human-lung-dendritic-cells-spatial-distribution-phenotypic

Human Lung Dendritic Cells: Spatial Distribution and Phenotypic Identification in Endobronchial Biopsies Using Immunohistochemistry and Flow Cytometry

The lungs are constantly exposed to the external environment, which in addition to harmless particles, also contains pathogens, allergens, and toxins. In ...

umeå university

A family of naturally occurring mycobacterial phosphatidylinositol (PI) and its dimannosides (PIM(2), AcPIM(2), and Ac(2)PIM(2)) that all possess the predominant natural 19:0/16:0 phosphatidyl acylation pattern were prepared to study their mass spectral fragmentations. Among these, the first synthesis of a fully lipidated PIM (i.e., (16:0,18:0)(19:0/16:0)-PIM(2)) was achieved from (±)-1,2:4,5-diisopropylidene-D-myo-inositol in 16 steps in 3% overall yield. A key feature of the strategy was extending the utility of the p-(3,4-dimethoxyphenyl)benzyl protecting group for its use at the O-3 position of inositol to allow installation of the stearoyl residue at a late stage in the synthesis. Mass spectral studies were performed on the synthetic PIMs and compared to those reported for natural PIMs identified from a lipid extract of M. bovis BCG. These analyses confirm that fragmentation patterns can be used to identify the structures of specific PIMs from the cell wall lipid extract.

Synthesis and mass spectral characterization of mycobacterial phosphatidylinositol and its dimannosides.

A reliable reagent system for the cleavage of 4-(3,4-dimethoxyphenyl)benzyl (DMPBn) ethers under acidic conditions has been established. Treatment of DMPBn-protected mono- and pseudodisaccharides with TFA in anhydrous CH2Cl2 and 3,4-(methylenedioxy)toluene as a cation scavenger resulted in the selective cleavage of the DMPBn ether giving the corresponding deprotected products in moderate to high yields. Examples are reported which show that allyl, benzyl, and p-bromobenzyl ethers, esters, and glycosidic linkages are stable to these reaction conditions. The selective cleavage of allyl, p-bromobenzyl, and PMB ethers in protected carbohydrates containing DMPBn ethers are also demonstrated. This work establishes the 4-(3,4-dimethoxyphenyl)benzyl ether as an effective and robust alternative to p-methoxybenzyl as a protecting group for alcohols.

The dimethoxyphenylbenzyl protecting group: an alternative to the p-methoxybenzyl group for protection of carbohydrates.

Oxylipins are oxidised fatty acids that can exert lipid mediator functions in inflammation, and several oxylipins derived from arachidonic acid are linked to asthma. This study quantified oxylipin profiles in different regions of the lung to obtain a broad-scale characterisation of the allergic asthmatic inflammation in relation to healthy individuals. Bronchoalveolar lavage fluid (BALF), bronchial wash fluid and endobronchial mucosal biopsies were collected from 16 healthy and 16 mildly allergic asthmatic individuals. Inflammatory cell counts, immunohistochemical staining and oxylipin profiling were performed. Univariate and multivariate statistics were employed to evaluate compartment-dependent and diagnosis-dependent oxylipin profiles in relation to other measured parameters. Multivariate modelling showed significantly different bronchial wash fluid and BALF oxylipin profiles in both groups (R(2)Y[cum]=0.822 and Q(2)[cum]=0.759). Total oxylipin concentrations and five individual oxylipins, primarily from the lipoxygenase (LOX) pathway of arachidonic and linoleic acid, were elevated in bronchial wash fluid from asthmatics compared to that from healthy controls, supported by immunohistochemical staining of 15-LOX-1 in the bronchial epithelium. No difference between the groups was found among BALF oxylipins. In conclusion, bronchial wash fluid and BALF contain distinct oxylipin profiles, which may have ramifications for the study of respiratory diseases. Specific protocols for sampling proximal and distal airways separately should be employed for lipid mediator studies.

Lipid mediator profiles differ between lung compartments in asthmatic and healthy humans.

