TELOCYTES – a case of serendipity: the winding way from Interstitial Cells of Cajal (ICC), via Interstitial Cajal-Like Cells (ICLC) to TELOCYTES

Ramon y Cajal discovered a particular cell type in the gut, which he named ‘interstitial neurons’ more that 100 years ago. In the early 1970s, electron microscopy/electron microscope (EM) studies showed that indeed a special interstitial cell type corresponding to the cells discovered by Cajal is localized in the gut muscle coat, but it became obvious that they were not neurons. Consequently, they were renamed ‘interstitial cells of Cajal’ (ICC) and considered to be pace-makers for gut motility. For the past 10 years many groups were interested in whether or not ICC are present outside the gastrointestinal tract, and indeed, peculiar interstitial cells were found in: upper and lower urinary tracts, blood vessels, pancreas, male and female reproductive tracts, mammary gland, placenta, and, recently, in the heart as well as in the gut. Such cells, now mostly known as interstitial Cajal-like cells (ICLC), were given different and confusing names. Moreover, ICLC are only apparently similar to canonical ICC. In fact, EM and cell cultures revealed very particular features of ICLC, which unequivocally distinguishes them from ICC and all other interstitial cells: the presence of 2–5 cell body prolongations that are very thin (less than 0.2 μm, under resolving power of light microscopy), extremely long (tens to hundreds of μm), with a moniliform aspect (many dilations along), as well as caveolae. Given the unique dimensions of these prolongations (very long and very thin) and to avoid further confusion with other interstitial cell types (e.g. fibroblast, fibrocyte, fibroblast-like cells, mesenchymal cells), we are proposing the term TELOCYTES for them, and TELOPODES for their prolongations, by using the Greek affix ‘telos’.     

TELOCYTES IN ENDOCARDIUM: Electron Microscope Evidence

The term TELOCYTES was very recently introduced, for replacing the name Interstitial Cajal‐Like Cells (ICLC). In fact, telocytes are not really Cajal‐like cells, they being different from all other interstitial cells by the presence of telopodes, which are cell‐body prolongations, very thin (under the resolving power of light microscopy), extremely long (tens up to hundreds of micrometers), with a moniliform aspect (many dilations along), and having caveolae. The presence of telocytes in epicardium and myocardium was previously documented. We present here electron microscope images showing the existence of telocytes, with telopodes, at the level of mouse endocardium. Telocytes are located in the subendothelial layer of endocardium, and their telopodes are interposed in between the endocardial endothelium and the cardiomyocytes bundles. Some telopodes penetrate from the endocardium among the cardiomyocytes and surround them, eventually. Telopodes frequently establish close spatial relationships with myocardial blood capillaries and nerve endings. Because we may consider endocardium as a ‘blood–heart barrier’, or more exactly as a ‘blood–myocardium barrier’, telocytes might have an important role in such a barrier being the dominant cell population in subendothelial layer of endocardium.     

Telocytes as supporting cells for myocardial tissue organization in developing and adult heart

Recent evidence indicates that the adult heart contains sub-epicardial cardiogenic niches where cardiac stem cells and stromal supporting cells reside together. Such stromal cells include a special population, previously identified as interstitial Cajal-like cells and recently termed telocytes because of their long, slender processes (telopodes) embracing the myocardial precursors. Specific stromal cells, presumptively originated from the epicardium, have been postulated to populate the developing heart where they are thought to play a role in its morphogenesis. This study is designed to investigate the occurrence of telocytes in the developing heart and provide clues to better understand their role as supporting cells involved in the architectural organization of the myocardium during heart development. Our results showed that stromal cells with the immunophenotypical (vimentin, CD34) and ultrastructural features of telocytes were present in the mouse heart since early embryonic to adult life, as well as in primary cultures of neonatal mouse cardiac cells. These cells formed an extended network of telopodes which closely embraced the growing cardiomyocytes and appeared to contribute to the aggregation of cardiomyocyte clusters in vitro. In conclusion, the present findings strongly suggest that, during heart development, stromal cells identifiable as telocytes could play a nursing and guiding role for myocardial precursors to form the correct three-dimensional tissue pattern and contribute to compaction of the embryonic myocardial trabeculae. It is tempting to speculate that telocytes could be a novel, possible target for therapeutic strategies aimed at potentiating cardiac repair and regeneration after ischemic injury.     

