Łukasz Mielańczyk

Electron microscopy of human fascia lata: focus on telocytes

From the histological point of view, fascia lata is a dense connective tissue. Although extracellular matrix is certainly the most predominant fascias feature, there are also several cell populations encountered within this structure. The aim of this study was to describe the existence and characteristics of fascia lata cell populations viewed through a transmission electron microscope. Special emphasis was placed on telocytes as a particular interstitial cell type, recently discovered in a wide variety of tissues and organs such as the heart, skeletal muscles, skin, gastrointestinal tract, uterus and urinary system. The conducted study confirmed the existence of a telocyte population in fascia lata samples. Those cells fulfil main morphological criteria of telocytes, namely, the presence of very long, thin cell processes (telopodes) extending from a relatively small cell body. Aside from telocytes, we have found fibroblasts, mast cells and cells with features of myofibroblastic differentiation. This is the first time it has been shown that telocytes exist in human fascia. Currently, the exact role of those cells within the fascia is unknown and definitely deserves further attention. One can speculate that fascia lata telocytes likewise telocytes in other organs may be involved in regeneration, homeostasis and intracellular signalling.

Role of Notch signaling pathway in gastric cancer pathogenesis

Notch signaling pathway is activated dynamically during evolution playing significant role in cell fate determination and differentiation. It has been known that alterations of this pathway may lead to human malignancies, including gastric cancer. Despite a decline in the overall incidence, this disease still remains an important global health problem. Therefore, a better understanding of the molecular alterations underlying gastric cancer may contribute to the development of rationally designed molecular targeted therapies. It has been reported that Notch1 receptor could become a prognostic marker of gastric cancer and novel target for gastric cancer therapy. Among the novel and targeted approaches for the treatment of gastric cancer is also the process of Notch receptors regulation by specific microRNA. γ-secretase inhibitors are also taken into consideration.

In Vitro Evaluation of Doxorubicin Conjugates Based on Sugar Core Nonlinear Polymethacrylates toward Anticancer Drug Delivery

V-shaped and star-shaped hydroxylamine-functionalized polymethacrylates designed as nanosized conjugates (<120 nm) with anticancer agent, namely, doxorubicin (DOX), were evaluated in vitro toward their potential usage as drug delivery systems in breast cancer (MCF-7) treatment. Statistical analysis of MTS assay results showed that the 4-arm conjugate (n(DOX) = 16) was the most effective polymeric system against MCF-7/W (wild type) and MCF-7/R (DOX resistant) cell lines. Apoptosis assay analysis showed that MCF-7/R cells cultured with nonlinear copolymers died due to necrosis and late apoptotis, whereas MCF-7/W cells were in early and late apoptosis. Among all tested conjugates, the most promising results with induction of apoptosis without inducing necrosis in both MCF-7 cell lines were obtained for conjugate based on 4-arm stars with low content of DOX. The cell cycle assay revealed that increase of MMA units in 4-arm copolymers induced MCF-7/R cell arrest in the SubG1 phase. In the same cell line, the corresponding conjugates triggered S and G2/M arrest. Gradual internalization of the chosen conjugate by MCF-7/R cells was monitored via fluorescence microscopy showing its main localization in the cytoplasm.

Changes in Subcellular Localization of Visfatin in Human Colorectal HCT-116 Carcinoma cell Line After Cytochalasin-B Treatment

The aim of the study was to assess the expression and subcellular localization of visfatin in HCT-116 colorectal carcinoma cells after cytokinesis failure using Cytochalasin B (CytB) and the mechanism of apoptosis of cells after CytB. We observed translocation of visfatin’s antigen in cytB treated colorectal carcinoma HCT-116 cells from cytosol to nucleus. Statistical and morphometric analysis revealed significantly higher area-related numerical density visfatin-bound nano-golds in the nuclei of cytB-treated HCT-116 cells compared to cytosol. Reverse relation to visfatin subcellular localization was observed in un-treated HCT-116 cells. The total amount of visfatin protein and visfatin mRNA level in HCT-116 cells was also decreased after CytB treatment. Additionally, CytB significantly decreased cell survival, increased levels of G2/M fractions, induced bi-nuclei formation as well as increased reactive oxygen species (ROS) level in HCT-116 cells. CytB treatment showed cytotoxic effect that stem from oxidative stress and is connected with the changes in the cytoplasmic/nuclear amount of visfatin in HCT-116 cells.