The selective inhibition of cancer-associated human carbonic anhydrase (CA) enzymes, specifically CA IX and XII, has been validated as a mechanistically novel approach toward personalized cancer management. Herein we report the design and synthesis of a panel of 24 novel glycoconjugate primary sulfonamides that bind to the extracellular catalytic domain of CA IX and XII. These compounds were synthesized from variably acylated glycopyranosyl azides and either 3- or 4-ethynyl benzene sulfonamide using Cu(I)-catalyzed azide alkyne cycloaddition (CuAAC). The CA enzyme inhibition profile for all compounds was determined, while in vitro metabolic stability, plasma stability, and plasma protein binding for a representative set of compounds was measured. Our findings demonstrate the influence of the differing acyl groups on these key biopharmaceutical properties, confirming that acyl group protected carbohydrate-based sulfonamides have potential as prodrugs for selectively targeting the extracellular cancer-associated CA enzymes.

A prodrug approach toward cancer-related carbonic anhydrase inhibition.

Carbonic anhydrase IX (CA IX) is a key modulator of aggressive tumor behavior and a prognostic marker and target for several cancers. Saccharin (SAC) based compounds may provide an avenue to overcome CA isoform specificity, as they display both nanomolar affinity and preferential binding, for CA IX compared to CA II (>50-fold for SAC and >1000-fold when SAC is conjugated to a carbohydrate moiety). The X-ray crystal structures of SAC and a SAC-carbohydrate conjugate bound to a CA IX-mimic are presented and compared to CA II. The structures provide substantial new insight into the mechanism of SAC selective CA isoform inhibition.

Saccharin: a lead compound for structure-based drug design of carbonic anhydrase IX inhibitors.

Vitamin C is an important low-molecular weight antioxidant at the air-lung interface. Despite its critical role as a sacrificial antioxidant, little is known about its transport into the respiratory tract lining fluid (RTLF), or the underlying airway epithelial cells. While several vitamin C transporters have been identified, such as sodium-ascorbate cotransporters (SVCT1/2) and glucose transporters (GLUTs), the latter transporting dehydroascorbate, knowledge of their protein distribution within the human lung is limited, in the case of GLUTs or unknown for SVCTs.

Identification of vitamin C transporters in the human airways: a cross-sectional in vivo study.

Inhibition of human carbonic anhydrase IX (hCA IX) has shown to be therapeutically advantageous for treating many types of highly aggressive cancers. However, designing selective inhibitors for hCA IX has been difficult due to its high structural homology and sequence similarity with off-target hCAs. Recently, the use of glucosyl sulfamate inhibitors has shown promise as selective inhibitors for hCA IX. In this study, we present five X-ray crystal structures, determined to a resolution of 1.7 Å or better, of both hCA II (a ubiquitous CA) and an engineered hCA IX-mimic in complex with selected glucosyl sulfamates and structurally rationalize mechanisms for hCA IX selectivity. Results from this study have allowed us, for the first time, to empirically "map" key interactions of the hCA IX active site in order to establish parameters needed to design novel hCA IX selective inhibitors.

Mapping Selective Inhibition of the Cancer-Related Carbonic Anhydrase IX Using Structure-Activity Relationships of Glucosyl-Based Sulfamates.

Chemical probes are small molecules designed to bind to a specific protein and disrupt the proteins function. Although many inhibitors are reported for human carbonic anhydrase (CA) enzymes, few may be considered useful as chemical probes as they exhibit broad action against the 12 catalytically active CA isozymes. In addition, most do not possess an appropriate physicochemical profile to discriminate intracellular CA activity from either global or extracellular CA activity. We report herein the synthesis of three monophosphate CA proinhibitors (compounds 2, 3, and 5) that are derived from cyclosaligenyl (cycloSal) phosphate and S-acyl-2-thioethyl (SATE) phosphate as protecting groups. The proinhibitors are designed as neutral, membrane-permeable compounds that once inside the cell may be hydrolyzed by pH-driven or enzymatic-driven mechanisms to release a negatively charged monophosphate. The resulting monophosphate compound is trapped intracellularly and available for locality specific inhibition of intracellular CAs.

Phosphate Chemical Probes Designed for Location Specific Inhibition of Intracellular Carbonic Anhydrases.

Smoke from combustion of biomass fuels is a major risk factor for respiratory disease, but the underlying mechanisms are poorly understood. The aim of this study was to determine whether exposure to wood smoke from incomplete combustion would elicit airway inflammation in humans.

Acute exposure to wood smoke from incomplete combustion--indications of cytotoxicity.

