CROCC-mutated rhabdoid colorectal carcinoma showing in intercellular spaces lamellipodia and cellular projections revealed by electron microscopy.

BACKGROUND: Rhabdoid colorectal carcinoma (RC) is a rare lesion localized to the proximal colon of patients with a mean age at diagnosis of around 70 years. This tumor shows an aggressive behavior with an overall survival period shorter than 12 months. The diagnostic hallmark is the presence of rhabdoid cells. Alterations in chromatin remodeling (SMARCB1) and in the centrosome structure (CROCC) are reported in RC usually BRAFmut and MSI-H. RKO intestinal neoplastic cells culture (BRAFmut, SMARCB1wt, MSI-H) with CROCC knockdown exhibit rhabdoid features and develop prominent projections from the edge of the cell. METHODS: Here, we investigated two cases of CROCCmutSMARCB1wt RC by scanning and transmission electron microscopy (SEM, TEM). RESULTS: TEM confirmed the diagnostic presence of intermediate cytoplasmic filaments and nucleolar margination. SEM showed cellular protrusions (lamellipodia) in the intercellular spaces not evident at light microscopy. CONCLUSIONS: These protrusions CROCC-related might represent the pathogenetic mechanism underlying the rhabdoid aggressive behavior, independently of tumor staging. To our knowledge, the SEM technique was applied in the study of this neoplasm for the first time.

TiO2-coated luminescent porous silicon micro-particles as a promising system for nanomedicine.

Porous silicon (pSi) is a sponge-like material obtained by electrochemical etching of a crystalline silicon wafer. Due to quantum confinement effects, this material is photoluminescent and this is a fundamental property from the perspective of bioimaging applications. Limitations in nanomedicine to the use of photoluminescent pSi structures are mainly due to optical quenching in an aqueous environment and to the adverse effects of reactive groups introduced by etching procedures. In this work, we exploited an inorganic TiO2 coating of pSi microparticles by Atomic Layer Deposition (ALD) that resulted in optical stability of pSi particles in a biological buffer (e.g. PBS). The use of a rotary reactor allows deposition of a uniform coating on the particles and enables a fine tuning of its thickness. The ALD parameters were optimized and the photoluminescence (PL) of pSi-TiO2 microparticles was stabilized for more than three months without any significant effect on their morphology. The biocompatibility of the coated microparticles was evaluated by analyzing the release of cytokines and superoxide anion (O2 -) by human dendritic cells, which play an essential role in the regulation of inflammatory and immune responses. We demonstrated that the microparticles per se are unable to significantly damage or stimulate human dendritic cells and therefore are suitable candidates for nanomedicine applications. However, a synergistic effect of the microparticles with bacterial products, which are known to stimulate immune-response, was observed, indicating that a condition unfavorable to the use of inorganic nanomaterials in biological systems is the presence of infection diseases. These results, combined with the proved PL stability in biological buffers, open the way for the use of pSi-TiO2 microparticles as promising materials in nanomedicine, but their ability to increase immune cell activation by other agonists should be considered and even exploited.

A High Frequency of HIV-Specific Circulating Follicular Helper T Cells Is Associated with Preserved Memory B Cell Responses in HIV Controllers.

