TL1A Promotes Lung Tissue Fibrosis and Airway Remodeling.

Lung fibrosis and tissue remodeling are features of chronic diseases such as severe asthma, idiopathic pulmonary fibrosis, and systemic sclerosis. However, fibrosis-targeted therapies are currently limited. We demonstrate in mouse models of allergen- and bleomycin-driven airway inflammation that neutralization of the TNF family cytokine TL1A through Ab blocking or genetic deletion of its receptor DR3 restricted increases in peribronchial smooth muscle mass and accumulation of lung collagen, primary features of remodeling. TL1A was found as a soluble molecule in the airways and expressed on the surface of alveolar macrophages, dendritic cells, innate lymphoid type 2 cells, and subpopulations of lung structural cells. DR3 was found on CD4 T cells, innate lymphoid type 2 cells, macrophages, fibroblasts, and some epithelial cells. Suggesting in part a direct activity on lung structural cells, administration of recombinant TL1A into the naive mouse airways drove remodeling in the absence of other inflammatory stimuli, innate lymphoid cells, and adaptive immunity. Correspondingly, human lung fibroblasts and bronchial epithelial cells were found to express DR3 and responded to TL1A by proliferating and/or producing fibrotic molecules such as collagen and periostin. Reagents that disrupt the interaction of TL1A with DR3 then have the potential to prevent deregulated tissue cell activity in lung diseases that involve fibrosis and remodeling.

Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage.

Tissue fibrosis is a major cause of mortality that results from the deposition of matrix proteins by an activated mesenchyme. Macrophages accumulate in fibrosis, but the role of specific subgroups in supporting fibrogenesis has not been investigated in vivo. Here, we used single-cell RNA sequencing (scRNA-seq) to characterize the heterogeneity of macrophages in bleomycin-induced lung fibrosis in mice. A novel computational framework for the annotation of scRNA-seq by reference to bulk transcriptomes (SingleR) enabled the subclustering of macrophages and revealed a disease-associated subgroup with a transitional gene expression profile intermediate between monocyte-derived and alveolar macrophages. These CX3CR1+SiglecF+ transitional macrophages localized to the fibrotic niche and had a profibrotic effect in vivo. Human orthologs of genes expressed by the transitional macrophages were upregulated in samples from patients with idiopathic pulmonary fibrosis. Thus, we have identified a pathological subgroup of transitional macrophages that are required for the fibrotic response to injury.

Platycodin D attenuates acute lung injury by suppressing apoptosis and inflammation in vivo and in vitro.

Platycodin D (PLD) is the major triterpene saponin in the root of Platycodon grandiflorum (Jacq.) with various pharmacological activities. The purpose of the present study was to evaluate the protective effects and possible mechanisms of PLD on acute lung injury (ALI) both in vivo and in vitro. In vivo, we used two ALI models, lipopolysaccharide (LPS)-induced ALI and bleomycin (BLE)-induced ALI to evaluate the protective effects and possible mechanisms of PLD. Female BALB/c mice were randomly divided into the following groups: control group, LPS group, LPS plus pre-treatment with dexamethasone (2 mg/kg) group, LPS plus pre-treatment with PLD groups (50 mg/kg, 100 mg/kg), LPS plus post-treatment with dexamethasone (2 mg/kg) group, LPS plus post-treatment with PLD groups (50 mg/kg, 100 mg/kg), BLE group, BLE plus pre-treatment with dexamethasone (2 mg/kg) group, BLE plus pre-treatment with PLD groups (50 mg/kg, 100 mg/kg), BLE plus post-treatment with dexamethasone (2 mg/kg) group, and BLE plus post-treatment with PLD groups (50 mg/kg, 100 mg/kg). PLD was orally administered before or after LPS or BLE challenge with mice. Mice were sacrificed, and lung tissues and bronchoalveolar fluid (BALF) were prepared for further analysis. Our results showed that PLD significantly decreased lung wet-to-dry weight ratio (lung W/D weight ratio), total leukocyte number and neutrophil percentage in the BALF, and myeloperoxidase (MPO) activity of lung in a dose-dependent manner. Besides, cytokine levels, including interleukin (IL)-6, tumor neurosis factor (TNF)-α were also found significantly inhibited in BALF. Furthermore, PLD effectively inhibited the expressions of nuclear factor κB (NF-κB), Caspase-3 and Bax in the lung tissues, as well as restored the expression of Bcl-2 in the lungs and improved the superoxide dismutase (SOD) activity in BALF. In vitro, we used LPS-challenged cell model to evaluate the protective effects and possible mechanisms of PLD. MLE-12 cells were stimulated with LPS in the presence and absence of PLD. The levels of TNF-α, IL-6 and the expressions of NF-κB, Caspase-3, and Bax were remarkably down-regulated, while the expression of bcl-2 was significantly up-regulated in PLD treatment groups in MLE-12 cells. These results showed that the administration of PLD improved ALI both in vivo and in vitro, possibly through suppressing apoptosis and inflammation.