Myocardial interstitial Cajal-like cells (ICLC) and their nanostructural relationships with intercalated discs: shed vesicles as intermediates

Intercalated discs (ID) are complex junctional units that connect cardiac myocytes mechanically and electrochemically. However, there is limited information concerning the cardiomyocyte interaction with interstitial non-muscle cells. Our previous studies showed that myocardial interstitial Cajal-like cells (ICLC) are located in between cardiomyocytes, blood capillaries and nerve fibres. Typically, ICLC have several very long, moniliform, cytoplasmic processes which establish closed contacts with nerve fibres, as well as each other. We report here ultrastructural evidence concerning the relationships of ICLC processes with ID. The ICLC cytoplasmic prolongations (tens micrometers length) preferentially pass by or stop nearby the ID. Transmission electron microscopy emphasized three distinct connecting features between the tips of ICLC extensions and myocytes at the 'mouth' of ID: free or budding shed vesicles, exocytotic multi-vesicular bodies and direct contacts. In the last case, electron-dense repetitive nanostructures ('pillars') (35-40 nm high and 100-150 nm wide, similar to adhesion molecules) fasten the ICLC to the myocytes. All these features suggest a juxtacrine and/or paracrine intercellular mutual modulation of ICLC and cardiomyocytes in the microenvironment of ID, possibly monitoring the cardiac functions, particularly the electrical activity.     

HMGB‐1 induces c‐kit+ cell microvascular rolling and adhesion via both toll‐like receptor‐2 and toll‐like receptor‐4 of endothelial cells

High-mobility group box 1 (HMGB-1) is a strong chemo-attractive signal for both inflammatory and stem cells. The aim of this study is to evaluate the mechanisms regulating HMGB-1–mediated adhesion and rolling of c-kit⁺ cells and assess whether toll-like receptor-2 (TLR-2) and toll-like receptor-4 (TLR-4) of endothelial cells or c-kit⁺ cells are implicated in the activation of downstream migration signals to peripheral c-kit⁺ cells. Effects of HMGB-1 on the c-kit⁺ cells/endothelial interaction were evaluated by a cremaster muscle model in wild-type (WT), TLR-2 (−/−) and Tlr4 (LPS-del) mice. The mRNA and protein expression levels of endothelial nitric oxide synthase were determined by quantitative real-time PCR and immunofluorescence staining. Induction of crucial adhesion molecules for rolling and adhesion of stem cells and leukocytes were monitored in vivo and in vitro. Following local HMGB-1 administration, a significant increase in cell rolling was detected (32.4 ± 7.1% in ‘WT’ versus 9.9 ± 3.2% in ‘control’, P < 0.05). The number of firmly adherent c-kit⁺ cells was more than 13-fold higher than that of the control group (14.6 ± 5.1 cells/mm² in ‘WT’ versus 1.1 ± 1.0 cells/mm² in ‘control’, P < 0.05). In knockout animals, the fraction of rolling cells did not differ significantly from control levels. Firm endothelial adhesion was significantly reduced in TLR-2 (−/−) and Tlr4 (LPS-del) mice compared to WT mice (1.5 ± 1.4 cells/mm² in ‘TLR-2 (−/−)’ and 2.4 ± 1.4 cells/mm² in ‘Tlr4 (LPS-del)’ versus 14.6 ± 5.1 cells/mm² in ‘WT’, P < 0.05). TLR-2 (−/−) and Tlr4 (LPS-del) stem cells in WT mice did not show significant reduction in rolling and adhesion compared to WT cells. HMGB-1 mediates c-kit⁺ cell recruitment via endothelial TLR-2 and TLR-4.     

Origin of the words denoting some of the most ancient old world pulse crops and their diversity in modern European languages

This preliminary research was aimed at finding the roots in various Eurasian proto-languages directly related to pulses and giving the words denoting the same in modern European languages. Six Proto-Indo-European roots were indentified, namely arnk('')- (‘a leguminous plant’), *bhabh- (‘field bean’), * (‘a kernel of leguminous plant’, ‘pea’), ghArs- (‘a leguminous plant’), *kek- (‘pea’) and *lent- (‘lentil’). No Proto-Uralic root was attested save hypothetically *kača (‘pea’), while there were two Proto-Altaic roots, *bŭkrV (‘pea’) and * (‘lentil’). The Proto-Caucasianx root * denoted pea, while another one, *hōwł(ā) (‘bean’, ‘lentil’) and the Proto-Basque root *iłha-r (‘pea’, ‘bean’, ‘vetch’) could have a common Proto-Sino-Caucasian ancestor, *hVwłV (‘bean’) within the hypothetic Dené-Caucasian language superfamily. The Modern Maltese preserved the memory of two Proto-Semitic roots, *''adaš- (‘lentil’) and *pūl- (‘field bean’). The presented results prove that the most ancient Eurasian pulse crops were well-known and extensively cultivated by the ancestors of all modern European nations. The attested lexicological continuum witnesses the existence of a millennia-long links between the peoples of Eurasia to their mutual benefit. This research is meant to encourage interdisciplinary concerted actions between plant scientists dealing with crop evolution and biodiversity, archaeobotanists and language historians.     

