Human breast milk cells are positive for the pioneer transcription factor ISL1.

OBJECTIVE: ISL1 is a pioneer transcription factor that plays important roles in cell lineage specification and differentiation, by programming the epigenome and recruiting additional regulatory factors. The aim of this study is to determine whether the human breastmilk contains ISL1-positive stem cells, and, if so, to describe the subcellular localization of ISL1. MATERIALS AND METHODS: Breast milk was obtained from fourteen healthy females during the first 2-6 months of lactation. Cell morphology was examined in the breast milk with the automatic ThinPrep® processor (Hologic® Inc.) in commercial Cytological ThinPrep® solution (Hologic® Inc.), followed by standard immunohistochemical staining of ISL1. RESULTS: ISL1 had a granular diffuse cytoplasmic localization, with varying intensity of staining in both single and grouped cells. Nuclear staining was also present, as was staining of intracellular and extracellular vesicles with ISL1 antibody. CONCLUSIONS: These preliminary results suggest that ISL1 could distinguish a readily available source of putative stem cells in human breast milk. These stem cells may complete the network created between the mother and the newborn during gestation, thereby improving the efficiency of programming and reprogramming postnatal events.

CD44 is highly expressed in stem/progenitor cells originating the intervertebral discs in the human notochord.

OBJECTIVE: The notochord acts as a patterning structure, playing a key role in the formation of the vertebral column, both indirectly by inducing sclerotome cell differentiation and directly by forming the nucleus pulposus of intervertebral discs. The abnormal development of the notochord results in an easy equation with a variety of birth defects. Therefore, we focused our attention on the analysis of the early stages of human notochord development by highlighting the role of progenitor stem cells involved in the origin of intervertebral discs (IVDs). MATERIALS AND METHODS: Eight human fetuses, ranging from 8 up to 21 weeks of gestational age, were obtained from spontaneous abortion or voluntary interruption of gestation. Samples were 10% formalin-fixed, routinely processed, and paraffin-embedded. Five micron-tick paraffin sections were obtained from each sample. Sections were stained with hematoxylin-eosin and PAS stain for a morphological examination. Tissue samples were immunostained with a commercial anti-human CD44 rabbit monoclonal antibody at 1:100 dilution. RESULTS: Immunoreactivity for CD44 was detected in six out of eight notochords examined in this study. Reactivity for CD44 was restricted to progenitor cells giving rise to the nucleus pulposus (NP) of the developing IVDs. Positive cells showed a membranous and/or cytoplasmic immunostaining, no reactivity was observed in the nuclear compartment. CD44 expression was always restricted to IVD precursor cells, whereas cartilage precursors were devoid of labelling. CONCLUSIONS: Our study shows, for the first time, that the stem cell marker CD44 selectively marks intervertebral disc progenitor cells, paralleling their differentiation toward a discogenic phenotype. Therefore, our results suggest that CD44 plays a key role in IVD development, allowing its differentiation from surrounding undifferentiated notochordal cells toward a IVD phenotype. Given the role of CD44 in IVD development, we may hypothesize that low CD44 levels might be associated with changes in IVD development and with susceptibility to develop back pain later in life.

Expression of L1 Cell Adhesion Molecule (L1CAM) in extracellular vesicles in the human spinal cord during development.

OBJECTIVE: L1  cell adhesion molecule (L1CAM) is a glycoprotein characterized by three components: an extracellular region, a transmembrane segment, and a cytoplasmic tail. L1CAM is expressed in multiple human cells, including neurons. The neural cell adhesion molecule L1 has been implicated in a variety of neurologic processes, including neuritogenesis and cerebellar cell migration. The presence of L1CAM on the surface of nerve cells allows the adhesion of neurons among them. Furthermore, when it is bound to itself or to other proteins, L1-CAM induces signals inside the cell. The aim of this work was to study L1CAM expression in the human spinal cord during development, at different gestational ages, through immunohistochemistry. MATERIALS AND METHODS: Immunohistochemical analysis for L1CAM was performed in five human spinal cord samples, including three embryos and two fetuses of different gestational ages, ranging from 8 to 12 weeks. RESULTS: L1CAM expression was detected in all 5 spinal cords examined in this study. The adhesion molecule was found in the vast majority of cells. The highest levels of immunoreactivity for L1CAM were detected at the periphery of the developing organs, in the spinal cord zones occupied by sensory and motor fibers. In the alar and basal columns, immunoreactivity for L1CAM was characterized by a reticular pattern, being mainly expressed in axons. Strong reactivity of L1CAM was also found in extracellular vesicles. This extracellular localization might indicate the ability of L1CAM to mediate the transduction of extracellular signals that support axon outgrowth. CONCLUSIONS: The high reactivity of L1cam in the axons of developing neurons in the fetal spinal cord confirms previous studies on the ability of L1CAM to promote axon sprouting and branching in the developing nervous system. In this work, a new actor is reported to have a role in the complex field of human spinal cord development: L1CAM, whose expression is highly found in the developing neuronal and glial precursors.

