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Kent J Johnson

Publications and source records attributed to Kent J Johnson.

15 recordsLinked to original sources
PubMed2007

Role of interleukin-6 in a glucan-induced model of granulomatous vasculitis.

The role of interleukin-6 (IL-6) in granulomatous vasculitis is not well understood. To investigate its involvement in this type of vasculitis a model of glucan-induced pulmonary vasculitis employed interleukin-6 deficient (IL-6-/-) mice. Briefly, IL-6-/- mice and C57B/J6 wild type (IL-6+/+) mice were injected intravenously with a suspension of glucan isolated from the cell wall of bakers yeast which results in a granulomatous vasculitis primarily in the pulmonary vasculature. Histological examination demonstrated no significant difference in the number of infiltrating leukocytes between the IL-6+/+ and IL-6-/- glucan-injured mice. Similar numbers of granulomas were noted in both the IL-6+/+ and IL-6-/- injured animals, while no granulomas were seen in saline injected control mice. Cells recovered from the bronchoalveolar lavage (BAL) fluid were differentially stained and counted. While there was a significant increase in infiltrating leukocytes recovered from the BAL following glucan-induced injury, there was no significant difference between the IL-6+/+ and IL-6-/- mice. In addition, no difference was demonstrated in total protein content in the BAL fluid between IL-6+/+ and IL-6-/- mice. However, myeloperoxidase (MPO) activity in the lungs of the IL-6-/- mice was less than in their IL-6+/+ counterparts suggesting that these animals have a partial defect in their ability to recruit neutrophils in this model. Studies done to look for levels of other cytokines/chemokines in these animals to compensate for the loss of IL-6 revealed that only IL-10 in the sera (p<0.016) and BAL fluid (p<0.05) of IL-6-/- mice was significantly higher then their IL-6+/+-injured counterparts. These studies suggest that IL-6, while possibly involved in early neutrophil accumulation in this model does not appear critical to the development of the TH-2 mediated granulomatous vasculitis.

Animals↗
PubMed2006

Matrix metalloproteinase-9 is an important factor in hepatic regeneration after partial hepatectomy in mice.

Partial hepatectomy triggers hepatocyte proliferation, hepatic matrix remodeling, and hepatocyte apoptosis, all of which are important processes in the regenerating liver. Previous studies have shown an increase in the levels of matrix metalloproteinases gelatinase A (MMP-2) and gelatinase B (MMP-9) after partial hepatectomy. The goal of this study was to investigate the role of MMP-9 in liver regeneration after partial hepatectomy. A 70% hepatectomy or sham laparotomy was performed in wild-type or MMP-9-deficient (MMP-9-/-) mice. Hepatic regeneration was determined by liver weight/total body weight ratios and BrdU staining, which was used to a calculate mitotic index at several times postoperatively. Cytokine and growth factor expression was evaluated by Luminex bead-based ELISA and Western blots. Finally, the effect of MMP-9 on apoptosis was measured using TUNEL and caspase expression. The MMP-9-/- animals had a delayed hepatic regenerative response when compared with wild-type controls. The MMP-9-deficient animals expressed significantly less VEGF, HGF, and TNF-alpha between days 2 and 3 post-hepatectomy. Apoptosis, as measured by TUNEL staining and caspase expression, was decreased in the MMP-9-/-. In conclusion, MMP-9 plays an important role in liver regeneration after partial hepatectomy by affecting matrix remodeling, as well as cytokine, growth factor, and caspase expression.

Animals↗
PubMed2006

Matrix metalloproteinases and matrix metalloproteinase inhibitors in acute lung injury.