Targeting Her-2+ breast cancer cells with bleomycin immunoliposomes linked to LLO.

Bleomycin is a membrane impermeable chemotherapeutic agent that is relatively innocuous extracellularly but highly cytotoxic when delivered directly to the cytoplasm. We report on the development of a liposome delivery system that targets Her-2 overexpressing breast cancer cells and breaches the endosomal barrier, delivering bleomycin to the cytoplasm. The liposomes are conjugated to the antibody trastuzumab, which results in specific binding and internalization of liposomes into Her-2 overexpressing cells. In addition, the liposomes are disulfide bonded to a pore-forming protein listeriolysin O (LLO) which forms pores in the endosome and allows the liposomal cargo to pass into the cytoplasm. We demonstrate specific delivery to Her-2 positive MCF-7/Her18 cells relative to Her-2 negative MCF-7 cells using a fluorescent probe calcein within the immunoliposomes. When calcein is replaced by bleomycin, the liposomes effectively reduce viability of five different Her-2 overexpressing cell lines (BT-474, SKBR-3, MCF-7/Her18, HCC-1954 and MDA-453) while harming to a much lesser extent Her-2 negative breast cell lines (MCF-7, MCF-12a and MCF-10a). The liposomes also affect trastuzumab-resistant cells, reducing MDA-453 cell number by 97% compared to untreated cells. Importantly, the concentration of drug needed to reduce tumor cell growth and viability using this liposome therapy is approximately 57,000-fold less than the concentration needed if drug is delivered extracellularly, raising the possibility of increased therapeutic specificity with decreased side effects.

Inhibition of pulmonary fibrosis in mice by CXCL10 requires glycosaminoglycan binding and syndecan-4.

Pulmonary fibrosis is a progressive, dysregulated response to injury culminating in compromised lung function due to excess extracellular matrix production. The heparan sulfate proteoglycan syndecan-4 is important in mediating fibroblast-matrix interactions, but its role in pulmonary fibrosis has not been explored. To investigate this issue, we used intratracheal instillation of bleomycin as a model of acute lung injury and fibrosis. We found that bleomycin treatment increased syndecan-4 expression. Moreover, we observed a marked decrease in neutrophil recruitment and an increase in both myofibroblast recruitment and interstitial fibrosis in bleomycin-treated syndecan-4-null (Sdc4-/-) mice. Subsequently, we identified a direct interaction between CXCL10, an antifibrotic chemokine, and syndecan-4 that inhibited primary lung fibroblast migration during fibrosis; mutation of the heparin-binding domain, but not the CXCR3 domain, of CXCL10 diminished this effect. Similarly, migration of fibroblasts from patients with pulmonary fibrosis was inhibited in the presence of CXCL10 protein defective in CXCR3 binding. Furthermore, administration of recombinant CXCL10 protein inhibited fibrosis in WT mice, but not in Sdc4-/- mice. Collectively, these data suggest that the direct interaction of syndecan-4 and CXCL10 in the lung interstitial compartment serves to inhibit fibroblast recruitment and subsequent fibrosis. Thus, administration of CXCL10 protein defective in CXCR3 binding may represent a novel therapy for pulmonary fibrosis.