Dissociation between schizophrenia-relevant behavioral profiles and volumetric brain measures after long-lasting social isolation in Roman rats.

Social isolation rearing of rodents is an environmental manipulation known to induce or potentiate psychotic-like symptoms and attentional and cognitive impairments relevant for schizophrenia. When subjected to a 28-week isolation rearing treatment, the Roman high-avoidance (RHA-I) rats display the common behavioral social isolation syndrome, with prepulse inhibition (PPI) deficits, hyperactivity, increased anxiety responses and learning/memory impairments when compared to their low-avoidance (RLA-I) counterparts. These results add face validity to the RHA-I rats as an animal model for schizophrenia-relevant behavioral and cognitive profiles and confirm previous results. The aim here was to further investigate the neuroanatomical effects of the isolation rearing, estimated through volume differences in medial prefrontal cortex (mPFC), dorsal striatum (dSt) and hippocampus (HPC). Results showed a global increase in volume in the mPFC in the isolated rats of both strains, as well as strain effects (RLA > RHA) in the three brain regions. These unexpected but robust results, might have unveiled some kind of compensatory mechanisms due to the particularly long-lasting isolation rearing period, much longer than those commonly used in the literature (which usually range from 4 to 12 weeks).

Immunocytochemical localization of MG1 mucin in human bulbourethral glands.

Salivary mucins MG1 and MG2 have been found in the oral cavity where they perform several functions such as the formation of the mucous layer covering the oral mucosa and teeth. Recent studies have demonstrated their presence in other organs and tissues. The aim of this study was to determine their expression in human bulbourethral (Cowper's) glands. Normal bulbourethral glands were obtained at surgery and fixed in a mixture of 1% paraformaldehyde-1.25% glutaraldehyde in 0.1 M cacodylate buffer and embedded in Epon resin. Thin sections were labeled with rabbit antibodies to MG1 or to an N-terminal synthetic peptide of MG2, followed by gold-labeled goat anti-rabbit IgG. The granules of all mucous cells were intensely reactive with anti-MG1, whereas no labeling was detected for MG2. These results indicate that MG1 is not exclusively a salivary component and furthermore show that bulbourethral glands represent a significant source of the MG1 detected in human seminal plasma.

Localization of MTT formazan in lipid droplets. An alternative hypothesis about the nature of formazan granules and aggregates.

MTT (3-(4, 5-dimethyl-2-thiazolyl)-2, 5-dihphenyltetrazolium bromide) assay is a widely used method to assess cell viability and proliferation. MTT is readily taken up by cells and enzymatically reduced to formazan, a dark compound which accumulates in cytoplasmic granules. Formazan is later eliminated by the cell by a mechanisms often indicated as exocytosis, that produces characteristic needle-like aggregates on the cell surface. The shape of formazan aggregates and the rate of exocytosis change in the presence of bioactive amyloid beta peptides (Abeta) and cholesterol. Though the cellular mechanisms involved in MTT reduction have been extensively investigated, the exact nature of formazan granules and the process of exocytosis are still obscure. Using Nile Red, which stains differentially neutral and polar lipids, and a fluorescent analog of cholesterol (NBD-cholesterol), we found that formazan localized in lipid droplets, consistent with the lipophilic nature of formazan. However, formazan granules and aggregates were also found to form after killing cells with paraformaldehyde fixation. Moreover, formazan aggregates were also obtained in cell-free media, using ascorbic acid to reduce MTT. The density and shape of formazan aggregates obtained in cell-free media was sensitive to cholesterol and Abeta. In cells, electron microscopy failed to detect the presence of secretory vesicles, but revealed unusual fibers of 50 nm of diameter extending throughout the cytoplasm. Taken together, these findings suggest that formazan efflux is driven by physico-chemical interactions at molecular level without involving higher cytological mechanisms.

Expression of BDNF and trkB in the hippocampus of a rat genetic model of vulnerability (Roman low-avoidance) and resistance (Roman high-avoidance) to stress-induced depression.

