Recreating Human Skin In Vitro: Should the Microbiota Be Taken into Account?

Skin plays crucial roles in the human body: besides protecting the organism from external threats, it acts as a thermal regulator, is responsible for the sense of touch, hosts microbial communities (the skin microbiota) involved in preventing the invasion of foreign pathogens, contains immunocompetent cells that maintain a healthy immunogenic/tolerogenic balance, and is a suitable route for drug administration. In the skin, four defense levels can be identified: besides the physical, chemical, and immune barriers that are inherent to the tissue, the skin microbiota (i.e., the numerous microorganisms living on the skin surface) provides an additional barrier. Studying the skin barrier function or the effects of drugs or cosmetic agents on human skin is a difficult task since snapshot evidence can only be obtained using bioptic samples where dynamic processes cannot properly be followed. To overcome these limitations, many different in vitro models of human skin have been developed that are characterized by diverse levels of complexity in terms of chemical, structural, and cellular composition. The aim of this review is to summarize and discuss the advantages and disadvantages of the different human skin models so far available and to underline how the insertion of a proper microbiota would positively impact an in vitro human skin model in an attempt to better mimic conditions in vivo.

1954-2024: 70 years of histochemical research with the European Journal of Histochemistry.

This Editorial celebrates the 70th anniversary of the European Journal of Histochemistry since its foundation as Rivista di Istochimica Normale e Patologica, and introduces a Special Collection of selected articles on the application of the histochemical approach for investigating cell biological features and processes in animals and plants, and under diseased conditions. The year 2024 is a special one for histochemists, as 100 years ago J.W. Robert Feulgen and H. Rossenbeck introduced the histochemical procedure for the specific stoichiometric staining of DNA in histological samples: to commemorate this influential publication, three papers in the present issue are devoted to the application of the Feulgen reaction at light and electron microscopy, and in cytometry.

L-Carnitine Functionalization to Increase Skeletal Muscle Tropism of PLGA Nanoparticles.

Muscular dystrophies are a group of rare genetic pathologies, encompassing a variety of clinical phenotypes and mechanisms of disease. Several compounds have been proposed to treat compromised muscles, but it is known that pharmacokinetics and pharmacodynamics problems could occur. To solve these issues, it has been suggested that nanocarriers could be used to allow controlled and targeted drug release. Therefore, the aim of this study was to prepare actively targeted poly(lactide-co-glycolide) (PLGA) nanoparticles (NPs) for the treatment of muscular pathologies. By taking advantage of the high affinity for carnitine of skeletal muscle cells due to the expression of Na+-coupled carnitine transporter (OCTN), NPs have been actively targeted via association to an amphiphilic derivative of L-carnitine. Furthermore, pentamidine, an old drug repurposed for its positive effects on myotonic dystrophy type I, was incorporated into NPs. We obtained monodispersed targeted NPs, with a mean diameter of about 100 nm and a negative zeta potential. To assess the targeting ability of the NPs, cell uptake studies were performed on C2C12 myoblasts and myotubes using confocal and transmission electron microscopy. The results showed an increased uptake of carnitine-functionalized NPs compared to nontargeted carriers in myotubes, which was probably due to the interaction with OCTN receptors occurring in large amounts in these differentiated muscle cells.

Twenty years of histochemistry in the third millennium, browsing the scientific literature.

Over the last twenty years, about 240,000 articles where histochemical techniques were used have been published in indexed journals, and their yearly number has progressively increased. The histochemical approach was selected by researchers with very different scientific interests, as the journals in which these articles were published fall within 140 subject categories. The relative proportion of articles in some of these journal categories did change over the years, and browsing the table of contents of the European Journal of Histochemistry, as an example of a strictly histochemical journal, it appeared that in recent years histochemical techniques were preferentially used to mechanistically investigate natural or experimentally induced dynamic processes, with reduced attention to purely descriptive works. It may be foreseen that, in the future, histochemistry will be increasingly focused on studying the molecular pathways responsible for cell differentiation, the maintenance or loss of the differentiated state, and tissue regeneration.