The objective of this study was to assess matrix metalloproteinase (MMP) and MMP inhibitor expression in the airspace of patients with acute lung injury (ALI) or acute respiratory distress syndrome (ARDS) and to determine the prognostic significance of MMP expression in this patient population. Twenty-eight patients with ALI or ARDS were prospectively enrolled in this study; bronchoalveolar lavage (BAL) fluid obtained from these patients was examined for expression of MMP-1 (interstitial collagenase), MMP-2 (gelatinase A), MMP-3 (stromelysin-1), MMP-8 (neutrophil collagenase), and MMP-9 (gelatinase B). Levels of MMP inhibitors (ie, tissue inhibitor of metalloproteinases-1 and -2 [TIMP-1 and TIMP-2]) were examined in parallel. Expression of MMPs was correlated with relevant clinical outcomes in patients with ALI/ARDS. In nearly all specimens obtained from patients with ALI/ARDS, there were high levels of MMP-2, MMP-8, MMP-9, and TIMP-1, but in only a small subset of patients (6/28) were there detectable levels of MMP-1 and/or MMP-3. In the patients with elevated MMP-1 and/or MMP-3, the mortality rate was higher (83%) than in the group without detectable levels of these enzymes (32%). Likewise, the overall severity of disease as indicated by Acute Physiology and Chronic Health Evaluation III scores was higher in this group (98 +/- 30) than in the group without detectable MMP-1 or MMP-3 (78 +/- 28). The percentage of individuals in whom lung disease was complicated by multiorgan failure was also higher in the group with detectable MMP-1 and/or MMP-3 (83%) than in the group without (64%), as was the number of organs that failed. In contrast, there was no correlation between MMP-1 and/or MMP-3 expression and impairment in gas exchange, as determined by the ratio of partial pressure of oxygen to fraction of inspired oxygen (Pao(2)/Fio(2)) on the day of BAL sample. Based on these findings, we conclude that elevated MMP-2, MMP-8, and MMP-9 in BAL fluid is a marker of acute lung injury (and, perhaps, a contributor to ALI) but is not necessarily an indicator of a poor outcome. On the other hand, the presence of detectable MMP-1 and/or MMP-3 is an indicator of more ominous disease progression.

Bronchoalveolar Lavage Fluid↗
PubMed2005

Comparison of antibody array substrates and the use of glycerol to normalize spot morphology.

Antibody microarrays are a high-throughput proteomic technology used to examine the expression of multiple proteins in complex solutions. Antibody microarrays can be manufactured on a variety of commercially available activated glass or coated slides. The goal of this study was to compare Hydrogeltrade mark, nitrocellulose, aldehyde-silane and epoxy-silane slides to determine the amount of antibody bound. The optimal substrate was defined as one that bound the greatest amount of antibody with minimal background. Our studies found that epoxy-silane enhanced surface (ES) slides gave the greatest degree of binding along with a minimal background. However, larger antibody microarrays showed variability in spot size, high intra-spot coefficient of variation and drying artifacts. Increasing the amount of glycerol in the spotting buffer caused a dose-dependent improvement in overall spot morphology. Glycerol was tested on 128 different antibodies and showed decreased: mean spot diameter, intra-spot coefficient of variation and drying artifacts. These studies revealed that the optimal slide substrate was epoxy-silane ES microarray slides. Furthermore, glycerol could normalize spot size, decrease intra-spot coefficient of variability, decrease drying artifacts and increase antibody-spotting density.

Animals↗
PubMed2005

Development of an internally controlled antibody microarray.

Antibody microarrays are a high throughput technology used to concurrently screen for protein expression. Most antibody arrays currently used are based on the ELISA sandwich approach that uses two antibodies to screen for the expression of a limited number of proteins. Also because antigen-antibody interactions are concentration-dependent, antibody microarrays need to normalize the amount of antibody that is used. In response to the limitations with the currently existing technology we have developed a single antibody-based microarray where the quantity of antibody spotted is used to standardize the antigen concentration. In addition, this new array utilizes an internally controlled system where one color represents the amount of antibody spotted, and the other color represents the amount of the antigen that is used to quantify the level of protein expression. When compared with median fluorescence intensity alone, normalization for antibody spot intensity decreased variability and lowered the limits of detection. This new antibody array was tested using standard cytokine proteins and also cell lysates obtained from mouse macrophages stimulated in vitro and evaluated for the expression of the cytokine proteins interleukin (IL)-1beta, IL-5, IL-6, and macrophage inflammatory proteins 1alpha and 1beta. The levels of protein expression seen with the antibody microarray was compared with that obtained with Western blot analysis, and the magnitude of protein expression observed was similar with both technologies with the antibody array actually showing a greater degree of sensitivity. In summary, we have developed a new type of antibody microarray to screen for protein expression that utilizes a single antibody and controls for the amount of antibody spotted. This type of array appears at least as sensitive as Western blot analysis, and the technology can be scaled up for high throughput screening for hundreds of proteins in complex biofluids such as blood.

Animals↗
PubMed2005

Role of interleukin-6 in immune complex induced models of vascular injury.