Follicular helper T cells (Tfh) play an essential role in the affinity maturation of the antibody response by providing help to B cells. To determine whether this CD4+ T cell subset may contribute to the spontaneous control of HIV infection, we analyzed the phenotype and function of circulating Tfh (cTfh) in patients from the ANRS CO21 CODEX cohort who naturally controlled HIV-1 replication to undetectable levels and compared them to treated patients with similarly low viral loads. HIV-specific cTfh (Tet+), detected by Gag-major histocompatibility complex class II (MHC-II) tetramer labeling in the CD45RA- CXCR5+ CD4+ T cell population, proved more frequent in the controller group (P = 0.002). The frequency of PD-1 expression in Tet+ cTfh was increased in both groups (median, >75%) compared to total cTfh (<30%), but the intensity of PD-1 expression per cell remained higher in the treated patient group (P = 0.02), pointing to the persistence of abnormal immune activation in treated patients. The function of cTfh, analyzed by the capacity to promote IgG secretion in cocultures with autologous memory B cells, did not show major differences between groups in terms of total IgG production but proved significantly more efficient in the controller group when measuring HIV-specific IgG production. The frequency of Tet+ cTfh correlated with HIV-specific IgG production (R = 0.71 for Gag-specific and R = 0.79 for Env-specific IgG, respectively). Taken together, our findings indicate that key cTfh-B cell interactions are preserved in controlled HIV infection, resulting in potent memory B cell responses that may play an underappreciated role in HIV control.IMPORTANCE The rare patients who spontaneously control HIV replication in the absence of therapy provide a unique model to identify determinants of an effective anti-HIV immune response. HIV controllers show signs of particularly efficient antiviral T cell responses, while their humoral response was until recently considered to play only a minor role in viral control. However, emerging evidence suggests that HIV controllers maintain a significant but "silent" antiviral memory B cell population that can be reactivated upon antigenic stimulation. We report that cTfh help likely contributes to the persistence of controller memory B cell responses, as the frequency of HIV-specific cTfh correlated with the induction of HIV-specific antibodies in functional assays. These findings suggest that T follicular help may contribute to HIV control and highlight the need for inducing such help in HIV vaccine strategies that aim at eliciting persistent B cell responses.

Molecules implicated in glucose homeostasis are differentially expressed in the trachea of lean and obese Zucker rats.

Recent studies indicate that the processes mediated by the (T1R2/T1R3) glucose/sugar receptor of gustatory cells in the tongue, and hormones like leptin and ghrelin contribute to the regulation of glucose homeostasis. Altered plasma levels of leptin and ghrelin are associated with obesity both in humans and rodents. In the present study, we evaluated the ultrastructure of the mucosa, and the expression of molecules implicated in the regulation of glucose homeostasis (GLUT2, SGLT1, T1R3, ghrelin and its receptor) in the trachea of an animal model of obesity (Zucker rats). We found that the tracheal epithelium of obese animals was characterized by the presence of poorly differentiated cells. Ciliated and secretory cells were the cell lineages with greatest loss of differentiation. Severe epithelial alterations were associated with marked deposit of extracellular matrix in the lamina propria. The expression pattern of GLUT2 and SGLT1 glucose transporters was similar in the trachea of both the Zucker rat genotypes, whereas that of T1R3 was reduced in ciliated cells of obese rats. A different immunolocalization for ghrelin was also found in the trachea of obese rats. In conclusion, the tracheal morphological alterations in obese animals seem to compromise the expression of molecules involved in the homeostasis of glucose.

Differences in glucose metabolic enzyme activities in human adipose tissue from abdominal and gluteal regions.

In order to verify whether intersite differences exist in glucose metabolism of subcutaneous human adipose tissue, basal and insulin-stimulated 14C-1-glucose incorporation into triglycerides and the activities of some enzymes of glucose disposal were tested in abdominal and gluteal adipose tissue of 31 nondiabetic obese otherwise healthy women during isocaloric diet and after 2 weeks of very-low-calorie-protein-sparing modified diet. Basal 14C-1-glucose incorporation into triglycerides was quite similar in the adipose tissue of the two sites, and it was not influenced by dietary restriction. Insulin stimulated this metabolic activity to the same extent in both sites during isocaloric diet; after hypocaloric diet this effect of insulin was slightly decreased in adipose tissue of the abdominal site and completely abolished in the gluteal site. No enzymatic activity was different between the examined subcutaneous regions during the isocaloric diet; after very-low-calorie intake, hexokinase activity decreased in both sites, once again more markedly in the gluteal one; glucose-6-P-dehydrogenase activity decreased in the adipose tissue of the gluteal region only. These data suggest that glucose metabolism of the adipose tissue of the gluteal region is particularly decreased by severe calorie restriction. Therefore, since lipolysis does not occur at a higher rate in gluteal adipose tissue during calorie restriction, this tissue seems to undergo a resting metabolic phase during hypocaloric diet.

