growth factor beta (TGFβ) is a potent immunosuppressive cytokine that is often expressed at high levels within the tumor microenvironment, potentially limiting T cell mediated anti-tumor activity. Here, we used a model of autochthonous murine prostate cancer to evaluate the effect of cell intrinsic abrogation of TGFβ signaling in self/tumor specific CD8 T cells used in ACT to target the tumor in situ. We found that persistence and anti-tumor activity of adoptively transferred effector T cells deficient in TGFβ signaling was significantly improved in the cancerous prostate. However, over time, despite persistence in peripheral lymphoid organs, the numbers of transferred cells in the prostate decreased and the residual prostate infiltrating T cells were no longer functional. These findings reveal that TGFβ negatively regulates the accumulation and effector function of transferred self/ tumor specific CD8 T cells and highlight that, when targeting a tumor antigen that is also expressed as a self-protein, additional substantive obstacles are operative within the tumor microenvironment, potentially hampering the success of ACT for solid tumors.
International Journal of Experimental Pathology, Nov 8, 2012
Melioidosis is a tropical disease caused by ingestion, percutaneous inoculation or inhalation of ... more Melioidosis is a tropical disease caused by ingestion, percutaneous inoculation or inhalation of the Gram-negative soil saprophyte Burkholderia pseudomallei. We developed a reproducible experimental murine model of pneumonic melioidosis induced by inhalation of aerosolized B. pseudomallei 1026b. In a series of experiments performed to bracket the lethal dose, we found that C57BL/6 mice were modestly more resistant than BALB/c mice (median lethal dose 334 CFU/lung vs 204 CFU/lung). We further characterized infection and pulmonary inflammation in C57BL/6 mice infected with a sublethal dose. We observed pulmonary replication and dissemination of bacteria to distant organs in the first days after infection, followed by bacterial containment by day 4 and no evidence of recrudescent infection for up to 2 months. We measured a robust host inflammatory response notable for a neutrophilic bronchoalveolar lavage fluid profile, elevated cytokines and chemokines in the lung and serum and scattered foci of neutrophilic infiltrates in the alveoli and in a perivascular distribution on histological analysis. We previously noted a similar pattern of inflammation in mice infected with aerosolized B. thailandensis. This report builds on the limited literature describing experimental murine pneumonic melioidosis induced by aerosol and characterizes pulmonary infection and resultant inflammation in C57BL/6 mice infected with aerosolized B. pseudomallei. This model has utility for the study of bacterial and host factors that contribute to the virulence of melioidosis.
Multiple sclerosis (MS) is an inflammatory disease of the CNS that causes the demyelination of ne... more Multiple sclerosis (MS) is an inflammatory disease of the CNS that causes the demyelination of nerve cells and destroys oligodendrocytes, neurons, and axons. Historically, MS has been thought to be a CD4 T cell-mediated autoimmune disease of CNS white matter. However, recent studies identified CD8 T cell infiltrates and gray matter lesions in MS patients. These findings suggest that CD8 T cells and CNS Ags other than myelin proteins may be involved during the MS disease process. In this article, we show that CD8 T cells reactive to glial fibrillary acidic protein (GFAP), a protein expressed in astrocytes, can avoid tolerance mechanisms and, depending upon the T cell-triggering event, drive unique aspects of inflammatory CNS autoimmunity. In GFAP-specific CD8 TCR-transgenic (BG1) mice, tissue resident memory-like CD8 T cells spontaneously infiltrate the gray matter and white matter of the CNS, resulting in a relapsing-remitting CNS autoimmunity. The frequency, severity, and remissions from spontaneous disease are controlled by the presence of polyclonal B cells. In contrast, a viral trigger induces GFAP-specific CD8 T effector cells to exclusively target the meninges and vascular/perivascular space of the gray and white matter of the brain, causing a rapid, acute CNS disease. These findings demonstrate that the type of CD8 T cell-triggering event can determine the presentation of distinct CNS autoimmune disease pathologies.
International Journal of Pharmaceutics, Aug 1, 1988
The absorption of recombinant methionyl human growth hormone (Met-hGH) from the nasal mucosa into... more The absorption of recombinant methionyl human growth hormone (Met-hGH) from the nasal mucosa into the systemic circulation was studied in anesthetized rats. Met-hGH was administered intranasally (i.n.), intramuscularly (i.m.) and intravenously (i.v.) to determine the relative and absolute bioavailability of an intranasal Met-hGH formulation. In the absence of detergent enhancers, the absolute bioavailability of met hGH was < 1%. Hypotonicity enhanced absorption slightly. The absolute bioavailability of Met-hGH increased markedly in the presence of the non-ionic surfactant, polyoxyethylene 9-1auryl ether (lauretb-9). The histological changes in the nasal mucosa of rats treated with 1% laureth-9 were severe, however, and included complete removal of the nasal epithelium in places. The bile salt sodium glycocholate was also evaluated for its permeation enhancing abilities. At 0.5% glycocholate there were few noticeable histological changes relative to controls and the absolute bioavailability of Met-hGH was approximately 7-8%. While absorption of Met-hGH from the nasal epithelium is demonstrated, the effects of permeation enhancing detergents on the delicate nasal mucosa must be better understood before the intranasal route of administration may be considered suitable for delivering Met-hGH to the systemic circulation.