Previous studies have suggested that Interleukin-6 (IL-6) acts as a marker of vasculitis. To determine the role of IL-6 in vasculitis we utilized two models of immune complex induced vascular injury (dermal Arthus and acute pulmonary alveolitis) in IL-6 deficient (IL-6(-/-)) and IL-6 sufficient (IL-6(+/+)) mice. Plasma and bronchoalveolar lavage (BAL) levels of IL-6 were elevated in the injured IL-6(+/+) mice with acute alveolitis and in the plasma of IL-6(+/+) mice with dermal Arthus vasculitis. While, IL-6 levels in IL-6(-/-) mice were near or below the levels of detection. Histological examination of the intensity of vascular injury response demonstrated no significant differences between IL-6(-/-) and IL6(+/+) mice. More specifically, lung permeability (total protein in the BAL) in the lung injury model in IL-6(-/-) mice was the same as injured IL-6(+/+) mice. As a corollary, assessment of vascular permeability in both models was the same in the IL-6(-/-) as the IL-6(+/+) mice. Quantification of leukocyte influx into the injured tissues in both models also revealed no differences between the IL-6(-/-) and IL-6(+/+) mice. These data demonstrate that while IL-6 is upregulated in acute vascular injury it does not appear to be critical in the development of the vascular inflammatory response.

Animals↗
PubMed2004

Matrix metalloproteinases in acute inflammation: induction of MMP-3 and MMP-9 in fibroblasts and epithelial cells following exposure to pro-inflammatory mediators in vitro.

Recent studies have demonstrated important pro-inflammatory roles for two matrix metalloproteinases (MMPs)-MMP-3 (stromelysin-1) and MMP-9 (gelatinase B)-in acute lung injury [Am. J. Respir. Cell Mol. Biol. 24 (2001) 1]. A role for MMP-3 in skin inflammation has also been demonstrated [Proc. Natl. Acad. Sci. U. S. A. 96 (1999) 6885]. While leukocytes (neutrophils and macrophages) are known to elaborate these tissue-destructive enzymes, parenchymal cells are also capable of synthesizing MMPs. In the present study, we examined the production of MMP-3 and MMP-9 by rodent lung fibroblasts, type II epithelial cells, and vascular endothelial cells. Dermal fibroblasts were also examined. Cells were examined under control conditions and in response to agonists that induce acute inflammatory tissue injury (IgG-containing immune complexes and lipopolysaccharide [LPS]). In the absence of stimulation, MMP-3 and MMP-9 were not detected or were present at low level. However, upon stimulation with either of the two pro-inflammatory agonists, production of both enzymes occurred in fibroblasts and epithelial cells (though not in endothelial cells). The observation that resident cells in the tissue parenchyma can elaborate MMPs in direct response to pro-inflammatory stimuli provides insight into possible mechanisms by which tissue damage occurs in acute inflammation.

Animals↗
PubMed2004

Marasmius oreades lectin induces renal thrombotic microangiopathic lesions.

The present studies demonstrate that infusion of a type B specific lectin derived from the mushroom Marasmius oreades (MOA) into mice binds selectively to the glomerular endothelial cells via surface carbohydrate moieties resulting in cell injury and death associated with platelet-fibrin thrombi. This selective MOA binding to the endothelial cells can be abrogated by a sugar specific for the carbohydrate sequence. Hemolytic-Uremic Syndrome (HUS) and the closely associated Thrombotic Thrombocytopenic Purpura (TTP) are diseases associated with widespread microvascular injury in various organs. Clinically, these diseases are associated with microangiopathic hemolytic anemia and thrombocytopenia. The kidney glomerulus is a primary target of this microvascular injury. There are many underlying etiologies including bacterial toxins. Experimentally, such toxins injure endothelial cells in vitro but in vivo studies have failed to reproduce the characteristic renal pathology. We suggest that MOA-induced glomerular microangiopathic injury could be used to study the pathophysiology of endothelial cell injury as related to glomerular microangiopathic injury.

Agaricales↗
PubMed2004

Role of metalloelastase in a model of allergic lung responses induced by cockroach allergen.