Notch signalling pathway as an oncogenic factor involved in cancer development.

Notch signalling is an evolutionarily conserved signalling pathway, which plays a significant role in a wide array of cellular processes including proliferation, differentiation, and apoptosis. Nevertheless, it must be noted that Notch is a binary cell fate determinant, and its overexpression has been described as oncogenic in a broad range of human malignancies. This finding led to interest in therapeutically targeting this pathway especially by the use of GSIs, which block the cleavage of Notch at the cell membrane and inhibit release of the transcriptionally active NotchIC subunit. Preclinical cancer models have clearly demonstrated that GSIs suppress the growth of such malignancies as pancreatic, breast, and lung cancer; however, GSI treatment in vivo is associated with side effects, especially those within the gastrointestinal tract. Although intensive studies are associated with the role of γ-secretase in pathological states, it should be pointed out that this complex impacts on proteolytic cleavages of around 55 membrane proteins. Therefore, it is clear that GSIs are highly non-specific and additional drugs must be designed, which will more specifically target components of the Notch signalling.

Expression of TRAIL and Fas in Primary Hyperparathyroidism

ABSTRACT Aim: Differentiating between parathyroid lesions is still difficult and ambiguous. In cases of primary hyperparathyroidism, appropriate and prompt diagnosis is of great importance for effective treatment and follow-up. A great amount of mechanisms contribute to the pathogenesis of primary hyperparathyroidism, such as disturbance in balance between pro- and anti-apoptotic factors. Therefore, we examined whether immunohistochemical expression of apoptotic factors, TNF-related apoptosis-inducing ligand (TRAIL) and Fas, could have clinical utility as a marker of proliferative lesions of parathyroid gland. Materials and methods: Parathyroid specimens of 58 consecutive patients who had undertaken surgery due to primary hyperparathyroidism were incubated with purified mouse monoclonal antihuman antibodies: anti-TRAIL and anti-Fas. Staining was considered positive when at least 5% of the cells showed immunoreactivity. Results: The percentage of cells which were positively stained for TRAIL in parathyroid hyperplasia was 9.65%, in parathyroid adenoma 8.31%, and in normal controls 2.24%. Immunoreactivity for TRAIL was detected in 91.89% of parathyroid hyperplasias, 85.71% of parathyroid adenomas, and none in healthy glands. The percentage of cells with a positive reaction to Fas in parathyroid hyperplasia was 8.92%, in parathyroid adenoma 8.09%, and in normal tissue 1.9%. The expression of Fas was found in 94.59% of parathyroid hyperplasias, 90.48% of parathyroid adenomas, and none in healthy glands. Conclusions: In our study, hyperplasias demonstrated the highest expression of TRAIL and Fas, whereas in adenomas it was increased compared to normal tissue, but lower than in hyperplasias. These factors could be an additive tool in the differential diagnosis of parathyroid lesions.