Interstitial Cajal-like cells (ICLC) in human atrial myocardium

We present here visual evidence for the existence of a new type of interstitial cells in human atrial myocardium: interstitial Cajal-like cells (ICLC). These cells fulfil the so-called 'platinum standard' (a set of 10 ultrastructural criteria for the positive diagnosis of ICLC). Conventional transmission electron microscopy (TEM), followed by reconstructions from serial photomicrographs, revealed typical ICLC with 2 or 3 long, moniliform processes (several tens of micrometers long and 0.1-0.5 microm thick), emerging from the (small) cell body. Cell processes dichotomously branch and have mitochondria (at the level of dilations), caveolae and Ca(2+) release units. Cell prolongations establish close spatial relationships between each other, as well as with capillaries, myocardial cells, and other connective tissue cells. Our preliminary data suggest that ICLC exist in rat ventricular myocardium, too.     

Relationships between telocytes and cardiomyocytes during pre‐ and post‐natal life

Evidence has been given that the adult heart contains a specific population of stromal cells lying in close spatial relationship with cardiomyocytes and with cardiac stem cells in sub‐epicardial cardiogenic niches. Recently termed ‘telocytes’ because of their long cytoplasmic processes embracing the parenchymal cells, these cells have been postulated to be involved in heart morphogenesis. In our opinion, investigating the occurrence and morphology of telocytes during heart histogenesis may shed further light on this issue. Our findings show that typical telocytes are present in the mouse heart by early embryonic to adult life. These cells closely embrace the growing cardiomyocytes with their long, slender cytoplasmic processes. Hence, in the developing myocardium, telocytes may play nursing and guiding roles for myocardial precursors to form the correct three‐dimensional tissue architectural pattern, as previously suggested.     

A canopy architectural model to study the competitive ability of chickpea with sowthistle

Background and Aims

Improving the competitive ability of crops is a sustainable method of weed management. This paper shows how a virtual plant model of competition between chickpea (Cicer arietinum) and sowthistle (Sonchus oleraceus) can be used as a framework for discovering and/or developing more competitive chickpea cultivars.

Methods

The virtual plant models were developed using the L-systems formalism, parameterized according to measurements taken on plants at intervals during their development. A quasi-Monte Carlo light-environment model was used to model the effect of chickpea canopy on the development of sowthistle. The chickpea–light environment–sowthistle model (CLES model) captured the hypothesis that the architecture of chickpea plants modifies the light environment inside the canopy and determines sowthistle growth and development pattern. The resulting CLES model was parameterized for different chickpea cultivars (viz. ‘Macarena’, ‘Bumper’, ‘Jimbour’ and ‘99071-1001’) to compare their competitive ability with sowthistle. To validate the CLES model, an experiment was conducted using the same four chickpea cultivars as different treatments with a sowthistle growing under their canopy.

Results and Conclusions

The growth of sowthistle, both in silico and in glasshouse experiments, was reduced most by ‘99071-1001’, a cultivar with a short phyllochron. The second rank of competitive ability belonged to ‘Macarena’ and ‘Bumper’, while ‘Jimbour’ was the least competitive cultivar. The architecture of virtual chickpea plants modified the light inside the canopy, which influenced the growth and development of the sowthistle plants in response to different cultivars. This is the first time that a virtual plant model of a crop–weed interaction has been developed. This virtual plant model can serve as a platform for a broad range of applications in the study of chickpea–weed interactions and their environment.Key words: Plant architecture, virtual plant modelling, L-systems formalism, crop/weed competition, integrated weed management, chickpea, Cicer arietinum, sowthistle, Sonchus oleraceus     

Fluctuating Helical Asymmetry and Morphology of Snails (Gastropoda) in Divergent Microhabitats at 'Evolution Canyons I and II,' Israel

Background

Developmental instability of shelled gastropods is measured as deviations from a perfect equiangular (logarithmic) spiral. We studied six species of gastropods at ‘Evolution Canyons I and II’ in Carmel and the Galilee Mountains, Israel, respectively. The xeric, south-facing, ‘African’ slopes and the mesic, north-facing, ‘European’ slopes have dramatically different microclimates and plant communities. Moreover, ‘Evolution Canyon II’ receives more rainfall than ‘Evolution Canyon I.’