Our laboratory and others have shown an important role of metalloelastase (MMP-12) in the pathogenesis of acute and chronic lung injury. Because chronic asthma is characterized by airway inflammation and alterations in the airway extracellular matrix, we explored the role of metalloelastase in a model of allergic airway inflammation induced by cockroach antigen (CRA). Using MMP-12-deficient mice we found a significant reduction in CRA-induced inflammatory injury, as evidenced by fewer peribronchial leukocytes, significantly less protein in the bronchoalveolar lavage (BAL) fluid, and a significant reduction in the number of infiltrating neutrophils, eosinophils, and macrophages, relative to wild-type mice. Although we did not find a significant reduction in the number of T cells in the injured MMP-12-deficient animals as compared to controls, levels of the chemotactic factors interleukin-5, macrophage inflammatory protein-1 alpha, monocyte chemoattractant protein-1, thymus activation regulated chemokine, and the proinflammatory cytokine tumor necrosis factor-alpha were significantly reduced in the bronchoalveolar lavage fluid of CRA-challenged MMP-12-deficient mice, relative to CRA-challenged control animals. These studies indicate that MMP-12 plays an important proinflammatory role in the development of allergic inflammation in the CRA model. Alterations in the levels of chemotactic factors and other proinflammatory cytokines in the MMP-12-deficient mice may underlie the decrease in leukocyte recruitment into inflamed lungs.

Allergens↗
PubMed2004

Complement determinations in human disease.

OBJECTIVE: To define techniques used for complement measurements and examine the clinical relevance of alterations of complement determinations in disease. DATA SOURCES: Data have been assembled from the authors' research, original articles, and reviews, as well as chapters and complete books on complement. STUDY SELECTION: Studies were chosen for inclusion by the opinions of the authors, relevant complement reviews, publications, and books. RESULTS: Complement has been shown to possess approximately 31 proteins, some of which are enzymes (C1r, C1s, C2, factor B, factor D), some cofactors, some inhibitors or inactivators, and others composed of membrane-integrated proteins. All of the complement proteins have been purified, and many of the respective genes have been identified. The complement cascade is a dual-edged sword, causing protection against bacterial and viral invasion by promoting phagocytosis and inflammation. Pathologically, complement can cause substantial damage to blood vessels (vasculitis), kidney basement membrane and attached endothelial and epithelial cells (nephritis), joint synovium (arthritis), and erythrocytes (hemolysis) if it is not adequately controlled. CONCLUSIONS: Definitive evidence is available that complement-mediated tissue destruction occurs after immune complex injury in the kidney and lung and may be important in lupus erythematosus and adult respiratory distress syndrome. Future studies on complement receptor structure and function may provide clues to treat effectively lupus, hemolytic anemias, and nephritis. In addition, gene therapy and antibody therapy need further refinement to treat immunodeficiency diseases.

Complement System Proteins↗
PubMed2003

Attenuation of autoimmune disease in Fas-deficient mice by treatment with a cytotoxic benzodiazepine.

OBJECTIVE: Elimination of autoreactive cells relies on Fas-dependent activation-induced cell death mechanisms, an important component of peripheral tolerance. Defects in Fas or its cognate ligand lead to inefficient activation-induced cell death and are specific causes of lymphoproliferative and autoimmune diseases. The present study was undertaken to investigate a novel 1,4-benzodiazepine (Bz-423) that induces apoptosis and limits autoimmune disease in NZB/NZW mice, to determine its activity against lupus-like disease associated with defective Fas expression. We investigated the Fas-dependence of its cytotoxic actions, its therapeutic potential in mice deficient in Fas, and its therapeutic mechanism of action. METHODS: Primary lymphocytes isolated from Fas-deficient MRL/MpJ-Fas(lpr) (MRL-lpr) mice were tested for sensitivity to Bz-423. Bz-423 was administered to MRL-lpr mice for short (1-week) or long (14-week) periods, and its effects on cell survival were determined along with measures of nephritis, arthritis, antibody titers, and Th subpopulations. BALB/c mice were similarly treated to determine if Bz-423 alters normal immune functions in vivo. RESULTS: Administration of Bz-423 to MRL-lpr mice significantly reduced autoimmune disease including glomerulonephritis and arthritis. Treatment was associated with decreases in CD4+ T cells and an alteration in the Th1/Th2 balance. At the therapeutic dosage, Bz-423 did not interfere with normal T and B cell responses in BALB/c mice, suggesting that this agent is not globally immunosuppressive. CONCLUSION: Bz-423 is a novel immunomodulatory agent that is active against disease even in the context of defective Fas signaling. It is a leading compound for further investigation into the development of selective therapies for lupus.

Adjuvants, Immunologic↗
PubMed2002

Partial liquid ventilation and positive end-expiratory pressure reduce ventilator-induced lung injury in an ovine model of acute respiratory failure.

