An acoustofluidic sputum liquefier.

We demonstrate the first microfluidic-based on-chip liquefaction device for human sputum samples. Our device is based on an acoustofluidic micromixer using oscillating sharp edges. This acoustofluidic sputum liquefier can effectively and uniformly liquefy sputum samples at a throughput of 30 μL min(-1).

IL13 activates autophagy to regulate secretion in airway epithelial cells.

Cytokine modulation of autophagy is increasingly recognized in disease pathogenesis, and current concepts suggest that type 1 cytokines activate autophagy, whereas type 2 cytokines are inhibitory. However, this paradigm derives primarily from studies of immune cells and is poorly characterized in tissue cells, including sentinel epithelial cells that regulate the immune response. In particular, the type 2 cytokine IL13 (interleukin 13) drives the formation of airway goblet cells that secrete excess mucus as a characteristic feature of airway disease, but whether this process is influenced by autophagy was undefined.

Long-term IL-33-producing epithelial progenitor cells in chronic obstructive lung disease.

Chronic obstructive lung disease is characterized by persistent abnormalities in epithelial and immune cell function that are driven, at least in part, by infection. Analysis of parainfluenza virus infection in mice revealed an unexpected role for innate immune cells in IL-13-dependent chronic lung disease, but the upstream driver for the immune axis in this model and in humans with similar disease was undefined. We demonstrate here that lung levels of IL-33 are selectively increased in postviral mice with chronic obstructive lung disease and in humans with very severe chronic obstructive pulmonary disease (COPD).

IL-13-induced airway mucus production is attenuated by MAPK13 inhibition.

Increased mucus production is a common cause of morbidity and mortality in inflammatory airway diseases, including asthma, chronic obstructive pulmonary disease (COPD), and cystic fibrosis. However, the precise molecular mechanisms for pathogenic mucus production are largely undetermined. Accordingly, there are no specific and effective anti-mucus therapeutics.

Self-cleavage of human CLCA1 protein by a novel internal metalloprotease domain controls calcium-activated chloride channel activation.

The chloride channel calcium-activated (CLCA) family are secreted proteins that regulate both chloride transport and mucin expression, thus controlling the production of mucus in respiratory and other systems. Accordingly, human CLCA1 is a critical mediator of hypersecretory lung diseases, such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis, that manifest mucus obstruction. Despite relevance to homeostasis and disease, the mechanism of CLCA1 function remains largely undefined.

Persistent activation of an innate immune response translates respiratory viral infection into chronic lung disease.

To understand the pathogenesis of chronic inflammatory disease, we analyzed an experimental mouse model of chronic lung disease with pathology that resembles asthma and chronic obstructive pulmonary disease (COPD) in humans. In this model, chronic lung disease develops after an infection with a common type of respiratory virus is cleared to only trace levels of noninfectious virus. Chronic inflammatory disease is generally thought to depend on an altered adaptive immune response.

Induction of high-affinity IgE receptor on lung dendritic cells during viral infection leads to mucous cell metaplasia.

Respiratory viral infections are associated with an increased risk of asthma, but how acute Th1 antiviral immune responses lead to chronic inflammatory Th2 disease remains undefined. We define a novel pathway that links transient viral infection to chronic lung disease with dendritic cell (DC) expression of the high-affinity IgE receptor (FcepsilonRIalpha). In a mouse model of virus-induced chronic lung disease, in which Sendai virus triggered a switch to persistent mucous cell metaplasia and airway hyperreactivity after clearance of replicating virus, we found that FceRIa(-/-) mice no longer developed mucous cell metaplasia.

Genetic segregation of airway disease traits despite redundancy of calcium-activated chloride channel family members.

Complex airway diseases such as asthma and chronic obstructive pulmonary disease exhibit stereotyped traits (especially airway hyperreactivity and mucous cell metaplasia) that are variably expressed in each patient. Here, we used a mouse model for virus-induced long-term expression of these traits to determine whether individual traits can be genetically segregated and thereby linked to separate determinants. We showed that an F2 intercross population derived from susceptible and nonsusceptible mouse strains can manifest individual phenotypic extremes that exhibit one or the other disease trait.

Characterization of HDJ-2, a human 40 kD heat shock protein.

Heat shock proteins (HSP) are a large and complex family of proteins that play important roles in cellular function and survival. In previous studies, cDNA for a 45 kD human HSP (HDJ-2) was cloned and shown to be homologous to DNA-J, a bacterial HSP [F.M.

Increased expression of HDJ-2 (heat shock protein 40) and heat shock protein 70 in biopsy specimens of transplanted human lungs.

Background: Heat shock proteins are expressed during several forms of stress and inflammation. This study was done to determine whether the expression of heat shock protein HDJ-2 (heat shock protein 40), heat shock protein 60, and heat shock protein 70 are increased during rejection in human pulmonary allografts.

