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.

Interindividual variability in L1CAM expression in the human kidney during development: are there implications for fetal programming of kidney diseases presenting in adulthood?

OBJECTIVE: L1 cell adhesion molecule (L1CAM) is a member of the immunoglobulin superfamily of cell adhesion molecules. The present study investigated the expression of L1CAM during the development in the fetal human kidney at different gestational ages, to reach a better knowledge on the role of L1CAM in renal morphogenesis. MATERIALS AND METHODS: The immunohistochemical analysis for L1CAM was performed in 24 fetal kidneys of different gestational ages, ranging from 10 to 38 weeks. L1CAM expression was observed in all 24 kidneys examined. RESULTS: Immunoreactivity for L1CAM was restricted to the collecting tubules, of the developing fetal kidneys. Moreover, L1CAM was detected in the ureteric bud tips, near the subcapsular metanephric mesenchymal stem/progenitor cells. L1CAM was also expressed in the collecting tubules undergoing fusion with the distal tubules of the developing nephrons. L1CAM was mainly expressed along the cell membrane. In fetal kidneys in which the renal pelvis was observed, epithelial cells of the renal pelvis showed strong membranous reactivity for L1CAM. CONCLUSIONS: Our study shows that L1CAM is expressed in all stages of human kidney nephrogenesis, being restricted to the renal structures derived from the ureteric bud. The expression of L1CAM in the cells of the ureteric bud tips suggests a major role for this adhesion molecule in the induction of metanephric mesenchymal cells to undergo mesenchymal-to-epithelial transition and differentiation into new nephrons. The interindividual variability in L1CAM expression observed in this study might be related to different levels of nephrogenesis, suggesting L1CAM involvement in the fetal programming of adult kidney diseases.

The role of fetal programming in human carcinogenesis - May the Barker hypothesis explain interindividual variability in susceptibility to cancer insurgence and progression?

The growing incidence of cancers is pushing oncologists to find out new explanations other than the somatic mutation theory, based on the accumulation of DNA mutations. In particular, the embryo-fetal exposure to an increasing number of environmental factors during gestation might represent a trigger able to influence the susceptibility of the newborn to develop cancer later in life. This idea agrees with the fetal programming theory, also known as the Barker hypothesis. Here the role of insulin-like growth factors, thymosin beta-4, and epigenome are discussed as mediators of cancer in prenatal human development. The role of epigenetic factors that during gestation increase the predisposition to develop cancer and the similarities in the gene expression (like MMP9, OPN, TP53 and CDKN2A) between embryonic development and cancer are key factors. Likewise, maternal obesity might be able to re-program embryo-fetal development with long-term changes, including an increased risk to develop neuroblastoma and acute leukemia. Birth weight alone and birth weight corrected for gestational age are proposed as important variables capable of predicting the vulnerability to develop cancers. According to the findings here reported, we hypothesize that cancer prevention should start during gestation by improving the quality of maternal diet. In conclusion, the Barker hypothesis should be applied to cancer as well. Therefore, the identification of the epigenetic factors of cancer appears mandatory, so that the cancer prevention might start in the womb before birth.

Strong ACE-2 expression in the choroidal vessels: do high choroid plexuses serve as a gateway for SARS-CoV-2 infection on the human brain?

OBJECTIVE: Previous studies have confirmed the key mechanism by which SARS-CoV-2 enters human cells. It is well established that ACE2 is the receptor that can mark the beginning of the infection. In light of this, the organs that express higher levels of ACE2 are generally considered at higher risk, while those with lower levels should be somehow more protected. This - if related to the scarcity of ace2-expressing cells in the brain - seems to contrast with the presence of a variety of neurological symptoms that follow infection with ace2.  The aim of this work was to analyze ACE2 expression in the human brain, focusing on the choroid plexuses. PATIENTS AND METHODS: Twenty brain samples were obtained at autopsy from ten human fetuses and from ten adult subjects. All samples were selected to contain the choroid plexus. Specimens were fixed in 10% formalin, routinely processed and paraffin embedded. 5-micron sections were stained with Hematoxylin and Eosin (H&E) and immunostained with a commercial anti-human ACE2 rabbit monoclonal antibody at 1:100 dilution. RESULTS: We analyzed 20 samples by immunohistochemistry, and we noted that, as far as fetal samples are concerned, a strong reactivity for ACE2 was detected in the myxoid stroma of the choroid plexuses and in the endothelial cells in fetuses. The complete absence of the ACE2 marker was detected in epithelial cells, neurons and glial cells of the cerebral cortex, both in fetuses and in adults. Whereas a  strong but selective reactivity for ACE2 was also detected in adult choroid plexuses, mainly localized in the endothelial cells of the choroid capillaries. CONCLUSIONS: Our study shows a strong expression of ACE in the fetal and adult brain choroid plexuses. This new histopathological finding may clarify the susceptibility of the human brain to SARS-COV-2 infection. Our data indicate the choroid plexus as the entry gate of virus for in the human brain; therefore, the entrance of SARS-CoV-2 into the cerebrospinal fluid through the choroid plexuses might represent the mechanism utilized by this coronavirus to cause direct injury to brain cells.