Ileal Transposition (IT) Surgery Changing the Ultrastructure of the Transposed Segment as well as Jejunum. Histomorphometric and Electron Microscopy Analysis

ObjectiveIleal transposition (IT) procedure leads to higher secretion of incretin hormones what is associated with a beneficial metabolic effect. However, IT will also have an influence on the related jejunum and ileum function. The aim of this research was to investigate the morphology of the jejunum and transposed ileum with the use of light and transmission electron microscopy (TEM) in order to determine the local alternations in the intestine resulting from the transposition.MethodsTwenty male, 8-week-old, obese Zucker rats underwent IT and six of them sham surgery. To compare both groups, the transection was made at all corresponding ileum positions among both groups of animals. The ileal anastomoses among the rats of sham procedure were subsequently formed accordingly without IT. Three months following the surgery, the tissue samples of jejunum and ileum were harvested.ResultsA significant increase in villus length, a decrease in the crypt depth, and an increased thickness of mucosa-muscularis-serosa (MMS) as well as cellular hyperplasia, with increased mitochondrial density of the transposed ileum segment, were observed among the group of rats which underwent IT comparing to the ones undergoing sham surgery. In rats undergoing IT, microvillus degeneration in jejunum regions was observed.ConclusionsIleal transposition alters the morphology and ultrastructure of the ileum as well as the jejunum. Given that the microvillus membrane represents an important aspect of the enterocyte functions, a further biochemical and molecular research is necessary in order to assess whether the observed changes are beneficial or not and to explore the phenomenon of gut adaptability after metabolic surgery.

Apoptosis in Primary Hyperparathyroidism

ABSTRACT Primary hyperparathyroidism (PHPT) is defined by inappropriate elevation of parathormone, caused by parathyroid hyperplasia, also known as multi-gland disease (MGD), parathyroid adenoma (PA), or parathyroid carcinoma (PC). Although several studies have already been conducted, there is a lack of a definite diagnostic marker, which could unambiguously distinguish MGD from PA or PC. The accurate and prompt diagnosis has the key meaning for effective treatment and follow-up. This review paper presents the role of apoptosis in PHPT. The comparison of the expression of Fas, TRAIL, BCL-2 family members, p53 in MGD, PA, and PC, among others, was described. The expression of described factors varies among proliferative lesions of parathyroid gland; therefore, these could serve as additional markers to assist in the diagnosis.

A different ultrastructural face of ribbon synapses in the rat retina

Ribbon synapses located exclusively within retinal, cochlear and vestibular connections belong to the most interesting cellular structures but their molecular nature and functions had remained unclear. The study has provided a descriptive morphological analysis of rat eye ribbon synapses using high‐resolution transmission electron microscopy (TEM). An original collection of untypical, rarely present in the literature sagittal or tangential sections through the single RIBEYE domain of the particular ribbon have been delivered.

Transmission Electron Microscopy of Biological Samples

During the last 70 years, transmission electron microscopy (TEM) has developed our knowledge about ultrastructure of the cells and tissues. Another aim is the determi‐ nation of molecular structure, interactions and processes including structure-function relationships at cellular level using a variety of TEM techniques with resolution in atomic to nanometre range. Even with the best transmission electron microscope, it is impossible to obtain real results without optimal sample preparation, respecting both the structure and the antigenicity preservation. Preparation techniques for highresolution study of both macromolecular complex and organelles within cellular complex are based on fast cryoimmobilisation process, where the sample is in the most native, hydrated state. Next, thin samples are directly visualised under cryotransmission electron microscopy (cryo-TEM), while thicker samples require a thinning step via cryo-electron microscopy of vitreous sections (CEMOVIS) or cryofocused ion beam (cryo-FIB) before visualisation. Alternatively, vitrified samples are freeze substituted and embedded in chosen resin for room temperature ultramicrot‐ omy. This preparation technique is suitable for morphological study, 3D analysis of cellular interior and immunoelectron microscopy. A different route for immunoloc‐ alisation study is cryosectioning according to the Tokuyasu technique that is a choice for rare or methacrylate-sensitive antigens. Most recently, new hybrid techniques have been developed for difficult-to-fix organisms and antigens or labile and anoxiasensitive tissues. Another preparation technique is, the oldest but still important, conventional chemical fixation dedicated in a wide range of research interest, involving morphological and immunolocalisation study. In this chapter, we present different sample preparation approaches for transmission electron microscopy of biological samples, including its methodological basis and applications.