Methods: Thirty-five transbronchial biopsy specimens were obtained from adult lung transplant recipients.

Peripheral blood microchimerism in human liver and renal transplant recipients: rejection despite donor-specific chimerism.

Background: Development of donor-specific microchimerism (DSM) has been proposed as one of the possible mechanisms for induction and maintenance of allograft tolerance. The aim of this study was to determine: (1) the state of DSM in liver transplant (LTx) and renal transplant (RTx) recipients, (2) whether the persistent presence of an allograft is a requirement for maintenance of chimerism, and (3) whether donor-specific blood transfusions (DST) facilitate chimerism development in RTx recipients and whether this correlates with allograft function.

Methods: Qualitative and quantitative analysis of DSM in peripheral blood of LTx and RTx recipients was assessed by polymerase chain reaction and competitive polymerase chain reaction using HLA-DR probes for mismatched antigens between the donor and recipient.

Increased expression of the HDJ-2 heat shock protein in biopsies of human rejected kidney.

The presence of donor-specific alloreactive helper and cytotoxic T cells has been described in allograft biopsies obtained from individuals undergoing acute allograft rejection of various solid organs. However, not all of these lymphocytes demonstrated specificity to mismatched donor HLA antigens. The identity of the antigens to which these T cells are directed to is still unknown at present.

Evidence that pediatric liver transplant recipients may undergo late rejection episodes in spite of donor-specific microchimerism.

Lymphocytes of donor origin can be demonstrated in the blood of many liver transplant recipients. It has been proposed that this chimerism may imply graft tolerance and permit withdrawal of immunosuppression. We report two children with liver transplants who had lymphocyte chimerism demonstrated at the time of late rejection episodes.

Cytokine gene transcripts for tumor necrosis factor-alpha, interleukin-2, and interferon-gamma in human pulmonary allografts.

Background: Cytokines participate in host responses to allografts, largely through recruiting and activating various regulatory and effector cells. We performed this study to determine the feasibility of using polymerase chain reaction methodology to define the expression of three important cytokines (tumor necrosis factor-alpha, interleukin-2, and interferon-gamma) in human pulmonary allografts.

Methods: Twenty-six graft-derived samples (11 transbronchial biopsy and 8 macrophage and 7 lymphocyte cell pellets isolated from bronchoalveolar lavage) were obtained from 13 lung transplant recipients and treated as follows: extraction of RNA; reverse transcription of RNA to complementary DNA; polymerase chain reaction amplification of cDNA with oligonucleotide primers specific for the three cytokines; gel electrophoresis of the polymerase chain reaction products; and verification of correct cytokine message by Dot blot technique (with specific 32P-labeled oligonucleotide probes).

CD32C (Fc gamma RIIC) mRNA expression and regulation.

The cell surface expression of the CD32 receptors for the Fc portion of immunoglobulin G (Fc gamma RII-CD32) is regulated by agents such as phorbol esters (PMA) and cytokines. In this study, we investigated the effects of PMA and interferon-gamma (IFN-gamma) on the expression of CD32C mRNA in U937 cells. When U937 (CD32+) cells are incubated with either PMA or IFN-gamma a significant enhancement of CD32C mRNA expression is observed with maximum enhancement at 18 hrs post-PMA and IFN-gamma addition.

CD32A (Fc gamma RIIa) mRNA expression and regulation in blood monocytes and cell lines.

The cell surface expression of the CD32 receptors for the Fc portion of immunoglobulin G (Fc gamma RII) is highly regulated by agents such as phorbol ester (PMA) and cytokines. In this study we investigated the regulatory effects of PMA, aggregated IgG (AIgG) and KuFc79 anti-CD32 monoclonal antibodies (mAb) on the expression of the CD32A isomer mRNA. When U937 (CD32+ cells) are incubated with PMA a significant enhancement of the CD32A isomer mRNA is observed.

Structure of the gene for human von Willebrand factor.

von Willebrand factor is a large multimeric plasma protein composed of identical subunits which contain four types of repeated domains. von Willebrand factor is essential for normal hemostasis, and deficiency of von Willebrand factor is the most common inherited bleeding disorder of man. Four human genomic DNA cosmid libraries and one bacteriophage lambda library were screened with von Willebrand factor cDNA probes.

Regulation of human IgE synthesis by soluble factors. Papain treatment of a FcE receptor-positive B-cell line (RPMI-1788) releases regulatory factors for IgE synthesis.

A novel mechanism for the release of helper and suppressor factors for human IgE synthesis is described. When FcE receptor-positive RPMI-1788 cells are treated with papain, a helper factor(s) for human IgE synthesis is released. At the same time a significant decrease in the number of cell surface FcE receptors is observed.

Induction of human immunoglobulin synthesis (IgG, IgA) by the novel, T cell independent mitogen Cytophaga allerginae endotoxin.