The combination of genetic and environmental factors that contribute to human autoimmune response... more The combination of genetic and environmental factors that contribute to human autoimmune responses has made potential triggers of these diseases difficult to identify. We examined how experimental allergic encephalomyelitis (EAE), an animal model of multiple sclerosis, is triggered using TCR-transgenic mice specific for myelin basic protein (MBP). In these TCR-transgenic mice, EAE can be actively induced and also occurs spontaneously. The incidence of spontaneous EAE in this model is largely confined to adolescence and early adulthood and is more prevalent among males than females, indicating that hormonal influences may contribute to triggering central nervous system autoimmune disease. Disease induction studies show that not all stimuli that activate MBP-specific T cells in vivo also induce EAE. Immunization with MBP peptide stimulates the transgenic T cells to produce Thl cytokines; however, the activated T cells do not accumulate in the central nervous system and induce EAE unless pertussis toxin is also administered. EAE can be induced by intrathecal injection of either stimulated or nonstimulated transgenic T cells into nontransgenic or transgenic recipients. Therefore, gaining access to the central nervous system appears to be the critical step in this model for the induction of EAE, regardless of the activation state of the T cells. The lournal of Immunology, 1997, 159: 497-507. T cell tolerance is induced by clonal deletion or induction of anergy following exposure to self Ags in the thymus and periphery (1-4). Peripheral tolerance is also dependent on maintaining a state of ignorance in T cells, in which Ags are ignored unless they are presented by cells with specific costimulatory activation signals ( 5 , 6). A breakdown in tolerance and the mechanisms that control activated T cell responses can result in autoimmune disease. One theory suggests that autoimmune disease is initiated when ignorant, self-reactive T cells are activated by pathogens whose structures mimic self Ags (7-9). Activated, selfreactive T cells may be difficult to regulate because of the chronic presence of self Ag. In addition, inflammatory conditions may exacerbate autoimmune responses in a target tissue through nonspecific recruitment of T cells that may fortuitously have specificities for other self Ags found at this site 11). Autoimmune responses to central nervous system (CNS)3 Ags have been studied using EAE, an animal model of the human disease multiple sclerosis. Like MS, EAE is an inflammatory, demyelinating disease manifested by acute, chronic, or relapsing paralysis (12). EAE is commonly induced either by
Figure S5 from Chimeric Antigen Receptor T Cell–Mediated Neurotoxicity in Nonhuman Primates
CSF and serum levels of 21 cytokines and chemokines measured before and at indicated days after a... more CSF and serum levels of 21 cytokines and chemokines measured before and at indicated days after adoptive CD20 CAR T cell transfer.
Figure S4 from Chimeric Antigen Receptor T Cell–Mediated Neurotoxicity in Nonhuman Primates
A, Immunofluorescence images for CD20 (green), CD3 (red) and DAPI (blue) in R.304 before (pre, da... more A, Immunofluorescence images for CD20 (green), CD3 (red) and DAPI (blue) in R.304 before (pre, day -7) and following CD20 CAR T cell transfer (day 7 (d7)), in lymph node (LN) and brain (day 8 (d8)) and in normal RM brain. B, H&amp;E CSF cytospin (day 8, R.304) shows marked lymphocytosis following CD20 CAR T cell transfer. C, Fraction (%) of EGFRt+ or EGFRt- CD4 an CD8 T cells expressing �4 and �1 integrins, in the blood of normal RM controls (n = 5), on day 0, pre-CD20 CAR infusion (d0, n = 4) and on day 7 or 8 (peak CD20 CAR expansion, (d7/8, n = 4)), in the CSF (on day 8 (R.304); d8, n = 1) and in the brain (on day 8 (R.304); d8, n = 1 ). * P &lt; 0.05.