OBJECTIVE: To examine the isolated and combined effects of positive end-expiratory pressure (PEEP) and partial liquid ventilation (PLV) on the development of ventilator-induced lung injury in an ovine model. DESIGN: Prospective controlled animal study. SETTING: University-based cardiovascular animal physiology laboratory. SUBJECTS: Thirty-eight anesthetized supine sheep weighing 22.3 +/- 2.2 kg. INTERVENTIONS: Animals were ventilated for 6 hrs (respiratory rate, 15; FIO2, 1.0, inspiratory/expiratory ratio, 1:1) with one of five pressure-controlled strategies, expressed as peak inspiratory pressure (PIP)/PEEP: low-PIP, 25/5 cm H2O (n = 8); high-PIP, 50/5 cm H2O (n = 8); high-PIP-PLV, 50/5 cm H2O-PLV (n = 8); high-PEEP, 50/20 cm H2O (n = 7); and high-PEEP-PLV, 50/20 cm H2O-PLV (n = 7). MEASUREMENTS AND MAIN RESULTS: Compared with the low-PIP control, high-PIP ventilation increased airleak, shunt, histologic evidence of lung injury, neutrophil infiltrates, and wet lung weight. Maintaining PEEP at 20 cm H2O or adding PLV reduced the development of physiologic shunt and dependent histologic injury indexes. Neither higher PEEP nor PLV reduced the high incidence of barotrauma observed in high-PIP animals. CONCLUSIONS: We conclude that application of PLV or PEEP at 20 cm H2O may improve gas exchange and afford lung protection from ventilator-induced lung injury during high-pressure mechanical ventilation in this model.

Acute Disease↗
PubMed2002

Benzodiazepine-induced superoxide signals B cell apoptosis: mechanistic insight and potential therapeutic utility.

The properties of a proapoptotic 1,4-benzodiazepine, Bz-423, identified through combinatorial chemistry and phenotype screening are described. Bz-423 rapidly generated superoxide (O(2)(-)) in transformed Ramos B cells. This O(2)(-) response originated from mitochondria prior to mitochondrial transmembrane gradient collapse and opening of the permeability transition pore. Bz-423-induced O(2)(-) functioned as an upstream signal that initiated an apoptotic program characterized by cytochrome c release, mitochondrial depolarization, and caspase activation. Pretreatment of cells with agents that either block the formation of Bz-423-induced O(2)(-) or scavenge free radicals attenuated the death cascade, which demonstrated that cell killing by Bz-423 depends on O(2)(-). Parallels between Ramos cells and germinal center B cells prompted experiments to determine whether Bz-423 had therapeutic activity in vivo. This possibility was tested using the (NZB x NZW)F(1) murine model of lupus, in which the pathologically enhanced survival and expansion of germinal center B cells mediate disease. Administration of Bz-423 for 12 weeks specifically controlled germinal center hyperplasia and reduced the histological evidence of glomerulonephritis. Collectively, these studies define a new structure-function relationship for benzodiazepines and point to a new target and mechanism that could be of value for developing improved drugs to manage systemic lupus erythematosus and related disorders.

Animals↗
PubMed2002

Effects of partial liquid ventilation on unilateral lung injury in dogs.

STUDY OBJECTIVE: The overall physiologic effect of partial liquid ventilation (PLV) in the setting of unilateral lung injury remains unclear. Therefore, we evaluated the effect of PLV on gas exchange in unilateral lung injury. DESIGN AND METHODS: Left unilateral lung injury was induced in 14 adult dogs by oleic acid instillation into a left pulmonary artery. The animals were divided into two groups: gas ventilation (GV) and PLV. During both GV and PLV, systemic blood gas levels were analyzed. Oxygen consumption (O(2)), carbon dioxide production (CO(2)) and pulmonary blood flow (Q) of both the right lung (uninjured lung) and left lung (injured lung) were measured. RESULTS: During PLV, O(2) of the injured left lung (o(2)-injured), CO(2) of the injured left lung (CO(2)-injured), and Q of the injured left lung (Q-injured) were greater than those in GV (O(2)-injured, 41.6 mL/min vs 23.4 mL/min, p = 0.006; CO(2)-injured, 34.4 mL/min vs 25.5 mL/min, p = 0.026; and Q-injured, 0.47 L/min vs 0.22 L/min, p = 0.002, respectively). However, overall PaO(2) during PLV was less than that during GV, likely due to either a redistribution of Q toward the injured lung (PLV Q-injured, 0.47 L/min vs GV Q-injured, 0.22 L/min; p = 0.002) or reduced gas exchange efficiency in the healthy lung. CONCLUSIONS: We conclude that in our model, PLV increases O(2) and VCO(2) in the injured lung. However, over all gas exchange efficiency is reduced.

Animals↗