Injection of bleomycin in newborn mice induces autoimmune sialitis that is transferred by CD4 T cells.

Bleomycin (BLM) induces cellular apoptosis or necrosis by producing reactive oxygen species, and has been used to induce scleroderma in adult mice. We wondered whether BLM induces the same pathological phenotype in newborn mice as in adult mice. BLM was subcutaneously administrated to newborn BALB/c mice. At 1 month of age, BLM-treated mice showed severe destruction of salivary glands with enlargement of nearby lymph nodes. These nodes contained CD4(+) T cells and B220(+)cells with high expression of MHC class II molecules. In addition, autoantibodies were detected by HEp-2 staining and western blotting. The cell transfer experiments were performed to evaluate the role of autoimmune phenomena in these pathological changes. Following the transfer of enriched CD4(+) T cells to 1-month-old BALB/c nude mice, the salivary glands were severely damaged with CD4(+) T cell and B220(+) cells infiltrations. The number of T-cell antigen receptor Vbeta 8.3(+) CD4(+) T cells was significantly increased in BLM-treated murine spleen. These findings will provide new insights into the causal factors of environment in autoimmunity and the relationship between autoreactive CD4(+) T cells and autoantibodies.

The need for routine bleomycin test dosing in the 21st century.

OBJECTIVE: To review the clinical evidence for routine use of bleomycin test dosing. DATA SOURCES: English-language review articles, references from retrieved articles, case reports, and clinical trials were identified from a MEDLINE literature search (1966-July 2005). Key search terms included bleomycin, test dose, anaphylactic reactions, and hypersensitivity. Information from an unpublished E-mail survey, the manufacturer, and the Internet was also used. DATA SYNTHESIS: Early clinical trials and isolated case reports suggest that bleomycin-induced acute hypersensitivity reactions occur in 1% of patients with lymphoma and <0.5% of those with solid tumors. The reactions are mainly characterized by high-grade fever, chills, hypotension, and in a few cases, cardiovascular collapse, which can lead to death. The exact mechanism of these reactions is unclear, but is thought to be related to the release of endogenous pyrogens from the host cells. Evidence does not suggest any correlation between doses and the onset or severity of the reactions. Supportive care, including hydration, steroids, antipyretics, and antihistamines, may resolve the symptoms. However, it may not completely prevent recurrences. CONCLUSIONS: The incidence of acute hypersensitivity or hyperpyrexic reactions associated with bleomycin is very low, but the reaction is potentially fatal. Clinicians should monitor their patients for any signs and symptoms of acute hyperpyrexic reactions during bleomycin administration. Since the onset of the reactions can occur with any dose of bleomycin and at any time, routine test dosing does not seem to predict when drug reactions may occur.

Monoclonal antibody mediated intracellular targeting of tallysomycin S(10b).

The potency of tallysomycin S(10b) (TLM S(10b)) an analogue bleomycin was enhanced by up to 875-fold when it was conjugated to the internalizing antibody BR96. Attachment to the antibody is achieved via a Cathepsin B cleavable linker. The enhancement in potency is believed to be a result of cellular uptake of the conjugate upon antigen binding followed by rapid release of the drug inside the lysosome. This method provides a novel approach for increasing the potency and therapeutic index of nominally moderately-active cytotoxic agents.

Expression of B7-1, B7-2, and interleukin 12 in bleomycin-induced pneumopathy in mice.