A journal of histochemistry as a forum for non-histochemical scientific societies.

Histochemical techniques are widely applied in biomedical research and, during the last twenty years, they were among the methods used in more than 590,000 scientific articles in indexed journals. However, a very small percentage of these papers were published in strictly histochemical journals. A possible strategy to widen the audience of the histochemical journals making them attractive to non-histochemist authors might be to publish and make open-access available the proceedings of the meetings and conferences of valued scientific societies whose fellows use microscopy and histochemistry in their experimental activity. In the last years' experience of the European Journal of Histochemistry, this approach was effective to increase the number of published articles on stem cells and development, connective tissue and nerve cell biology.

Histochemistry as a versatile research toolkit in biological research, not only an applied discipline in pathology.

The impressive progress of histochemistry over the last 50 years has led to setting up specific and sensitive techniques to describe dynamic events, through the detection of specific molecules in the very place where they exist in live cells. The scientific field where histochemistry has most largely been applied is histopathology, with the aim to identify disease-specific molecular markers or to elucidate the etiopathological mechanisms. Numerous authors did however apply histochemistry to a variety of other research fields; their interests range from the microanatomy of animal and plant organisms to the cellular mechanisms of life. This is especially apparent browsing the contents of the histochemical journals where the articles on subjects other than pathology are the majority; these journals still keep a pivotal role in the field of cell and tissue biology, while being a forum for a diverse range of biologists whose scientific interests expand  the research horizon of histochemistry to ever novel subjects. Thus, histochemistry can always receive inspiring stimuli toward a continuous methodological refinement.

Adapted physical exercise enhances activation and differentiation potential of satellite cells in the skeletal muscle of old mice.

During ageing, a progressive loss of skeletal muscle mass and a decrease in muscle strength and endurance take place, in the condition termed sarcopenia. The mechanisms of sarcopenia are complex and still unclear; however, it is known that muscle atrophy is associated with a decline in the number and/or efficiency of satellite cells, the main contributors to muscle regeneration. Physical exercise proved beneficial in sarcopenia; however, knowledge of the effect of adapted physical exercise on the myogenic properties of satellite cells in aged muscles is limited. In this study the amount and activation state of satellite cells as well as their proliferation and differentiation potential were assessed in situ by morphology, morphometry and immunocytochemistry at light and transmission electron microscopy on 28-month-old mice submitted to adapted aerobic physical exercise on a treadmill. Sedentary age-matched mice served as controls, and sedentary adult mice were used as a reference for an unperturbed control at an age when the capability of muscle regeneration is still high. The effect of physical exercise in aged muscles was further analysed by comparing the myogenic potential of satellite cells isolated from old running and old sedentary mice using an in vitro system that allows observation of the differentiation process under controlled experimental conditions. The results of this ex vivo and in vitro study demonstrated that adapted physical exercise increases the number and activation of satellite cells as well as their capability to differentiate into structurally and functionally correct myotubes (even though the age-related impairment in myotube formation is not fully reversed): this evidence further supports adapted physical exercise as a powerful, non-pharmacological approach to counteract sarcopenia and the age-related deterioration of satellite cell capabilities even at very advanced age.

Is there still room for novelty, in histochemical papers?

Histochemistry continues to be widely applied in biomedical research, being nowadays mostly addressed to detect and locate single molecules or molecular complexes inside cells and tissues, and to relate structural organization and function at the high resolution of the more advanced microscopical techniques. In the attempt to see whether histochemical novelties may be found in the recent literature, the articles published in the European Journal of Histochemistry in the period 2014-2016 have been reviewed. In the majority of the published papers, standardized methods have been preferred by scientists to make their results reliably comparable with the data in the literature, but  many papers (approximately one fourth of the published articles) described novel histochemical methods and procedures.  It is worth noting that there is a growing interest for minimally-invasive in vivo techniques (magnetic resonance imaging, autofluorescence spectroscopy) which may parallel conventional  histochemical analyses to obtain information not only on the morphological features of living organs and tissues, but also on their functional, biophysical and molecular characteristics. Thanks to this unceasing methodological refinement, histochemistry will continue to provide innovative applications in the biomedical field.