Every breath we take contains potentially harmful pathogens or allergens. Dendritic cells (DCs), monocytes, and macrophages are essential in maintaining a delicate balance of initiating immunity without causing collateral damage to the lungs because of an exaggerated inflammatory response. To document the diversity of lung mononuclear phagocytes at steady-state, we performed bronchoscopies on 20 healthy subjects, sampling the proximal and distal airways (bronchial wash and bronchoalveolar lavage, respectively), as well as mucosal tissue (endobronchial biopsies). In addition to a substantial population of alveolar macrophages, we identified subpopulations of monocytes, myeloid DCs (MDCs), and plasmacytoid DCs in the lung mucosa. Intermediate monocytes and MDCs were highly frequent in the airways compared with peripheral blood. Strikingly, the density of mononuclear phagocytes increased upon descending the airways. Monocytes from blood and airways produced 10-fold more proinflammatory cytokines than MDCs upon ex vivo stimulation. However, airway monocytes were less inflammatory than blood monocytes, suggesting a more tolerant nature. The findings of this study establish how to identify human lung mononuclear phagocytes and how they function in normal conditions, so that dysregulations in patients with respiratory diseases can be detected to elucidate their contribution to immunity or pathogenesis.

Dendritic Cells and Monocytes with Distinct Inflammatory Responses Reside in Lung Mucosa of Healthy Humans.

Carbonic anhydrase XII (CAXII) is a membrane enzyme that maintains pH homeostasis and sustains optimum P-glycoprotein (Pgp) efflux activity in cancer cells. Here, we investigated a panel of eight CAXII inhibitors (compounds 1-8), for their potential to reverse Pgp mediated tumor cell chemoresistance. Inhibitors (5 nM) were screened in human and murine cancer cells (colon, lung, breast, bone) with different expression levels of CAXII and Pgp. We identified three CAXII inhibitors (compounds 1, 2 and 4) that significantly (≥ 2 fold) increased the intracellular retention of the Pgp-substrate and chemotherapeutic doxorubicin, and restored its cytotoxic activity. The inhibitors lowered intracellular pH to indirectly impair Pgp activity. Ca12-knockout assays confirmed that the chemosensitizing property of the compounds was dependent on active CAXII. Furthermore, in a preclinical model of drug-resistant breast tumors compound 1 (1900 ng/kg) restored the efficacy of doxorubicin to the same extent as the direct Pgp inhibitor tariquidar. The expression of carbonic anhydrase IX had no effect on the intracellular doxorubicin accumulation. Our work provides strong evidence that CAXII inhibitors are effective chemosensitizer agents in CAXII-positive and Pgp-positive cancer cells. The use of CAXII inhibitors may represent a turning point in combinatorial chemotherapeutic schemes to treat multidrug-resistant tumors.

Article	Year
Synthesis and mass spectral characterization of mycobacterial phosphatidylinositol and its dimannosides. The Journal of organic chemistry\| PubMed ID: 22845613	2012
The dimethoxyphenylbenzyl protecting group: an alternative to the p-methoxybenzyl group for protection of carbohydrates. The Journal of organic chemistry\| PubMed ID: 23678997	2013
Lipid mediator profiles differ between lung compartments in asthmatic and healthy humans. The European respiratory journal\| PubMed ID: 24036245	2014
A prodrug approach toward cancer-related carbonic anhydrase inhibition. Journal of medicinal chemistry\| PubMed ID: 24200125	2013
Saccharin: a lead compound for structure-based drug design of carbonic anhydrase IX inhibitors. Bioorganic & medicinal chemistry\| PubMed ID: 25614109	2015
Identification of vitamin C transporters in the human airways: a cross-sectional in vivo study. BMJ open\| PubMed ID: 25854967	2015
Mapping Selective Inhibition of the Cancer-Related Carbonic Anhydrase IX Using Structure-Activity Relationships of Glucosyl-Based Sulfamates. Journal of medicinal chemistry\| PubMed ID: 26203869	2015
Phosphate Chemical Probes Designed for Location Specific Inhibition of Intracellular Carbonic Anhydrases. Journal of medicinal chemistry\| PubMed ID: 26347950	2015
Acute exposure to wood smoke from incomplete combustion--indications of cytotoxicity. Particle and fibre toxicology\| PubMed ID: 26511835	2015
Dendritic Cells and Monocytes with Distinct Inflammatory Responses Reside in Lung Mucosa of Healthy Humans. Journal of immunology (Baltimore, Md. : 1950)\| PubMed ID: 27183618	2016
P-glycoprotein-mediated chemoresistance is reversed by carbonic anhydrase XII inhibitors. Oncotarget\| PubMed ID: 27811376	2016

Gregory Rankin

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