Thymosin ß4 and ß10 are highly expressed at the deep infiltrative margins of colorectal cancer - A mass spectrometry analysis.

OBJECTIVE: Colorectal cancer (CRC) is a complicated tumor, involving several oncogenic signaling pathways, and with a molecular mechanism not fully understood yet. The implication of thymosin ß4 (Tß4) with tumor insurgence and in migration of CRC cells was evidenced in the past with different methodologies, while Tß10 connection with CRC has been sporadically investigated. This study focused on the implication of both types of thymosin in CRC progression and invasion by analyzing the changes in their levels according to different zones of the tumor, and to Dukes stage and budding index. PATIENTS AND METHODS: Tß4 and Tß10 were analyzed in deep and superficial tumor samples, and normal mucosa from 18 patients. Concentrations of Tß4 and Tß10 have been measured by high-pressure liquid chromatography (HPLC) coupled to electrospray-ion trap mass spectrometry (ESI-IT-MS). MS data were compared by t-test and ANOVA statistical analysis. Identification of thymosin and their proteoforms has been performed by HPLC-high resolution-ESI-IT-MSMS. RESULTS: Both Tß4 and Tß10, exhibited intra-tumoral quantitative differences, being upregulated in the deep part of the CRC. They exhibited, moreover, strong association with the Dukes stage and the budding grade, being more concentrated in patients at Dukes stage B and with budding index "2". CONCLUSIONS: The results obtained in the present investigation encouraged the hypothesis that the two thymosin are involved in colorectal cancer progression, and in promoting cancer invasion. Thus, they are good candidates to be diagnostic/prognostic biomarkers and therapy targets.

Fetal programming of COVID-19: may the barker hypothesis explain the susceptibility of a subset of young adults to develop severe disease?

The risk stratification of young adults between subjects who will develop a mild form COVID-19 and subjects who will undergo a severe disease remains inaccurate. In this review, we propose that the Barker hypothesis might explain the increased susceptibility to severe forms of COVID-19 in subjects who underwent intrauterine growth restriction (IUGR). In this paper evidence indicating an association between a low birth weight and an adult phenotype which might favor a severe outcome of SARS-CoV-2 infection are presented: lower lung functional capacity; increased respiratory morbidity; changes in fibrinogen and Factor VII serum levels and dysregulation of the hemostasis and thrombosis system; acquisition of a pro-thrombotic phenotype; low nephron number, with decreased ability to sustain renal function and increased renal morbidity; heart remodeling, with a less efficient cardiac function; endothelial dysfunction, a risk factor for the insurgence of the multiple organ failure; remodeling of arteries, with changes in the elastic properties of the arterial wall, predisposing to the insurgence and progression of atherosclerosis; dysfunction of the innate immune system, a risk factor for immune diseases in adulthood. These data suggest that young and adult subjects born too small (IUGR) or too early (pre-terms) might represent a subgroup of "at risk subjects", more susceptible toward severe forms of COVID-19. Given that LBW may be considered a surrogate of IUGR, this phenotypic marker should be included among the indispensable clinical data collected in every patient presenting with SARS-COV-2 infection, irrespectively of his/her age.

Thymosin beta-4 prenatal administration improves fetal development and halts side effects due to preterm delivery.

OBJECTIVE: Thymosin beta 4 (TB4) is the most abundant member of the beta-thymosin family in humans. The main physiological role of TB4 is the regulation of actin polymerization. TB4 is also involved in angiogenesis, cell survival, cell migration and fetal development. The aim of this study was to evaluate the activity of TB4 as a fetal growth promoter when administered during pregnancy. MATERIALS AND METHODS: Our protocols have been carried out in full conformity with the rules and guidelines expected for this kind of trial. 10 pregnant mice received the same injection regimen. Only 6 of these 10 are part of this experiment because they were pregnant. At 10:00 a.m. on day E14 and E17 of gestation mice were weighed and treated with an intraperitoneal injection of TB4 (Regene RX, Rockville, MD, USA; 6 mg/kg in PBS). RESULTS: The mothers treated with TB4 for two days precisely E14 and E17, showed a higher cranio-caudal length when compared to control newborns. At histology, maternal TB4 treatment was associated with more advanced development of lungs, heart, kidney, cerebral cortex and notochord. CONCLUSIONS: Our study shows that TB4 administration during gestation may act as a powerful fetal growth promoter, by accelerating the development of newborn organs and tissues.