BACKGROUND: The bleomycin-induced pneumopathy involves a T cell-mediated immune response. T cell activation requires both antigen/MHC recognition and costimulatory signals. The CD28 receptor on T cells with its ligand B7 represents one of the most important examples of this costimulation. Interleukin 12 (IL-12) has a strong synergistic effect with the B7-1/CD28 interaction on inducing proliferation and cytokine production in T cells. METHODS: In this study, we investigated the expression of B7-1, B7-2, and IL-12 in bleomycin-induced pneumopathy in mice using reverse transcription polymerase chain reaction (RT-PCR), RT in situ PCR, and immunohistochemistry. RESULTS: We observed concurrent upregulation of B7-1, B7-2, and IL-12p40 mRNA in the lung tissues at 1 h to 7 days after bleomycin instillation into the trachea. B7-1 mRNA and protein were found in bronchiolar epithelial cells as well as macrophages, B7-2 and IL-12p40 mRNA appeared to be expressed in mononuclear cells. CONCLUSIONS: These findings indicate that T cell-mediated immune response in this model involves the upregulation of B7-1, B7-2, and IL-12p40 mRNA, and also demonstrate the aberrant expression of B7-1 in bronchiolar epithelial cells.

Occurrence of an anti-peplomycin IgE antibody cross-reacting with bleomycin in a patient with cervical uterine cancer.

An allergic reaction to peplomycin was observed in a patient with cervical uterine cancer who had previously been treated with peplomycin. A positive Prausnitz-Küstner test and its elimination after heat treatment of the serum showed the production of anti-peplomycin IgE antibody. Peplomycin was coupled to a paper disc and a sensitive radioallergosorbent test for peplomycin was developed to quantitate the antibody. Patient serum IgE and IgG were purified by DE52 column chromatography; the IgE fraction contained binding activity to peplomycin. A competition test revealed that the antibody bound to both peplomycin and bleomycin. DNA, RNA, and mononucleotides had no effect on antibody binding, but the antibody inhibited peplomycin's activity.

Interaction of antibody-bearing small unilamellar liposomes with antigen-coated cells. The effect of antibody and antigen concentration on the liposomal and cell surface respectively.

Human blood lymphocytes were coated with increasing amounts of human kappa chain (2-85mug/10(7) cells) through the linking reagent CrCl(3). These cells were then exposed to small unilamellar liposomes composed of egg phosphatidylcholine, cholesterol and phosphatidic acid (molar proportions 7:7:1) containing carboxyfluorescein and/or (111)In-labelled bleomycin and bearing (131)I-labelled affinity chromatography-purified or non-purified anti-(kappa-chain) immunoglobulin G (IgG) [see the preceding paper, Gregoriadis, Meehan & Mah (1981) Biochem. J.200, 203-210]. In some experiments liposomes contained [(14)C]phosphatidylcholine. (1) Lymphocytes (10(7)) coated with 2-85mug of kappa chain and exposed to liposomes devoid of IgG or bearing non-purified anti-(kappa chain) IgG bound only a small proportion of the liposomal markers. Even with liposomes bearing the purified anti-(kappa chain) IgG, uptake of the labels improved only slightly for cells coated with up to 10mug of kappa chain. However, with higher concentrations of the antigen on the cell surface, binding was improved considerably to reach values of 31% ((111)In-labelled bleomycin) and 43% ((131)I-labelled IgG) of added liposomes for cells coated with 85mug of kappa chain. (2) Lymphocytes coated with kappa chain were exposed to liposomes bearing increasing amounts (0-180mug/0.9mg of egg phosphatidylcholine) of purified anti-(kappa chain) IgG. It was found that under the present conditions, binding of all three markers ((111)In-labelled bleomycin, (131)I-labelled IgG and [(14)C]phosphatidylcholine) was directly proportional to the concentration of IgG on the liposomal surface. However, uptake values remained unchanged above 90mug of IgG. (3) Antibody-mediated uptake of liposomes by cells coated with the corresponding antigen without loss of their metabolic activities may provide a method of efficient targeting.