Histochemistry in biology and medicine: a message from the citing journals.

Especially in recent years, biomedical research has taken advantage of the progress in several disciplines, among which microscopy and histochemistry. To assess the influence of histochemistry in the biomedical field, the articles published during the period 2011-2015 have been selected from different databases and grouped by subject categories: as expected, biological and biomedical studies where histochemistry has been used as a major experimental approach include a wide of basic and applied researches on both humans and other animal or plant organisms. To better understand the impact of histochemical publications onto the different biological and medical disciplines, it was useful to look at the journals where the articles published in a multidisciplinary journal of histochemistry have been cited: it was observed that, in the five-years period considered, 20% only of the citations were in histochemical periodicals, the remaining ones being in journals of Cell & Tissue biology,  general and experimental Medicine, Oncology, Biochemistry & Molecular biology, Neurobiology, Anatomy & Morphology, Pharmacology & Toxicology, Reproductive biology, Veterinary sciences, Physiology, Endocrinology, Tissue engineering & Biomaterials,  as well as in multidisciplinary journals.It is easy to foresee that also in the future the histochemical journals will be an attended forum for basic and applied scientists in the biomedical field. It will be crucial that these journals be open to an audience as varied as possible, publishing articles on the application of refined techniques to very different experimental models: this will stimulate non-histochemist scientists to approach histochemistry whose application horizon could expand to novel and possibly exclusive subjects.

Regulated forms of cell death are induced by the photodynamic action of the fluorogenic substrate, Hypocrellin B-acetate.

The addition of chemical groups to a photosensitizer makes it to act as a fluorogenic substrate, increasing its ability to enter the cells. In this work, the cytotoxic efficacy of Hypocrellin B modified by addition of two acetate groups (HypB-Ac) was investigated in HeLa cells. Using transmission electron microscopy, cytochemical and immunocytochemical techniques, and flow cytometry we demonstrated that light irradiation of HypB-Ac-loaded cells resulted in either necrosis or apoptosis, depending on the HypB-Ac concentration. Administration of Hyp-Ac at high concentration (1×10(-)(5) M) resulted in massive necrosis, while at low concentration (2.5×10(-)(7) M) apoptosis along with autophagy were induced. Focusing on cells still exhibiting non-apoptotic features, we provide the evidence of early involvement of different organelles in the photodamage, with the frequent presence of autophagic vacuoles already at very short post-irradiation times (30 min, when ultrastructural apoptotic features are rarely found). These findings suggest that the widespread photodamage rather than the target organelle(s) involved is crucial for inducing either a catastrophic or a regulated form of cell death. Fluorogenic substrates such as HypB-Ac have an increased capability to accumulate in cancer cells compared to the native photosensitizing molecules: this would allow to use lower drug doses in vivo, thus decreasing the risk of systemic cytotoxicity in the absence of irradiation improving the efficacy of photodynamic therapy. The ability of HypB-Ac at very low concentration to induce autophagy and apoptosis would additionally be advantageous for therapeutic application, as the preferential induction of regulated forms of cell death entails the rapid phagocytotic removal of dying cells without affecting the tissue and organ structure.

Human myoblasts from skeletal muscle biopsies: in vitro culture preparations for morphological and cytochemical analyses at light and electron microscopy.

We describe protocols for the isolation of satellite cells from human muscle biopsies, for the in vitro culture of proliferating and differentiating myoblasts, and for the preparation of cell samples suitable for morphological and cytochemical analyses at light and electron microscopy. The procedures described are especially appropriate for processing small muscle biopsies, and allow obtaining myoblast/myotube monolayers on glass coverslips, thus preserving good cell morphology and immunoreactivity for protein markers of myoblast proliferation, differentiation, and senescence.These cell preparations are suitable for cytochemical, immunocytochemical, and FISH procedures at light microscopy, and can be observed not only in bright field, phase contrast, and differential interference contrast but also in fluorescence (which can hardly be used for cells grown on conventional plastic surfaces, which generally exhibit intense autofluorescence). In their ultrastructural cytochemical application, the protocols are intended for post-embedding techniques, by which ultrathin sections from a single sample may be used for detecting a wide variety of molecular markers.