Identification of two new repetitive elements and chromosomal mapping of repetitive DNA sequences in the fish Gymnothorax unicolor (Anguilliformes: Muraenidae).

Muraenidae is a species-rich family, with relationships among genera and species and taxonomy that have not been completely clarified. Few cytogenetic studies have been conducted on this family, and all of them showed the same diploid chromosome number (2n=42) but with conspicuous karyotypic variation among species. The Mediterranean moray eel Gymnothorax unicolor was previously cytogenetically studied using classical techniques that allowed the characterization of its karyotype structure and the constitutive heterochromatin and argyrophilic nucleolar organizer regions (Ag-NORs) distribution pattern. In the present study, we describe two new repetitive elements (called GuMboI and GuDdeI) obtained from restricted genomic DNA of G. unicolor that were characterized by Southern blot and physically localized by in situ hybridization on metaphase chromosomes. As they are highly repetitive DNA sequences, they map in heterochromatic regions. However, while GuDdeI was localized in the centromeric regions, the GuMboI fraction was distributed on some centromeres and was co-localized with the nucleolus organizer region (NOR). Comparative analysis with other Mediterranean species such as Muraena helena pointed out that these DNA fractions are species-specific and could potentially be used for species discrimination. As a new contribution to the karyotype of this species, we found that the major ribosomal genes are localized on acrocentric chromosome 9 and that the telomeres of each chromosome are composed of a tandem repeat derived from a poly-TTAGGG DNA sequence, as it occurs in most vertebrate species. The results obtained add new information useful in comparative genomics at the chromosomal level and contribute to the cytogenetic knowledge regarding this fish family, which has not been extensively studied.

Some remarks on the use of TaqI to detect highly repetitive DNA sequences in human chromosomes.

In the attempt to conclude investigation of the action of restriction endonucleases on eukaryote chromosomes, we carried out a series of experiments digesting in situ human metaphase chromosomes with AluI/TaqI followed by Giemsa staining. We focused on the centromeric regions of chromosomes1, 2 and 16 and noted that those areas appeared as intensely stained blocks after AluI digestion, but were dramatically reduced in size or completely destroyed after subsequent TaqI treatment. These results permitted us to draw some conclusions on the highly repetitive DNA composition of these regions, in terms of alphoid and classical satellite DNAs.

The organization of classical satellite DNAs in human chromosomes: an approach using AluI and TaqI restriction endonucleases.

Human classical satellite DNAs were used as probes to investigate the molecular mechanism(s) of AluI/TaqI attack in situ on specific centromeric areas. The biochemical results obtained show that the majority of such highly repetitive DNAs are not solubilized from chromosomes, in spite of a cleavage pattern identical to that shown in naked genomic DNA digested with the same enzymes. Moreover, when digestion in situ with restriction enzymes precedes in situ hybridization, it is possible to observe an increased signal in the centromeres of some chromosomes as compared to that shown in standard undigested chromosomes and, on the other hand, hybridization labelling in centromeres which are difficult to detect by in situ hybridization using standard undigested chromosomes. Lastly, our results show that centromeric heterochromatin is not a homogeneous class in regard to organizational structure.

The impact of StuI digestion in situ on FISH to human chromosomes with satellite DNA probes.

Human metaphase chromosomes were digested with StuI and subsequently hybridized in situ using chromosome 9 alphoid DNA and classical satellite III DNA as probes. The data obtained suggest that it is not possible to establish a general rule regarding the cytological effects induced by restriction enzymes in particular chromosome regions and that a number of factors, such as DNA sequences, DNA-protein interaction and enzyme structure, play a role in determining such effects.

The efficiency of in-situ hybridization on human chromosomes with alphoid DNAs is enhanced by previous digestion with AluI and TaqI.