Enzyme immunoassay for pepleomycin, a new bleomycin analog.

An antibody directed toward pepleomycin, a new antitumor antibiotic related structurally to bleomycin, has been produced in rabbits by immunization with a pepleomycin-protein conjugate which was prepared by a novel procedure of coupling pepleomycin to mercaptosuccinylated bovine serum albumin using N-(gamma-maleimidobutyryloxy)succinimide as a coupling agent. The antiserum was monospecific to pepleomycin and showed almost no cross-reactivity with a variety of other bleomycin analogs. An enzyme immunoassay for pepleomycin has been developed utilizing this antiserum and beta-D-galactosidase-labeled pepleomycin. The lower limit of detection by this assay, which involves a double antibody technique for the separation of antibody-bound and free antigen, was 50 pg of pepleomycin per tube. Using this assay, drug levels were easily determined in blood and urine of rabbits following administration of pepleomycin in a single dose of 1.2 mg/kg i.v. This assay is also suitable for measuring pepleomycin in the presence of other drugs since the assay is not significantly affected by any other antineoplastic agents tested. Since pepleomycin is now undergoing clinical trial, the enzyme immunoassay of the drug will be a valuable tool in clinical pharmacological studies.

[Effect of 57Co-bleomycetin on the immune response induced in mice by ram erythrocytes]. / Vliianie 57Co-bleomitsetina na immunnyi otvet, indutsirovannyi u myshei éritrotsitami barana.

The effect of 57Co-bleomycin on the immune response induced by sheep red cells was studied on mice. It was found that the immune response was not suppressed when the labeled antibiotic was used in a single dose of 5-20 mg/kg. By the 5th day the level of 57Co-bleomycin in the skin, thymus and blood was 3-10 times higher than that of 57CoCl2 and in the spleen it was 1.3-1.5 times higher.

[Bleomycin-induced tumor cell lysis caused by antibodies and complement]. / Bleomycin-induzierte Tumorzell-Lyse durch Antikörper und Komplement.

The well-known cytotoxic mode of action of bleomycin up to now has been clarified exclusively by intracellular processes. Our findings however indicate that an immunologic effect induced by bleomycin can also lead to the destruction of tumor cells.

A radioimmunoassay for tallysomycin.

Antibodies were raised against a new antineoplastic agent, tallysomycin, for use in a radioimmunoassay. The assay was sensitive to 2.5 ng of tallysomycin, had a coefficient of variation of 10.5% at 50 ng/ml, and was highly specific for tallysomycin A. The antibody exhibited reduced cross-reactivity with closely related tallysomycin analogs and little or no immunoreactivity with other antitumor antibiotics including bleomycin and phleomycin. Increased immunoreactivity occurred following copper chelation of tallysomycin B as compared with the non-chelated forms, thus indicating possible conformational alterations upon metal binding.

Disposition of the pulmonary toxin, bleomycin.

The disposition of bleomycin in rats was investigated with the use of a 57Co-labeled bleomycin radioimmunoassay. Bleomycin (1 mg/kg, ip) reached a peak serum level of 0.7-1.0 microgram/ml in 54 +/- 4 min (mean +/- SE) and was eliminated with a t 1/2 of 32 +/- 1 min. The apparent volume of distribution in rats was 184 +/- 11 ml/kg. No change in t 1/2 was observed when rats were pretreated with phenobarbital (1 mg/ml in drinking water for 4 days) or exposed to ozone (3 ppm, 24 hr). Probenecid (0.2 g/kg, ip) given 20 min prior to bleomycin administration increased the t 1/2 2-fold. The order of tissue distribution of 57Co-labeled bleomycin 2 hr after ip administration was: kidney greater than liver greater than lungs greater than spleen greater than heart greater than thymus. The affinity of 57Co-labeled bleomycin for homogenates of lungs, liver, or kidney was equal.