Intra-epithelial lipids in the dorsoposterior area of the tongue in new-born rats.

The mucosa covering the posterior surface of the tongue in new-born rats was studied by scanning and transmission electron microscopy. The results demonstrated that in the first days of extra-uterine life in the epithelium of this mucosa there is an accumulation of lipidic material, which appeared as round or oval droplets with a mean diameter of about 0.8-1.0 microm. Their density was homogeneously low and they did not contain fibrillar or granular material. In the first day of extra-uterine life, the droplets were scarce and mainly localized in the dorsal surface of the vallate papilla. In the following days, the lipid deposit was present in large areas of the posterior surface of the tongue. At the end of the first week of extra-uterine life, as well as in adult animals, the lipids were rather scarce. A multilocular lipid deposit was also visible in superficial cells detaching from the mucosa. With magnetic resonance spectroscopy, lipids extracted from the posterior mucosa of the tongue appeared mainly saturated. Small amounts of unsaturated and polyunsaturated lipid were also detectable. These findings suggest that in the first week of extra-uterine life, specialized areas of the oral epithelium store lipid.

An ultrastructural study of brown adipose tissue in pre-term human new-borns.

The ultrastructure of brown adipose tissue (BAT), the thermogenic type of adipose tissue, was investigated in biopsies from 4 pre-term human new-borns delivered at 25-27 week's gestational age and compared with peri-renal brown fat from 2 adult patients with phaeochromocytoma (a condition of brown fat activation). The cell size of brown adipocytes was smaller in pre-term new-borns than in adult patients; adipocytes were almost exclusively multilocular, suggesting active thermogenesis. In 3 of the pre-term new-borns, brown adipocyte ultrastructure indicated a good to high degree of differentiation (in particular at the level of mitochondria) as compared with activated brown fat cells found in adult patients; in one pre-term infant the tissue morphology was obviously suggestive of an earlier, proliferative phase of development and the differentiation process of brown adipocytes could be traced in some detail. The results suggest that (a) brown adipose tissue may be fairly well-differentiated and thermogenetically active in pre-term human new-borns weighing about 750 g at birth; (b) brown adipocytes apparently develop from vessel-associated cells, the early signs of adipocyte differentiation being glycogen and lipid accumulation; (c) the ultrastructural morphology of mitochondria of mitochondria in well-differentiated BAT from pre-term infants can strictly resemble that found in active brown adipose tissue of adult phaeochromocytoma patients.

The post-adipocytic phase of the adipose cell cycle.

Subcutaneous white adipose tissue harvested by liposuction has been studied with the aim to understand how the adipocytes modify their morphology when subjected to the passage in a needle for liposuction and to cryopreservation. The work try to clarify the ultrastructural aspects of adipose tissue, in the conditions described before, examining samples of body fat employed in fat graft procedures, and samples after cryopreservation. Scanning and transmission electron microscopy show that the first event that occur in the adipocytes is a lesion of mild degree detectable early in the samples fixed immediately after liposuction. The sequence of events following the adipocyte stress appeared composed by different phases: plasmatic membrane interruption, loss of lipid charge, formation of cup-like adipocytes and formation of post-adipocytes (i.e. cells that survive to traumatic events and restart to internalize lipid droplets). In conclusion, the study suggests that the loss of lipid charge in adipose cell is an active process that can be due to a small hole in the cytoplasmic membrane with the preservation of a large part of the cytoplasmatic content and that at the end of the process of lipid extrusion the cell can maintain viability.

The trochanteric fat pad.