Centromeric alphoid DNAs of human chromosomes 6, 9, 16 and Y were employed to obtain information on the molecular mechanism(s) determining cytological effects produced by digestion in situ with AluI and TaqI restriction enzymes, possibly related to the structure of the above-cited areas. The following cytological and biochemical experiments were carried out using the above-mentioned alphoid sequences as probes: (1) standard in-situ hybridization and in-situ hybridization after chromosome cleavage with AluI/TaqI, and (2) filter hybridization on the DNA fractions obtained from the material solubilized and that retained on the slides after digestion in situ with AluI/TaqI. Biochemical data show that cleavage of alphoid DNAs is not prevented by the peculiar organization of centromeric heterochromatin, but such cleavage is not necessarily followed by complete DNA solubilization. The analysis of alphoid sequence cleavage in naked genomic DNA as well as during digestion of fixed chromosomes shows that (1) AluI cuts more efficiently than TaqI, (2) DNA fragments as large as 3-5 kb can be solubilized, and (3) DNA fragments of the same size are found in both fractions of DNA, i.e. that retained on the chromosomes as well as that solubilized from chromosomes. Cytological data show that previous chromosome digestion, mostly with TaqI, increases the hybridization signal area, suggesting that this fact might be due to (1) chromatin reorganization produced by enzyme attack and/or (2) the presence of alphoid DNAs which might be restricted not only to the kinetochore area but also to para/peri-centromeric heterochromatin. Lastly, centromere DNA solubilization as a consequence of restriction enzyme cleavage seems to vary from chromosome to chromosome, thus suggesting that centromeric regions do not represent a homogeneous class of constitutive heterochromatin.

The molecular characterization of the genome of Muraena helena L. I. Isolation and hybridization of two MboI-restricted DNA fractions.

To investigate the genome of the anguilliform fish Muraena helena at the molecular level we characterized total DNA by agarose gel electrophoresis after cleavage with AluI, HaeIII, MboI, and DdeI restriction endonucleases. Subsequently, we isolated the DNA from two specific electrophoretic fractions to be used as probes for Southern and in situ hybridization experiments. One such fraction showed an electrophoretic pattern typical of highly repetitive DNA localized in the centromeres of many chromosomes. The other fraction was shown to be located in the nucleolar organizer region, partially coincident with 45S rDNA, and to be composed of highly repetitive sequences.

Sister chromatid differentiation in 5-bromo-2'-deoxyuridine-substituted chromosomes: a study with DNA-specific ligands and monoclonal antibody to histone H2B.

Human and Chinese hamster chromosomes were obtained from cells grown in the presence of 5-bromodeoxyuridine (BrdU) for (a) one replicative round, (b) two replicative rounds, (c) one replicative round followed by another round in thymidine and (d) the last period of synthetic phase. Untreated chromosomes and chromosomes treated with UV radiation after previous staining with 33258 Hoechst as photosensitizer were studied in order to investigate the mechanism(s) responsible for BrdU-induced sister chromatid differentiation (SCD). Metaphases were prepared by (1) standard methanol-acetic acid fixative for subsequent investigation with Giemsa or DNA-specific dyes such as ethidium bromide, acridine orange and monoclonal antibodies to double- or single-stranded DNA; (2) the procedure for observation under phase-contrast or electron microscopy; and (3) the cytospin method for subsequent immunoreaction with a monoclonal antibody to histone H2B. Our data exclude the possibility that the presence/absence of BrdU in the template strand might affect chromatin organization and thus resistance, while confirming that UV-induced DNA alteration is not sufficient, by itself, to explain SCD mechanism(s). That molecules other than DNA play a role in explaining SCD production is indicated by the fact that BrdU incorporation induces alterations in DNA-histone H2B interactions which, in turn, seem to produce structural variations in chromatin, possibly at the level of condensation, as monitored by phase-contrast and electron microscopy.

Studies on the kinetics of expression of cell cycle dependent proto-oncogenes during mitogen-induced liver cell proliferation.

The present study was undertaken to determine the kinetics of DNA synthesis and expression of cell cycle dependent proto-oncogenes in response to two types of cell proliferative stimuli in male Wistar rat liver. The peak of DNA synthesis was approximately 24 h after a compensatory cell proliferative stimulus induced by 2/3 partial hepatectomy and approximately 36 h following a mitogenic stimulus obtained with a single dose of lead nitrate (10 micromol/100 g body wt, through femoral vein). Even though both proliferative stimuli induced the expression of c-fos, c-myc and c-Ha-ras, the extent of the increase in c-fos expression was 4- to 5-fold less in mitogen-induced cell proliferation. In addition, while the expression of c-myc, following partial hepatectomy returned to basal level by 4 h, the induced expression of c-myc persisted for up to 40 h during the lead nitrate-induced liver cell proliferation.