Morphological Features of Organelles during Apoptosis: An Overview.

An apoptotic program leading to controlled cell dismantling implies perturbations of nuclear dynamics, as well as changes affecting the organelle structure and distribution. In human cancer cells driven to apoptosis by different stimuli, we have recently investigated the morphological properties of several organelles, including mitochondria, lysosomes, endoplasmic reticulum and Golgi apparatus. In this review, we will discuss the body of evidence in the literature suggesting that organelles are generally relocated and/or degraded during apoptosis, irrespectively of the apoptogenic stimulus and cell type.

Physical training modulates structural and functional features of cell nuclei in type II myofibers of old mice.

Aging is associated with a progressive loss of muscle mass, strength, and function, a condition known as sarcopenia, which represents an important risk factor for physical disability in elderly. The mechanisms leading to sarcopenia are still largely unknown, and no specific therapy is presently available to counteract its onset or progress. Many studies have stressed the importance of physical exercise as an effective approach to prevent/limit the age-related muscle mass loss. This study investigated the effects of physical training on pre-mRNA pathways in quadriceps and gastrocnemius muscles of old mice by ultrastructural cytochemistry: Structural and in situ molecular features of myonuclei and satellite cell nuclei of type II fibers were compared in exercised versus sedentary old mice, using adult individuals as control. Our results demonstrated that in myonuclei of old mice physical exercise stimulates pre-mRNA transcription, splicing, and export to the cytoplasm, likely increasing muscle protein turnover. In satellite cells, the effect of physical exercise seems to be limited to the reactivation of some factors involved in the transcriptional and splicing apparatus without increasing RNA production, probably making these quiescent cells more responsive to activating stimuli.

RNA processing is altered in skeletal muscle nuclei of patients affected by myotonic dystrophy.

Myotonic dystrophies (DMs) are characterised by highly variable clinical manifestations consisting of muscle weakness and atrophy, and a wide spectrum of extramuscular manifestations. In both DM1 and DM2 forms, expanded nucleotide sequences cause the accumulation of mutant transcripts in the nucleus, thus deregulating the function of some RNA-binding proteins and providing a plausible explanation for the multifactorial phenotype of DM patients. However, at the skeletal muscle level, no mechanistic explanation for the muscle wasting has so far been proposed. We therefore performed a study in situ by immunoelectron microscopy on biceps brachii biopsies from DM1, DM2 and healthy subjects, providing the first ultrastructural evidence on the distribution of some nuclear ribonucleoprotein (RNP)-containing structures and molecular factors involved in pre-mRNA transcription and maturation in dystrophic myonuclei. Our results demonstrated an accumulation of splicing and cleavage factors in myonuclei of both DM1 and DM2 patients, suggesting an impairment of post-transcriptional pre-mRNA pathways. The transcription of the expanded sequences in DM myonuclei would therefore hamper functionality of the whole splicing machinery, slowing down the intranuclear molecular trafficking; this would reduce the capability of myonuclei to respond to anabolic stimuli thus contributing to muscle wasting.

Pre-mRNA processing is partially impaired in satellite cell nuclei from aged muscles.

Satellite cells are responsible for the capacity of mature mammalian skeletal muscles to repair and maintain mass. During aging, skeletal muscle mass as well as the muscle strength and endurance progressively decrease, leading to a condition termed sarcopenia. The causes of sarcopenia are manifold and remain to be completely elucidated. One of them could be the remarkable decline in the efficiency of muscle regeneration; this has been associated with decreasing amounts of satellite cells, but also to alterations in their activation, proliferation, and/or differentiation. In this study, we investigated the satellite cell nuclei of biceps and quadriceps muscles from adult and old rats; morphometry and immunocytochemistry at light and electron microscopy have been combined to assess the organization of the nuclear RNP structural constituents involved in different steps of mRNA formation. We demonstrated that in satellite cells the RNA pathways undergo alterations during aging, possibly hampering their responsiveness to muscle damage.

