Breast cancer chemotherapy treatment monitoring based on serum sample Raman spectroscopy.

In this paper, breast cancer patients were monitored throughout their chemotherapy treatments (CHT), with blood serum sample Raman spectroscopy and multivariate analysis, approximately for a year. First of all, we discriminate between healthy and clinically diagnosed breast cancer patients. Breast cancer detection in terms of sensitivity and specificity were 87.14% and 90.55% respectively. Although no shifts of peaks in mean spectrum of samples from breast cancer patients were found with respect to the mean spectrum from control patients, some peaks did show clear differences in intensity, the greatest disparities found at 509, 545, 1063, 1103, 1338, 1556, 1083 and 1449 cm- 1 are associated with amino acids and phospholipid, 1246 and 1654 cm- 1, corresponding to amide III and I, respectively. Other peaks of interest encountered at 450, 661, 890, 917 and 1405 cm- 1 are associated to glutathione. Then, 6 breast cancer patients were monitored during their chemotherapy treatments, the results were in complete correspondence with their medical records, enabling a detailed study of the evolution of each patient's cancer. A special interest arose in the possible correlation between the intensity of Raman peak, 450 cm- 1, corresponding to glutathione and evolution of cancer throughout CHT, i.e., glutathione appears to be a good candidate as breast cancer biomarker. The results confirmed that Raman spectroscopy and PCA are, not only a good support to current breast cancer detection techniques, but could also be excellent techniques to monitor more efficiently breast cancer patients undergoing CHT, using blood serum samples which are a lot less invasive than other methods.

Type 2 diabetes detection based on serum sample Raman spectroscopy.

In this work, we propose to the Raman spectroscopy as a new technique for the detection of the type 2 diabetes using blood serum samples. The serum samples were obtained from 15 patients who were clinically diagnosed with type 2 diabetes mellitus and 20 healthy volunteers. The average spectra showed equally intense peaks as, 695 cm-1, the doublet of tyrosine at 828 and 853 cm-1, phenylalanine at 1002 and 1028 cm-1, the phospholipid shoulder at 1300-1345 cm-1, and proteins (amide I) at 1654 cm-1. The major differences were found at 661 and 1404 cm-1 (glutathione), 714 (polysaccharides), 605 (Phe), 545 cm-1 (tryptophan), and the shoulder of amide III at 1230-1282 cm-1, where seem to disappear in the diabetes spectrum. On the contrary, the region that is more highlighted due to that diabetes peaks are clearly more intense was 897-955 cm-1. Principal component analysis and linear discriminate analysis were employed for developing discrimination method. The first three principal components provided a classification of the samples from healthy and diabetes patients with high sensitivity and specificity. In addition, when the first principal component was plotted as a function of the Raman shift, it revealed these shifts accounted for the greatest differences between control and diabetes samples, which coincided with the shifts of spectral differences shown by mean spectra. Our results demonstrated that serum sample Raman spectroscopy promises to become a non-invasive support tool of the currently applied techniques for type 2 diabetes detection, decreasing the false-positive cases.

Acute phase lipocalin Ex-FABP is involved in heart development and cell survival.

Ex-FABP is an extracellular fatty acid binding protein, expressed during chicken embryo development in cartilage, muscle fibers, and blood granulocytes. Transfection of chondrocytes and myoblasts with anti-sense Ex-FABP cDNA results in inhibition of cell proliferation and apoptosis induction. Ex-FABP expression is dramatically enhanced by inflammatory stimuli and in pathological conditions. In this paper, by in situ whole mount and immunohistochemistry analysis we show that, at early developmental stage, Ex-FABP is diffuse in all tissues of chick embryos. Particularly high level of transcript and protein are expressed in the heart. During acute phase response (APR) induced by endotoxin LPS injection, a marked increase of Ex-FABP mRNA was observed in embryos, highest Ex-FABP expression being in heart and liver. To investigate in vivo the biological role of Ex-FABP, we have directly microinjected chicken embryos with antibody against Ex-FABP. Almost 70% of chicken embryos died and the target tissue was the heart. We detected in heart of the treated embryos a significant increase of apoptotic cells and high level of fatty acids. We propose that the accumulation of fatty acid, specific ligand of Ex-FABP, in the cell microenvironment is responsible of heart cell death, and we suggest that Ex-FABP may act as a survival protein by playing a role as scavenger for fatty acids.

Pilot study on lipocalin expression into extracellular fluids of women in fertile age.