Exposure to fine particulate air pollution (PM 2.5 ) is strongly associated with cardiovascular m... more Exposure to fine particulate air pollution (PM 2.5 ) is strongly associated with cardiovascular morbidity and mortality. Exposure to PM 2.5 during pregnancy promotes reduced birthweight, and the associated adverse intrauterine conditions may also promote adult risk of cardiovascular disease. Here, we investigated the potential for in utero exposure to diesel exhaust (DE) air pollution, a major source of urban PM 2.5 , to promote adverse intrauterine conditions and influence adult susceptibility to disease. We exposed pregnant female C57Bl/6J mice to DE (<300 mg/m 3 PM 2.5 , 6 hrs/day, 5 days/week) from embryonic day (E) 0.5 to 17.5. At E17.5 embryos were collected for gravimetric analysis and assessed for evidence of resorption. Placental tissues underwent pathological examination to assess the extent of injury, inflammatory cell infiltration, and oxidative stress. In addition, some dams that were exposed to DE were allowed to give birth to pups and raise offspring in filtered air (FA) conditions. At 10-weeks of age, body weight and blood pressure were measured. At 12-weeks of age, cardiac function was assessed by echocardiography. Susceptibility to pressure overload-induced heart failure was then determined after transverse aortic constriction surgery. We found that in utero exposure to DE increases embryo resorption, and promotes placental hemorrhage, focal necrosis, compaction of labyrinth vascular spaces, inflammatory cell infiltration and oxidative stress. In addition, we observed that in utero DE exposure increased body weight, but counterintuitively reduced blood pressure without any changes in baseline cardiac function in adult male mice. Importantly, we observed these mice to have increased susceptibility to pressure-overload induced heart failure, suggesting this in utero exposure to DE 'reprograms' the heart to a heightened susceptibility to failure. These observations provide important data to suggest that developmental exposure to air pollution may strongly influence adult susceptibility to cardiovascular disease.
American Journal of Physiology-lung Cellular and Molecular Physiology, Jul 1, 2004
To determine the role of respiratory epithelial cells in the inflammatory response to inhaled end... more To determine the role of respiratory epithelial cells in the inflammatory response to inhaled endotoxin, we selectively inhibited NF-B activation in the respiratory epithelium using a mutant IB-␣ construct that functioned as a dominant negative inhibitor of NF-B translocation (dnIB-␣). We developed two lines of transgenic mice in which expression of dnIB-␣ was targeted to the distal airway epithelium using the human surfactant apoprotein C promoter. Transgene expression was localized to the epithelium of the terminal bronchioles and alveoli. After inhalation of LPS, nuclear translocation of NF-B was evident in bronchiolar epithelium of nontransgenic but not of transgenic mice. This defect was associated with impaired neutrophilic lung inflammation 4 h after LPS challenge and diminished levels of TNF-␣, IL-1, macrophage inflammatory protein-2, and KC in lung homogenates. Expression of TNF-␣ within bronchiolar epithelial cells and of VCAM-1 within peribronchiolar endothelial cells was reduced in transgenic animals. Thus targeted inhibition of NF-B activation in distal airway epithelial cells impaired the inflammatory response to inhaled LPS. These data provide causal evidence that distal airway epithelial cells and the signals they transduce play a physiological role in lung inflammation in vivo.
Staphylococcus aureus is an important cause of acute bacterial pneumonia. Toll-like receptor 2 (T... more Staphylococcus aureus is an important cause of acute bacterial pneumonia. Toll-like receptor 2 (TLR2) recognizes multiple components of the bacterial cell wall and activates innate immune responses to gram-positive bacteria. We hypothesized that TLR2 would have an important role in pulmonary host defense against S. aureus. TLR null (TLR2 À/À ) mice and wild type (WT) C57BL/6 controls were challenged with aerosolized S. aureus at a range of inocula for kinetic studies of cytokine and antimicrobial peptide expression, lung inflammation, bacterial killing by alveolar macrophages, and bacterial clearance. Survival was measured after intranasal infection. Pulmonary induction of most pro-inflammatory cytokines was significantly blunted in TLR2 À/À mice 4 and 24 h after infection in comparison with WT controls. Bronchoalveolar concentrations of cathelicidin-related antimicrobial peptide also were reduced in TLR2 À/À mice. Lung inflammation, measured by enumeration of bronchoalveolar neutrophils and scoring of histological sections, was significantly blunted in TLR2 À/À mice. Phagocytosis of S. aureus by alveolar macrophages in vivo after low-dose infection was unimpaired, but viability of ingested bacteria was significantly greater in TLR2 À/À mice. Bacterial clearance from the lungs was slightly impaired in TLR2 À/À mice after low-dose infection only; bacterial elimination from the lungs was slightly accelerated in the TLR2 À/À mice after high-dose infection. Survival after high-dose intranasal challenge was 50-60% in both groups. TLR2 has a significant role in early innate immune responses to S. aureus in the lungs but is not required for bacterial clearance and survival from S. aureus pneumonia.
Figure S1 from Chimeric Antigen Receptor T Cell–Mediated Neurotoxicity in Nonhuman Primates
A, Schema of the CD20 CAR construct. B, Cytolytic activity of human CD19 CAR T cells (solid lines... more A, Schema of the CD20 CAR construct. B, Cytolytic activity of human CD19 CAR T cells (solid lines) and mock (dashed lines) T cells against 51Cr-labeled human K562 and CD19-K562 and RM B-LCL1 and B-LCL2.
Figure S3 from Chimeric Antigen Receptor T Cell–Mediated Neurotoxicity in Nonhuman Primates
Serum levels of 24 cytokines and chemokines measured before and at indicated days after adoptive ... more Serum levels of 24 cytokines and chemokines measured before and at indicated days after adoptive GFP or CD20 CAR T cell transfer in the blood.
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