INTRODUCTION: The selective breeding of Roman High- (RHA) and Low-Avoidance (RLA) rats for, respectively, rapid versus poor acquisition of the active avoidance response has generated two distinct phenotypes differing in many behavioral traits, including coping strategies to aversive conditions. Thus, RLA rats are considered as a genetic model of vulnerability to stress-induced depression whereas RHA rats are a model of resilience to that trait. Besides the monoamine hypothesis of depression, there is evidence that alterations in neuronal plasticity in the hippocampus and other brain areas are critically involved in the pathophysiology of mood disorders. MATERIALS AND METHODS: Western blot (WB) and immunohistochemistry were used to investigate the basal immunochemical occurrence of brain-derived neurotrophic factor (BDNF) and its high-affinity tyrosine-kinase receptor trkB in the dorsal and ventral hippocampus of adult RHA and RLA rats. RESULTS: WB analysis indicated that the optical density of BDNF- and trkB-positive bands in the dorsal hippocampus is, respectively, 48% and 25% lower in RLA versus RHA rats. Densitometric analysis of BDNF- and trkB-like immunoreactivity (LI) in brain sections showed that BDNF-LI is 24% to 34% lower in the different sectors of the Ammon's horn of RLA versus RHA rats, whereas line-related differences are observed in the dentate gyrus (DG) only in the ventral hippocampus. As for trkB-LI, significant differences are observed only in the dorsal hippocampus, where density is 23% lower in the DG of RLA versus RHA rats, while no differences across lines occur in the Ammon's horn. CONCLUSION: These findings support the hypothesis that a reduced BDNF/trkB signaling in the hippocampus of RLA versus RHA rats may contribute to their more pronounced vulnerability to stress-induced depression.

Cyclic AMP-receptor proteins in human salivary glands.

In mammalian species, cyclic AMP receptor proteins (cARP) are the regulatory (R) subunits of cyclic AMP-dependent protein kinase (PKA), the cellular effector of cyclic AMP-mediated signal transduction. An isoform of the PKA type II R subunit (RII), cARP, is a polyfunctional protein, present in most tissues and cells. It is expressed in salivary and other glands of rodents, and secreted into the saliva of rats and Man. The aim of the present study was to determine the expression of cARP in human salivary glands using immunoelectron microscopy. Thin sections of normal salivary glands embedded in LR Gold resin were labeled with anti-cARP primary antibody, then with gold-conjugated secondary antibody. Labeling was present in the secretory granules and cytoplasm of parotid, submandibular (SMG) and sublingual gland serous cells. Quantitative analysis showed considerable variability in granule labeling from sample to sample, indicating shifts in expression and cellular location of cARP. Unlike rodent salivary glands, the granules of intercalated and striated duct cells also were labeled. The cytoplasm and granules of mucous cells of the SMG and sublingual glands were unlabeled, while the Golgi complex and filamentous bodies in these cells showed moderate reactivity. Mitochondria and nuclei of both serous and mucous cells were unlabeled. Labeling also was present in the connective tissue adjacent to the epithelial cells. The results indicate that serous cells of the parotid and SMG are the major source of salivary cARP. They also reveal significant species differences in the glandular distribution of RII. RII binds to cytoskeletal and nuclear proteins, and may function to regulate extracellular cyclic AMP levels. Thus, the tissue and cellular distribution of RII may serve as an index of regulation of gene expression and cell differentiation.

Ultrastructural localization of epidermal growth factor receptor in human parotid gland.

The epidermal growth factor receptor (EGFR) is widely distributed in several organs in which, following interaction with its ligand, it can affect development and differentiation. The aim of this study was to define the distribution of EGFR in human parotid gland by means of a post-embedding immunogold staining method. Normal human parotid glands obtained at surgery were routinely prepared for electron microscopy. Semithin and ultrathin sections were treated for immunocytochemistry using a mouse monoclonal antibody specific for EGFR and a goat anti-mouse gold conjugated secondary antiserum. At the light microscope level, EGFR reactivity was revealed by a specific dark staining in both acinar and ductal cells. At the electron microscope level, EGFR was strongly stained in the cytoplasmic compartments and occasionally labeled on cell surfaces. In acinar cells, it appeared to be associated with small vesicles of uncertain nature that were scattered among the secretory granules. EGFR-positive vesicles were also observed in the ductal cells, with the most intense labeling being localized in striated ducts. Since cytoplasmic vesicles were previously found to be EGF-positive, these results may be due to the presence of the EGF-EGFR complex that is internalized after binding of EGF to the surface EGFR.

Localisation of epidermal growth factor receptor in mucous cells of human salivary glands.