Technological developments based on the use of autologous white adipose tissue (WAT) attracted attention to minor fat depots as possible sources of adipose tissue. In plastic surgery, the trochanteric fatty pad is one of the most used WAT depots for its location and organoleptic characteristics that make it particularly suitable for reconstructive procedures. Despite its wide use in clinic, the structure of this depot has never been studied in detail and it is not known if structural differences exist among trochanteric fat and other subcutaneous WAT depots. The present study was performed on trochanteric fat pad with the aim to clarify the morphology of its adipocytes, stroma and microcirculation, with particular reference to the stem niches. Histological and ultrastructural studies showed that the main peculiar feature of the trochanteric fat concerns its stromal component, which appears less dense than in the other subcutaneous WATs studied. The intra-parenchymal collagen stroma is poor and the extracellular compartment shows large spaces, filled with electron-light material, in which isolated collagen bundles are present. The adipocytes are wrapped in weak and easily detachable collagen baskets. These connective sheaths are very thin compared to the sheaths in other subcutaneous WAT depots. The capillaries are covered by large, long and thin elements surrounded by an external lamina; these perivascular cells are poor in organelles and mainly contain poly-ribosomes. In conclusion, when compared to other WAT deposits, the trochanteric fatty pad shows structural peculiarities in its stroma and microcirculation suggesting a high regenerative potential. Resistance, dissociability, microvascular weft and high regenerative potential make the trochanteric fatty pad a privileged source for harvesting in autologous WAT-based regenerative procedures.

Subcutaneous adipose tissue classification.

The developments in the technologies based on the use of autologous adipose tissue attracted attention to minor depots as possible sampling areas. Some of those depots have never been studied in detail. The present study was performed on subcutaneous adipose depots sampled in different areas with the aim of explaining their morphology, particularly as far as regards stem niches. The results demonstrated that three different types of white adipose tissue (WAT) can be differentiated on the basis of structural and ultrastructural features: deposit WAT (dWAT), structural WAT (sWAT) and fibrous WAT (fWAT). dWAT can be found essentially in large fatty depots in the abdominal area (periumbilical). In the dWAT, cells are tightly packed and linked by a weak net of isolated collagen fibers. Collagenic components are very poor, cells are large and few blood vessels are present. The deep portion appears more fibrous then the superficial one. The microcirculation is formed by thin walled capillaries with rare stem niches. Reinforcement pericyte elements are rarely evident. The sWAT is more stromal; it is located in some areas in the limbs and in the hips. The stroma is fairly well represented, with a good vascularity and adequate staminality. Cells are wrapped by a basket of collagen fibers. The fatty depots of the knees and of the trochanteric areas have quite loose meshes. The fWAT has a noteworthy fibrous component and can be found in areas where a severe mechanic stress occurs. Adipocytes have an individual thick fibrous shell. In conclusion, the present study demonstrates evident differences among subcutaneous WAT deposits, thus suggesting that in regenerative procedures based on autologous adipose tissues the sampling area should not be randomly chosen, but it should be oriented by evidence based evaluations. The structural peculiarities of the sWAT, and particularly of its microcirculation, suggest that it could represent a privileged source for regenerative procedures based on autologous adipose tissues.

Membrane-bounded intratubular bodies in rat taste buds.

The ultrastructural features of membrane-bounded bodies contained in the tubulo-vesicular system in the outer segment of taste bud cells are described. Each body showed a round, fusiform or oval shape, was surrounded by a trilaminar membrane and enclosed an electron dense matrix sometimes containing inclusions. These bodies were found at all ages studied. Similar structures were also found embedded in the material plugging the taste pore. Our findings suggest that these bodies could be secreted at the free surface of the cells and be involved in the concentration of divalent cations.

Solitary chemosensory cells in the developing chemoreceptorial epithelium of the vallate papilla.

The solitary chemosensory cells are considered typical of aquatic vertebrates and have never been described in the oral cavity of terrestrial vertebrates. We describe elements with ultrastructural characteristics of the solitary chemosensory cell in the gustatory epithelium of rats in the first week of extrauterine life. These elements appeared isolated, located among keratinocytes, and wrapped by glial-like elements. They showed a bipolar shape with a round cell body, a thin apical process, and a thicker basal one. Nerve fibers contacted the cell body and the processes. The cells showed small dense granules mainly located near nerve contacts. Small bundles of tonofilaments were visible in the perinuclear cytoplasm. Similar elements were not found in rats after the first week of extrauterine life. The present study demonstrates in a mammal that the development of taste buds is preceded by the presence of epithelial elements with ultrastructural characteristics of the solitary chemosensory cells described in lower vertebrates. This finding suggests that the oral chemoreception in the suckling rats may be mediated by three different pathways (i.e., gustatory system, common chemical sense, and solitary chemosensory cell system).