BACKGROUND: To verify the expression of neutrophil gelatinase associated lipocalin (NGAL), molecule now arising great interest because of its proposed involvement in cell-cycle regulation, acute phase response and immunomodulation, into extracellular fluids of female reproductive tract, in order to provide useful data to understand its biological functions. The data collected are purely qualitative, just meant to reveal the presence of lipocalin into the assayed fluids, and they have to be considered as preliminary for a quantitative study (in progress at the moment) based on a double antibody radioimmunoassay. METHODS: Three kinds of extracellular fluid were randomly sampled: amniotic fluid (13 samples), cervical mucus (10 samples), coloster (20 samples). The inclusion criteria concerning the selection of the donor women were: age (fertile period), healthy state and pregnancy state. All the samples underwent protein assay, electrophoresis and western blot. RESULTS: All the samples examined revealed NGAL's presence. CONCLUSIONS: The positive results of this study seem to strengthen the hypothesis related to NGAL biological functions, specifically the ones that suggest its role into cell differentiation, embryonic development and inflammation. Therefore the female reproductive tract is suggested as a new promising study object in this research field.

Extracellular fatty acid binding protein (ex-FABP) is a stress protein expressed during chondrocyte and myoblast differentiation.

OBJECTIVE: We have isolated and characterized in our laboratory a lipocalin specifically binding unsaturated long chain fatty acids (Ex-FABP). In developing chicken embryo long bones, Ex-FABP first appears at the boundary of the cone of hypertrophic cartilage. 'In vitro' EX-FABP is highly expressed by differentiating hypertrophic chondrocytes. Ex-FABP is expressed also in the forming myotubes both 'in vivo' and 'in vitro'. In cultured chondrocytes, Ex-FABP expression is strongly induced by treatment with inflammatory agents such as the bacterial liposaccharide LPS or interleukin-6. The possible mechanism for this induction was investigated. Expression of Ex-FABP was studied in other stress conditions. DESIGN: To investigate a possible mechanism for Ex-FABP induction by LPS or interleukin-6, we have cultured the cells in the presence of either hydrogen peroxide or the NO donor SNAP (S-nitrosil-acetil-D, L-penicillamine), two agents known to produce cellular stresses through the activation of specific signalling pathways. To investigate Ex-FABP expression in other stress conditions, chondrocytes were cultured for 3 days in the presence of alpha,alpha-dipyridyl, an agent inhibiting prolyl hydroxylase activity and collagen secretion. Supplement of this agent to the culture medium results in an impairment of collagen secretion and assembly and the consequent altered interaction of the cell with the surrounding extracellular matrix. In addition Ex-FABP expression was studied also in chondrocytes cultured in the absence of serum, a stress condition activating cell defence mechanisms. RESULTS: We have excluded that induction of Ex-FABP expression by inflammatory agents is mediated by oxidative stress or NO production. Ex-FABP expression was induced also by changes in the hypertrophic chondrocyte microenvironment, considered either as extracellular matrix surrounding the cell in culture or as nature and concentration of growth factor in the culture medium. CONCLUSIONS: No definitive data are so far available on the possible role of Ex-FABP when induced by cellular stresses. The capacity of the protein to specifically bind and transport unsaturated long chain fatty acids suggests that lipid metabolism and fatty acid utilization by the cells may be involved. Based on literature data the NRL/N-GAL (neu-related lipocalin/neutrophil gelatinase-associated lipocalin) protein was proposed as a possible mammal counterpart of the chick Ex-FABP. We have suggested that Ex-FABP and NRL/NGAL expression in forming bones and muscles is part of a 'physiological' acute phase response. Interestingly the expression of Ex-FABP and NRL/NGAL is also activated in osteoarthritic cartilage and in the case of NRL/N-GAL during neoplastic transformation of chondrogenic lineage cells.

Avidin expression during chick chondrocyte and myoblast development in vitro and in vivo: regulation of cell proliferation.

Avidin is a major [35S]methionine-labeled protein induced by bacterial lipopolysaccharide (LPS) and interleukin 6 (IL-6) in cultured chick embryo myoblasts and chondrocytes. It was identified by N-terminal sequencing of the protein purified from conditioned culture medium of LPS-stimulated myoblasts. In addition, avidin was secreted by unstimulated myoblasts and chondrocytes during in vitro differentiation; maximal expression being observed in differentiated myofibers and hypertrophic chondrocytes. In developing chick embryos, immunohistochemistry revealed avidin in skeletal muscles and growth plate hypertrophic cartilage. Avidin was secreted into culture as a biologically active tetramer. Exogenous avidin added to the medium of proliferating chondrocytes progressively inhibited cell proliferation, whereas addition of avidin to differentiating chondrocytes in suspension allowed full cell differentiation. No toxic effects for the cells were observed in both culture conditions. Western blots of samples from cytosolic extracts using alkaline-phosphatase-conjugated streptavidin showed three biotin-containing proteins. Acetyl-CoA carboxylase was identified by specific antibodies. Based on these data, we propose that avidin binds extracellular biotin and regulates cell proliferation by interfering with fatty acid biosynthesis during terminal cell differentiation and/or in response to inflammatory stimuli.