Cytophaga allerginae endotoxin (CAE) has been purified from C. allerginae, a newly discovered bacterial species isolated from a chilled water spray humidification system. The present study was undertaken in order to determine whether CAE can induce immunoglobulin synthesis by human peripheral blood lymphocytes (PBL) in culture.

An avidin-biotin ELISA assay for the measurement of de novo human IgE synthesis in culture supernatants.

A very sensitive (100 pg/ml) solid-phase enzyme immunoassay (ELISA) for the determination of human IgE has been developed. This assay incorporates the avidin-biotin system to increase sensitivity and can detect as little as 100 pg/ml (10 pg/test) of human IgE. The assay is highly specific and allows quantitative determination of human IgE in supernatants of peripheral blood lymphocytes as well as in serum.

Immune response in experimentally induced uremia. VII. Uremic thymocytes amplify the response of control lymph node cells to alloantigens.

The effect of experimentally induced uremia in the rat on the synergistic response between thymus cells (TC) and lymph node cells (LNC) was examined. It was found that: (1) Uremic TC and LNC interact in a synergistic fashion which is greater than that observed for control cells; (2) The response of uremic LNC to alloantigens is suppressed when compared to the response of control LNC; (3) Uremic TC provide more help to control LNC in their response to alloantigens than do control TC; and (4) Treatment of uremic rats with cortisone acetate (CA) enhances their TC ability to amplify control LNC response to alloantigens. Thus, it appears that, while the response of uremic LNC to alloantigens is markedly suppressed, there are potent amplifier cells present in the thymus of uremic rats which have the ability to act in synergism with control LNC in response to alloantigens.

Suppression of mixed lymphocyte culture by uremic adherent spleen cells and peritoneal macrophages is dependent on a cyclophosphamide-sensitive cell.

A model of experimentally induced uremia in the rat has been used to study the effect of uremia on the response of spleen cells to alloantigens. The proliferative ability of uremic spleen cells in mixed lymphocyte culture is significantly suppressed when compared to that of cells from control animals. This suppression appears to be due to both adherent suppressor cells which can be eliminated by adherence to rayon wool and to the inability of uremic T cells to respond to alloantigens.

The role of monocytes and responder cells in suppression of antigen-specific T cell proliferation in chronic renal failure.

The response of peripheral blood lymphocytes (PBL) to the antigen tetanus toxoid (TT) in patients with chronic renal failure being maintained on hemodialysis was examined. We have found that: (1) the response of patients' unfractionated PBL to TT is markedly suppressed when compared to the response of control PBL; (2) the response of patients' purified T cells and T4+ cells to TT co-cultured with 5% autologous monocytes is suppressed when compared to the response of comparable control cultures; (3) the response of patients' purified T lymphocytes co-cultured with 5% autologous monocytes is significantly enhanced over the response of patients' unfractionated PBL, and (4) the suppressed proliferation of patients' PBL to TT is not reversed by hemodialysis. Thus, the presence of suppressor monocytes and the inability of the responding T cells and accessory monocytes to react to antigen contribute to the suppressed antigen-specific T cell proliferation observed in chronic renal failure patients.

Immune response in experimentally induced uremia. VI. Uremic macrophages are defective in their ability to present antigen to T cells.

The ability of uremic lymphocytes to respond to antigens was examined. We have found that (1) The ability of uremic unfractionated lymph node cells to respond to antigens is severely diminished when compared to the response of control cells; (2) Uremic macrophages are defective in their ability to present antigen to T cells; (3) Treatment of control splenic macrophages with monoclonal anti-Ia antibodies diminishes these macrophages' ability to present antigen, while uremic macrophages so treated show little change in their already diminished accessory cell function; and (4) The uremic splenic macrophage population has more small cells and less Ia determinants per cell than do control splenic macrophages as determined by cytofluorographic analysis. The percentage of Ia+ splenic macrophages is similar in control and uremic rats.

Immune response in experimentally induced uremia. V. Description of a model for chronic uremia in the rat that produces a long-term suppressive effect on T cell responses to mitogens.

This study describes a model for chronic uremia in the rat that produces a long-term suppressive effect on T cell responses to mitogens. We have found that (1) chronically uremic rats have significantly higher serum creatine levels than control rats and lymphocytopenia at all intervals tested after induction of uremia up to 4 1/2 months; (2) at all times tested after induction of uremia, the response of spleen cells from uremic rats to the T cell mitogens ConA and PHA was significantly suppressed as compared to control rats; (3) the severely suppressed response to PHA at all intervals tested after induction of uremia was eliminated by the removal of adherent spleen cells; and (4) adherent spleen cells, PMø, and AMø from chronically uremic rats are significantly more suppressive to control T cells than the corresponding control cells at all intervals tested after induction of uremia. Thus this animal model for chronic uremia is stable for at least 4 1/2 months and the effect of uremia on the response of lymphocytes to mitogens as well as on regulatory cells is not transient.