Metformin enhances insulin binding to "in vitro" down regulated human fat cells.

Insulin binding to human adipose tissue from surgical patients was determined after three different preincubation conditions: a) 24 hrs in the presence or absence of 80 ng/ml insulin; b) 24 hrs in the presence of 80 ng/ml insulin or insulin plus 4 micrograms/ml metformin; c) 48 hrs pre-incubation as in b). We found that insulin down regulated its own receptor after 24 hours pre-incubation; when metformin was present in the pre-incubation medium together with insulin, insulin binding to adipose tissue was significantly higher than in tissue exposed to insulin alone after 48 hrs pre-incubation; a similar effect of metformin was already seen after 24 hrs, but was not statistically significant. We suggest that metformin can correct down regulation of the insulin receptor. This finding could explain discrepant results among studies dealing with the influence of metformin on insulin binding. Moreover, these results could be useful in understanding the mechanism of action of metformin in insulin-resistant states, e.g. type II diabetes.

Postnatal development of the intrinsic nervous system in the circumvallate papilla-vonEbner gland complex.

We have studied the postnatal development of the intrinsic nervous system in the circumvallate papilla-vonEbner gland complex using NADPH-diaphorase cytochemistry, immunocytochemistry (for nitric oxide synthase-1 and alpha-internexin) and electron microscopy. In rats sacrificed in their first day post partum (1 p.p.), only isolated NADPH-diaphorase positive neurons were visible in the organ. At 2 p.p., a small group of neurons was visible at the base of the papillae and positive neurons formed short chains close to the developing glandular tubules. In the following days, the NADPH-diapharase positive cells increased in number and nerve fibres were associated to small ganglia located at the base of the papilla or in the gland. After the first week of extrauterine life, the intrinsic nervous system was similar to the intrinsic system of adult animals. An immunocytochemical positivity for nitric oxide synthase-1 appeared at 4 p.p. in neurons located in the gland and at 7 p.p. in cells located at the base of the papilla. Immunocytochemical staining for alpha-internexin showed that at 1 p.p. developing nerve fibres were present in the connective tissue of the tongue's muscle layer. At 2-3 p.p., developing nerve fibres were also present at the bases and in the core of the papilla. In the following days, the positivity for alpha-internexin was reduced and one week after birth was virtually absent. Ultrastructural examination revealed that since 1 p.p. isolated neurons can be found at the base of the papilla. In conclusion, the intrinsic nervous system originates from neurons present in the organ at the birth which, in the first days, undergo a biochemical and morphological maturation while the nerve fibres rapidly grow. These findings support the hypothesis that the intrinsic nervous system of the circumvallate papilla has a role in the maturation of the vonEbner gland.

A brief survey of the modifications in sensory-secretory organs of the neonatal rat tongue.

Recent data obtained on rats suggest that in the days immediately following birth several events take place in the circumvallate papillae of the oral cavity. A phylogenetically primitive system of solitary chemosensory cells develops and is rapidly replaced by taste buds. The lipase-secreting von Ebner gland, which is associated with taste organs, begins to develop by forming short tubules. The intrinsic nervous system of the gustatory organs rapidly completes its maturation showing fast proliferation of fibers and immunocytochemical maturation. Intraepithelial lipid accumulation is visible in the non-receptorial mucosa of the tongue, showing aspects which suggest an active lipid secretion. These data demonstrate that in the rat the structure of the sensory-secretory organs of the newborn's tongue shows a typical conformation with respect to the adult and rapidly changes its organization in the first week after the birth. At the present level of knowledge, it is difficult to link the anatomical structures to peculiar functional roles but the rather simple organization of the neonatal gustatory epithelium could be in relation to the dietary regimen. The data obtained in laboratory animals underline the necessity of studies on human newborns to update the anatomical knowledge of the oral chemoceptive system.

In vivo octreotide administration acutely reduces exocrine granule size in the human pancreas.

