Clinical utility of diagnostic guidelines and putative biomarkers in lymphangioleiomyomatosis

Background

Lymphangioleiomyomatosis is a rare disease occurring almost exclusively in women. Diagnosis often requires surgical biopsy and the clinical course varies between patients with no predictors of progression. We evaluated recent diagnostic guidelines, clinical features and serum biomarkers as diagnostic and prognostic tools.

Methods

Serum vascular endothelial growth factor-D (VEGF-D), angiotensin converting enzyme (ACE), matrix metalloproteinases (MMP) -2 and -9, clinical phenotype, thoracic and abdominal computerised tomography, lung function and quality of life were examined in a cohort of 58 patients. 32 healthy female controls had serum biomarkers measured.

Results

Serum VEGF-D, ACE and total MMP-2 levels were elevated in patients. VEGF-D was the strongest discriminator between patients and controls (median = 1174 vs. 332 pg/ml p < 0.0001 with an area under the receiver operating characteristic curve of 0.967, 95% CI 0.93-1.01). Application of European Respiratory Society criteria allowed a definite diagnosis without biopsy in 69%. Adding VEGF-D measurement to ERS criteria further reduced the need for biopsy by 10%. VEGF-D was associated with lymphatic involvement (p = 0.017) but not the presence of angiomyolipomas.

Conclusions

Combining ERS criteria and serum VEGF-D reduces the need for lung biopsy in LAM. VEGF-D was associated with lymphatic disease but not lung function.     

Spectroscopic studies on the interaction of pradimicin BMY-28864 with mannose derivatives

Pradimicin BMY-28864 (Pm) is an antibiotic effective against yeasts and fungi, and is known to bind mannose in the presence of Ca2+. We examined spectroscopically the mode of interactions among Pm, Ca2+, and glycosides of mannose and mannose oligosaccharides (Manalpha1-OMe, Manalpha1-2Manalpha1-OMe, Manalpha1-3Manalpha1-OMe, Manalpha1- 4Manalpha1-OMe, Manalpha1-6Manalpha1-OMe, Manalpha1-6(Manalpha1- 3)Manalpha1-OMe, and Man9GlcNAc2-Asn, a high mannose type N-linked oligosaccharide). All the mannosides interacted with Pm in the presence of Ca2+ and caused absorbance changes. The absorbance changes occurred nonlinearly with respect to the carbohydrate concentration and do not follow a simple binding isotherm equation, suggesting a unique multistep interaction mode. The concentrations that induced half the maximum absorbance change were approximately 10 mM for the mono- and di- mannosides and around 1.5 mM for the trimannoside and Man9GlcNAc2-Asn. Methyl alpha-D-glucopyranoside, methyl alpha-D-galactopyranoside, lactose, and myo-inositol did not affect the absorbance of Pm up to 50 mM. Ca2+ alone also influenced the absorbance of Pm. The absorbance between 200 and 700 nm decreased hypochromically when Ca2+ was added. The concentration that gave half the maximum absorbance decrease caused by Ca2+was around 15 microM. Our results suggest that two Pm molecules bind one C a2+, and each Pm binds two mannosyl residues.      

Substructural specificity and polyvalent carbohydrate recognition by the Entamoeba histolytica and rat hepatic N- acetylgalactosamine/galactose lectins

Both the Entamoeba histolytica lectin, a virulence factor for the causative agent of amebiasis, and the mammalian hepatic lectin bind to N-acetylgalactosamine (GalNAc) and galactose (Gal) nonreducing termini on oligosaccharides, with preference for GalNAc. Polyvalent GalNAc- derivatized neoglycoproteins have >1000-fold enhanced binding affinity for both lectins (Adler,P., Wood,S.J., Lee,Y.C., Lee,R.T., Petri,W.A.,Jr. and Schnaar,R.L.,1995, J. Biol. Chem ., 270, 5164-5171). Substructural specificity studies revealed that the 3-OH and 4-OH groups of GalNAc were required for binding to both lectins, whereas only the E.histolytica lectin required the 6-OH group. Whereas GalNAc binds with 4-fold lower affinity to the E.histolytica lectin than to the mammalian hepatic lectin, galactosamine and N-benzoyl galactosamine bind with higher affinity to the E. histolytica lectin. Therefore, a synthetic scheme for converting polyamine carriers to poly-N-acyl galactosamine derivatives (linked through the galactosamine primary amino group) was developed to test whether such ligands would bind the E.histolytica lectin with high specificity and high affinity. Contrary to expectations, polyvalent derivatives including GalN6lys5, GalN4desmosine, GalN4StarburstTMdendrimer, and GalN8StarburstTMdendrimer demonstrated highly enhanced binding to the mammalian hepatic lectin but little or no enhancement of binding to the E.histolytica lectin. We propose that the mammalian hepatic lectin binds with greatest affinity to GalNAc "miniclusters," which mimic branched termini of N-linked oligosaccharides, whereas the E.histolytica lectin binds most effectively to "maxiclusters," which may mimic more widely spaced GalNAc residues on intestinal mucins.      

2-acetylaminofluorene up-regulates rat mdr1b expression through generating reactive oxygen species that activate NF-kappa B pathway

Overexpression of multidrug resistance genes and their encoded P-glycoproteins is a major mechanism for the development of multidrug resistance in cancer cells. The hepatocarcinogen 2-acetylaminofluorene (2-AAF) efficiently activates rat mdr1b expression. However, the underlying mechanisms are largely unknown. In this study, we demonstrated that a NF-kappa B site on the mdr1b promoter was required for this induction. Overexpression of antisense p65 and I kappa B alpha partially abolished the induction. We then delineated the pathway through which 2-AAF activates NF-kappa B. 2-AAF treatment led to the increase of intracellular reactive oxygen species (ROS) which causes activation of IKK kinases, degradation of I kappa B beta (but not I kappa B alpha), and increase in NF-kappa B DNA binding activity. Consistent with the idea that ROS may participate in mdr1b regulation, antioxidant N-acetylcysteine inhibited the induction of mdr1b by 2-AAF. Overproduction of a physiological antioxidant glutathione (GSH) blocked the activation of IKK kinase complex and NF-kappa B DNA binding. Based on these results, we conclude that 2-AAF up-regulates mdr1b through the generation of ROS, activation of IKK kinase, degradation of I kappa B beta, and subsequent activation of NF-kappa B. This is the first report that reveals the specific cis-elements and signaling pathway responsible for the induction of mdr1b by the chemical carcinogen 2-AAF.     

mRNA-dependent synthesis of rat apolipoprotein E in vitro: Cotranslational processing and identification of an endoglycosidase H-sensitive glycopeptide intermediate

Total polyadenylated enriched mRNA was prepared from rat liver by guanidine-HCl extraction and oligo(dT)-cellulose chromatography. It was translated $\text{in vitro}$ in an mRNA-dependent wheat germ system and rabbit reticulocyte lysate system, using radiolabeled leucine or methionine as amino acid precursor. A product, designated preapoE, was specifically precipitated by a rabbit anti-rat apoE serum and accounted for 1.5% of the total radioactive peptides. It migrated as a single band of radioactivity on SDS gels with an apparent molecular weight similar to that of mature plasma apoE. Inclusion of dog pancreatic microsomal membranes in the translation reaction resulted in a slightly smaller product (by 500 daltons). It also converted the preapoE from an endoglycosidase H-resistant to an enzyme-sensitive species. This suggests that processing of preapoE takes place by the cotranslational removal of a signal peptide and core glycosylation of the mature protein.