Ex-FABP: a fatty acid binding lipocalin developmentally regulated in chicken endochondral bone formation and myogenesis.

Extracellular fatty acid binding protein (Ex-FABP) is a 21 kDa lipocalin specifically binding fatty acids, expressed during chicken embryo development in hypertrophic cartilage, in muscle fibers and in blood granulocytes. In chondrocyte and myoblast cultures Ex-FABP expression is increased by inflammatory agents and repressed by anti-inflammatory agents. In adult cartilage Ex-FABP is expressed only in pathological conditions such as in dyschondroplastic and osteoarthritic chickens. The possible mammalian counterpart is the Neu-related lipocalin (NRL), a lipocalin overexpressed in rat mammary cancer; NRL is homologous to the human neutrophil gelatinase associated lipocalin (NGAL) expressed in granulocytes and in epithelial cells in inflammation and malignancy and to the Sip24 (super-inducible protein 24), an acute phase lipocalin expressed in mouse after turpentine injection. Immunolocalization and in situ hybridization showed that NRL/NGAL is expressed in hypertrophic cartilage, in forming skeletal muscle fibers and in developing heart. In adult cartilage NRL/NGAL was expressed in articular cartilage from osteoarthritic patients and in chondrosarcoma. Moreover, NRL was induced in chondrocyte and myoblast cultures by an inflammatory agent. We propose that these lipocalins (Ex-FABP, NRL/NGAL, Sip24) represent stress proteins physiologically expressed in tissues where active remodeling is taking place during development and also present in tissues characterized by an acute phase response due to pathological conditions.

Multiple functions of Dlx genes.

Dlx genes comprise a highly conserved family of homeobox genes homologous to the distal-less (Dll) gene of Drosophila. They are thought to act as transcription factors. All Dlx genes are expressed in spatially and temporally restricted patterns in craniofacial primordia, basal telencephalon and diencephalon, and in distal regions of extending appendages, including the limb and the genital bud. Most of them are expressed during morphogenesis of sensory organs and during migration of neural crest cells and interneurons. In addition, Dlx5 and Dlx6 are expressed in differentiating osteoblasts. Gene targeting of Dlx1, Dlx2, Dlx3 and Dlx5 in the mouse germ-line has revealed functions in craniofacial patterning, sensory organ morphogenesis, osteogenesis and placental formation. However, no effect on limb development has yet been revealed from gene inactivation studies. A role for these genes in limb development is however suggested by the linkage of the Split Foot/Hand Malformation human syndrome to a region containing DLX5 and DLX6. As for most transcription factors, these genes seem to have multiple functions at different stages of development or in different tissues and cell types.

Extracellular fatty acid binding protein (Ex-FABP) modulation by inflammatory agents: "physiological" acute phase response in endochondral bone formation.

Ex-FABP, extracellular fatty acid binding protein, is a 21 kDa lipocalin expressed in hypertrophic cartilage, muscle and heart during chick embryo development and in granulocytes. Ex-FABP synthesis was increased in chondrocyte and myoblast cultures by inflammatory agents (LPS; IL6) and repressed by antiinflammatory agents. Expression of Ex-FABP and specific gelatinases is paralleled in hypertrophic cartilage; LPS specifically induced high molecular weight gelatinase ( > 200 kDa). LPS-treated hypertrophic chondrocytes showed increased chemotactic activity for endothelial cells paralleled by increased expression of transferrin. A high amount of Ex-FABP was expressed in adult pathological cartilage both in dyschondroplastic and osteoarthritic chickens. Controls were negative. Ex-FABP could represent a stress protein physiologically expressed in tissues where active remodelling is taking place during development and in tissues characterized by an acute phase response due to pathological conditions. We also suggest that during endochondral bone formation other responses characteristic of a local inflammatory status, such as gelatinase production and angiogenic factor secretion, are "physiologically" activated.

Expression of NRL/NGAL (neu-related lipocalin/neutrophil gelatinase-associated lipocalin) during mammalian embryonic development and in inflammation.