EGFR activation has been related to an increase in synthesis and secretion of mucins in epithelial cells, so that the use of EGFR tyrosine kinase inhibitors has been proposed in the therapy of mucin hypersecretory diseases. In this paper, we describe the ultrastructural localisation of EGFR in the mucous elements of human major and minor salivary glands and relate it to mucin distribution. A post-embedding immunogold staining method has been applied to normal surgical samples of human submandibular, sublingual, and labial glands, using a mouse monoclonal antibody specific for the intracellular domain of human EGFR. In mucous cells of all the glands examined, specific reactivity was detected in the cytoplasmic basolateral portions and near the mucous droplets, but not on cell surfaces. Since this pattern of labelling must be related to the internalisation process of the ligand-GFR complex, our results support the hypothesis that EGFR activation takes place in mucous cells and affects mucin production in human salivary glands.

The coupling of RP-HPLC and ESI-MS in the study of small peptides and proteins secreted in vitro by human salivary glands that are soluble in acidic solution.

The aim of this study was the development of a method based on the coupling of RP-HPLC and ESI-MS for identifying and quantifying proteins and peptides secreted by human salivary glands in vitro. Salivary gland specimens, obtained from informed patients undergoing surgical resection, were incubated in an optimized medium. Incubation media of glandular specimens, selected on the basis of cytomorphological and ultrastructural analysis, were investigated by HPLC-MS. Several salivary peptides/proteins, previously recognized in human whole saliva, were searched for along the chromatogram by the selected ion monitoring (SIM) strategy. Analysis of the incubation media of parotid glands revealed the presence of basic PRPs PC, PD, PH, IB-1, II-2, and acidic PRP-1 and PRP-3 in all of the investigated samples. Basic PRPs PB and PA, acidic PRPs, and cystatins SN and S1 were detected in all of the incubation media of submandibular glands, whereas histatin 1 was detected in only one sample. Moreover, the method allowed detection of some post-translational derivatives of known salivary proteins, as well as of several previously unidentified small peptides. The present method represents a sensitive and powerful instrument to detect peptides and proteins secreted by human salivary glands in vitro.

Ultrastructural localization of glycodelin oligosaccharides Le-x and Le-y in human seminal vesicles by immunogold staining.

Histo-blood group antigens Le-x and Le-y are oligosaccharidic terminals that characterize many glycoproteins in the human tissues. In seminal plasma, they are expressed as part of the so-called glycodelin S, which is suggested to regulate sperm capacitation/decapacitation. It has recently been demonstrated that the core protein of glycodelin S is secreted by seminal vesicles. Here we show that epithelial cells of human seminal vesicles also release the Le-x and Le-y antigens. The presence of these substances in secretory material was revealed by means of an immunogold staining method in normal surgical samples. The results suggest that glycodelin S is secreted by seminal vesicles in its finished glycosylated form. Moreover, antigen reactivity was also revealed associated with plasma membranes.

Subcellular localization of epidermal growth factor receptor in human submandibular gland.

The subcellular distribution of the epidermal growth factor receptor (EGFr) was demonstrated in the normal human submandibular gland by means of immunogold cytochemistry. EGFr labelling appeared in both acinar and ductal cells, where strong immunoreactivity was associated with a tubulovesicular system near the basolateral surfaces. In addition, groups of reactive vesicles were highlighted among secretory granules of both serous and mucous cells and at the apex of ductal cells. Basolateral vesicles were interpreted as being a result of EGFr internalization after activation by an exogenous ligand, although the functional meaning of those located apically remains unclear.

Subcellular localization of epidermal growth factor in human parotid gland.

The intracellular distribution of epidermal growth factor was investigated in human parotid gland by immunogold cytochemistry at the electron-microscopy level. Epidermal growth factor immunoreactivity was demonstrated in both acini and ducts. In acinar cells, secretory granules appeared moderately stained, clearly indicating that parotid gland contributes to salivary epidermal growth factor through granule exocytosis. In ductal cells, gold particles were found to decorate numerous cytoplasmic vesicles, particularly abundant in striated duct cells. Since epidermal growth factor reactive vesicles were seen not only at the cellular apex, but nearby lateral plasma membranes as well, it leads to the hypothesis that epidermal growth factor may be discharged both apically into the saliva, and basally into the interstitium.

Subcellular localization of epidermal growth factor in human submandibular gland.

Epidermal growth factor in human submandibular gland was localized at the subcellular level by means of an immunogold staining method. Labelling was observed in serous acini and ducts. In the acini, gold particles were found within secretory granules, indicating that the growth factor is released into the saliva through granule exocytosis. In the ductal system, the most intense reactivity was revealed in the principal cells of striated ducts. In these cells, an abundant population of small cytoplasmic vesicles was specifically stained. Immunoreactive vesicles were found both apically and basally, suggesting that ductal cells can release their products not only into the saliva but also into the interstitium.