Changes of mitochondria and relocation of the apoptosis-inducing factor during apoptosis.

During apoptosis, apoptosis-inducing factor (AIF) is released from the mitochondrial intermembrane space to the cytosol and to the nucleus. We analyzed AIF in HeLa cells driven to apoptosis by either etoposide or actinomycin D, and we observed changes in the structure and function of mitochondria as well as the translocation of cytochrome c and AIF from mitochondria to the nucleus in early apoptosis. In cells with fragmented chromatin (i.e., in late apoptosis), the immunolabeling for AIF appeared to be distinct from chromatin, being mainly confined to mitochondria.

Distribution of centromeric proteins and PARP-1 during mitosis and apoptosis.

A large complex of proteins, called CENPs, are associated with centromeric DNA. Some of them exhibit a cell cycle-related expression (e.g., CENP-E and -F) and are required for the transition from interphase to mitosis, whereas constitutive proteins (e.g., CENP-A, -B, -C, -G, and -H) reside permanently at the centromere and are essential for the correct kinetochore assembly. Poly(ADP-ribose) polymerase-1 (PARP-1), which plays an active role in many basic processes, was described as a possible regulator of CENPs. By multicolor immunofluorescence we therefore analyzed the distribution of PARP-1 and its interaction with CENP-B, -E, and -F during mitosis and apoptosis.

Enzyme-assisted photosensitization activates different apoptotic pathways in Rose Bengal acetate treated HeLa cells.

Photosensitization of tumor cells after incubation with Rose Bengal acetate (RB-Ac) induces multiple organelle photodamage followed by apoptotic cell death. We used immunocytochemical techniques in multicolor fluorescence microscopy to elucidate whether this occurs through the simultaneous activation of different apoptotic pathways, in HeLa cells. We detected in situ the activated forms of caspases 9 and 3, and the translocation from the mitochondria to the nucleus of the apoptosis inducing factor; DNA electrophoretic techniques were also used to assess the occurrence of nuclear DNA cleavage into either high- or low-molecular-weight fragments. Both the caspase-dependent and caspase-independent apoptotic pathways are activated. The genomic DNA is degraded into high molecular weight molecules only, without the formation of oligonucleosome-sized fragments. The ability of RB-Ac to induce the simultaneous release of apoptogenic signals from different photodamaged organelles makes it an especially powerful cytotoxic agent.

Dynamic relocation of nuclear proteins during the execution phase of apoptosis.

In the apoptotic program of controlled cell dismantling, the most characteristic nuclear changes involve chromatin, which condenses and often collapses against the nuclear envelope in the form of crescents. A severe reorganization also occurs in ribonucleoprotein (RNP)-containing structures which are involved in the synthesis and processing of transcripts: already during early apoptosis, the nucleoplasmic RNPs (namely, perichromatin fibrils, perichromatin granules, and interchromatin granules) coalesce in the interchromatin space where they associate with segregated nucleolar components, to ectopically form fibro-granular heterogeneous clusters. This was found to occur in cell systems in vivo and in cultured cell lines, after different apoptogenic stimuli. These RNP aggregates we have called heterogeneous ectopic RNP-derived structures (HERDS) move from the nucleus to the cytoplasm, and may be found in apoptotic bodies, in late apoptosis. Immunolabeling experiments demonstrated that several other proteins which are normally located inside the nucleus also move into the cytoplasm, during apoptosis, independently from HERDS. Apoptotic cells have been suggested to be a powerful source of nuclear auto-antigens, which are produced by the partial proteolytic or nucleolytic cleavage of a wide variety of nuclear substrates. In the presence of defective phagocytosis (or when massive apoptosis overwhelms the clearance capability of the tissue scavenger cells), the disposal of apoptotic cells becomes insufficient and unphagocytosed late apoptotic cells may accumulate in the tissue where they may be engulfed by antigen-presenting cells (such as dendritic cells); an autoimmune response may thus be elicited, by which apoptosis-derived auto-antigens are recognized and presented to the immune system.