The neu-related lipocalin (NRL) is a protein overexpressed in rat mammary cancer induced by activated neu (HER-2/c-erbB2). This protein belongs to the family of the lipocalins or low molecular weight proteins able to bind and transport small hydrophobic molecules. The NRL homologue in mouse is SIP24, an acute phase protein induced in the animal by turpentine injection; the human homologous protein is NGAL expressed in granulocytes and epithelial cells in pathological conditions, such as inflammation and malignancy. We have investigated NRL expression in developing rat embryos. By immunolocalization we have shown localization of the protein in the hypertrophic region of growth plate cartilage. NRL was particularly enriched in prehypertrophic chondrocytes. In addition, we observed localization of the protein in forming skeletal muscle fibres and in the myocardium of developing heart. In agreement with the immunolocalization data, by in situ hybridization we have demonstrated the presence of the specific mRNA in the same tissues. At an early stage of differentiation, cultured rat embryo-derived chondrocytes did not express NRL; nevertheless expression of the protein was induced in these cells by treatment with an inflammatory agent, such as LPS. By Western blot analysis with specific antibodies we showed protein synthesis by cultured myoblasts also in the absence of LPS treatment, but only when forming myotubes were observed in culture. Stimulation of myoblast cultures with LPS resulted in an enhancement of the NRL expression in well formed myotubes. Our data suggest a role of NRL in cartilage and muscle differentiation. NRL expression was induced by inflammatory agents. We wish to propose that the expression of NRL in hypertrophic chondrocytes and forming myotubes is part of a "physiological" acute phase response occurring during cartilage and muscle development. In this manuscript we also report that NRL is not detectable by immunolocalization in adult cartilage (both articular and tracheal) from normal subjects. On the contrary articular cartilage from osteoarthritic patients was highly positive for the presence of NRL/NGAL. Interestingly the expression of this protein is also activated during neoplastic transformation of chondrogenic lineage cells.

Craniofacial, vestibular and bone defects in mice lacking the Distal-less-related gene Dlx5.

The Dlx5 gene encodes a Distal-less-related DNA-binding homeobox protein first expressed during early embryonic development in anterior regions of the mouse embryo. In later developmental stages, it appears in the branchial arches, the otic and olfactory placodes and their derivatives, in restricted brain regions, in all extending appendages and in all developing bones. We have created a null allele of the mouse Dlx5 gene by replacing exons I and II with the E. coli lacZ gene. Heterozygous mice appear normal. Beta-galactosidase activity in Dlx5+/- embryos and newborn animals reproduces the known pattern of expression of the gene. Homozygous mutants die shortly after birth with a swollen abdomen. They present a complex phenotype characterised by craniofacial abnormalities affecting derivatives of the first four branchial arches, severe malformations of the vestibular organ, a delayed ossification of the roof of the skull and abnormal osteogenesis. No obvious defect was observed in the patterning of limbs and other appendages. The defects observed in Dlx5-/- mutant animals suggest multiple and independent roles of this gene in the patterning of the branchial arches, in the morphogenesis of the vestibular organ and in osteoblast differentiation.

Parathyroid hormone [PTH(1-34)] and parathyroid hormone-related protein [PTHrP(1-34)] promote reversion of hypertrophic chondrocytes to a prehypertrophic proliferating phenotype and prevent terminal differentiation of osteoblast-like cells.

The effects of parathyroid hormone/parathyroid hormone-related protein (PTH/PTHrP) on late events in chondrocyte differentiation were investigated by a dual in vitro model where conditions of suspension versus adhesion culturing are permissive either for apoptosis or for the further differentiation of hypertrophic chondrocytes to osteoblast- like cells. Chick embryo hypertrophic chondrocytes maintained in suspension synthesized type II and type X collagen and organized their extracellular matrix, forming a tissue highly reminiscent of true cartilage, which eventually mineralized. The formation of mineralized cartilage was associated with the expression of alkaline phosphatase (ALP), arrest of cell growth, and apoptosis, as observed in growth plates in vivo. In this system, PTH/PTHrP was found to repress type X collagen synthesis, ALP expression, and cartilage matrix mineralization. Cell proliferation was resumed, whereas apoptosis was blocked. Hypertrophic chondrocytes cultured in adherent conditions in the presence of retinoic acid underwent further differentiation to osteoblast-like cells (i.e., they resumed cell proliferation, switched to type I collagen synthesis, and produced a mineralizing bone-like matrix). In this system, PTH addition to culture completely inhibited the expression of ALP and matrix mineralization, whereas cell proliferation and expression of type I collagen were not affected. These data indicate that PTH/PTHrP inhibit both the mineralization of a cartilage-like matrix and apoptosis (mimicked in the suspension culture) and the production of a mineralizing bone-like matrix, characterizing further differentiation of hypertrophic chondrocytes to osteoblasts like cells (mimicked in adhesion culture). Treatment of chondrocyte cultures with PTH/PTHrP reverts cultured cells in states of differentiation earlier than hypertrophic chondrocytes (suspension), or earlier than mineralizing osteoblast-like cells (adhesion). However, withdrawal of hormonal stimulation redirects cells toward their distinct, microenvironment-dependent, terminal differentiation and fate.