BACKGROUND: Octreotide has been found to be effective in the prevention of postoperative complications of pancreatic surgery, but the benefit of preoperative octreotide administration has not been assessed. AIMS: To evaluate the ability of octreotide in reducing the amount of digestive enzymes in the pancreas before surgery, a morphometric ultrastructural study of the gland was undertaken in patients undergoing demolitive pancreatic surgery. METHODS: Twenty-three inpatients received saline (n = 8) or octreotide (100 micrograms s.c.) before surgery either once (n = 5), or three (n = 5) or six (n = 5) times at 8-hour intervals. At surgery, biopsies of the pancreas were taken and processed for electron microscopy. Several parameters were assessed in exocrine cells by means of ultrastructural morphometry. RESULTS: A single administration of octreotide significantly reduced the exocrine granule number and the mean and total granular surface sectional area, and the ratio between granule area and cytoplasmic area. Repeated octreotide administrations were associated with partial (3 administrations) and complete (6 administrations) recovery of all parameters to control values. CONCLUSION: Preoperative administration of octreotide, the synthetic analogue of somatostatin, acutely reduces exocrine granule number and size in the pancreatic cell. This finding can partially explain the prophylactic effect of the drug on early complications of pancreatic surgery. Such an effect is not maintained over multiple administrations of the somatostatin analogue. Possible explanations for this latter finding are discussed.

Starvation-induced insulin resistance: influence on 3-O-methylglucose transport.

Starvation causes insulin resistance which is partly due to decrease of insulin action on glucose transport in target cells. Preliminary data from other authors suggest that starvation is associated with an increase of the time necessary for insulin to exert its maximum effect on glucose transport. In order to verify this finding, 3-O-methylglucose (3OMG) transport was studied in isolated rat adipocytes from starved and fed rats. The total intracellular space for 3OMG was not modified by 48h starvation. After 48h starvation, 3OMG transport into fat cells was less responsive to maximum insulin concentration than in controls: 33 +/- 2% (controls) vs 23 +/- 4% (starved) of total glucose space, p less than 0.05; the sensitivity to insulin was normal or even increased in comparison to controls: 50% of maximum effect was reached at 42 microU/ml insulin in controls and at 26 microU/ml insulin in starved animals. We could not find any effect of starvation on the time-response curves of insulin action.

Ultrastructural phenotype of "intestinal-type" cells in columnar-lined esophagus.

An intestinal-type epithelium is often present at columnar-lined esophagus, gastroesophageal junction or within the so-called short segment Barrett's esophagus, but ultrastructural study failed to detect enterocytes in columnar-lined esophagus. The authors have analyzed the intestinal aspects present in areas of columnar-lined esophagus in a population of patients with reflux esophagitis to better understand the morphology and histogenesis of the proliferating elements. Columnar-lined mucosa was studied in 35 patients. Columnarsurface cells displayed a wide spectrum of ultrastructural features. Well-differentiated columnar secretory cells, secretory-absorptive cells, poorly differentiated columnar cells, atypical columnar cells, and goblet cells were detected. Well-differentiated absorptive cells were never found, These results demonstrate that the areas of intestinal metaplasia show a wide spectrum of ultrastructural phenotypes, ranging from poorly to well-differentiated cells. However, true enterocytes were not found and the most represented phenotype is that of secretory-absorptive cells, whose principal characteristic is the presence of secretory and absorptive aspects together. They can be described as secretory enterocytes or cells with double specialization. To the authors' knowledge, similar cells were not previously described in normal intestinal mucosa, and ultrastructural studies are consistent in describing a broad spectrum of ultrastructural features, suggesting that Barrett's specialized metaplasia is derived from cells with the capacity for a wide range of differentiation. Therefore, despite the wide use of term intestinal metaplasia in the medical literature, experimental data clearly failed to detect enterocytes in the columnar-lined esophagus, and ultrastructural data do not support the concept of intestinal metaplasia. The cellular heterogeneity seems to be the result of a "phenotypic shift" of undifferentiated elements, which show a different pattern of evolution. The result of this process is the formation of new cell types dissimilar from those normally present in esophageal, gastric, or duodenal mucosa.