Methodology/Principal Findings

We examined fluctuating asymmetry, rate of whorl expansion, shell height, and number of rotations of the body suture in six species of terrestrial snails from the two ‘Evolution Canyons.’ The xeric ‘African’ slope should be more stressful to land snails than the ‘European’ slope, and ‘Evolution Canyon I’ should be more stressful than ‘Evolution Canyon II.’ Only Eopolita protensa jebusitica showed marginally significant differences in fluctuating helical asymmetry between the two slopes. Contrary to expectations, asymmetry was marginally greater on the ‘European’ slope. Shells of Levantina spiriplana caesareana at ‘Evolution Canyon I,’ were smaller and more asymmetric than those at ‘Evolution Canyon II.’ Moreover, shell height and number of rotations of the suture were greater on the north-facing slopes of both canyons.

Conclusions/Significance

Our data is consistent with a trade-off between drought resistance and thermoregulation in snails; Levantina was significantly smaller on the ‘African’ slope, for increasing surface area and thermoregulation, while Eopolita was larger on the ‘African’ slope, for reducing water evaporation. In addition, ‘Evolution Canyon I’ was more stressful than Evolution Canyon II’ for Levantina.     

Foliar Mn accumulation in eastern Australian herbarium specimens: prospecting for ��new�� Mn hyperaccumulators and potential applications in taxonomy

Background and Aims

The analysis of herbarium specimens has previously been used to prospect for ‘new’ hyperaccumulators, while the use of foliar manganese (Mn) concentrations as a taxonomic tool has been suggested. On the basis of their geographic and taxonomic affiliations to known Mn hyperaccumulators, six eastern Australian genera from the Queensland Herbarium collection were sampled for leaf tissue analyses.

Methods

ICP-OES was used to measure Mn and other elemental concentrations in 47 species within the genera Austromyrtus, Lenwebbia, Gossia (Myrtaceae), Macadamia (Proteaceae), Maytenus and Denhamia (Celastraceae).

Key Results

The resulting data demonstrated (a) up to seven ‘new’ Mn hyperaccumulators, mostly tropical rainforest species; (b) that one of these ‘new’ Mn hyperaccumulators also had notably elevated foliar Ni concentrations; (c) evidence of an interrelationship between foliar Mn and Al uptake among the Macadamias; (d) considerable variability of Mn hyperaccumulation within Gossia; and (e) the possibility that Maytenus cunninghamii may include subspecies.

Conclusions

Gossia bamagensis, G. fragrantissima, G. sankowsiorum, G. gonoclada and Maytenus cunninghamii were identified as ‘new’ Mn hyperaccumulators, while Gossia lucida and G. shepherdii are possible ‘new’ Mn hyperaccumulators. Of the three Myrtaceae genera examined, Mn hyperaccumulation appears restricted to Gossia, supporting its recent taxonomic revision. In the context of this present investigation and existing information, a reassesment of the general definition of Mn hyperaccumulation may be warranted. Morphological variation of Maytenus cunninghamii at two extremities was consistent with variation in Mn accumulation, indicating two possible ‘new’ subspecies. Although caution should be exercised in interpreting the data, surveying herbarium specimens by chemical analysis has provided an effective means of assessing foliar Mn accumulation. These findings should be followed up by field studies.Key words: Gossia bamagensis, G. fragrantissima, G. sankowskiorum, G. gonoclada, G. lucida, G. shepherdii, Maytenus cunninghamii, Mn hyperaccumulator     

Differential expression of anthocyanin biosynthetic genes in relation to anthocyanin accumulation in the pericarp of Litchi chinensis Sonn

Litchi has diverse fruit color phenotypes, yet no research reflects the biochemical background of this diversity. In this study, we evaluated 12 litchi cultivars for chromatic parameters and pigments, and investigated the effects of abscisic acid, forchlorofenron (CPPU), bagging and debagging treatments on fruit coloration in cv. Feizixiao, an unevenly red cultivar. Six genes encoding chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS) and UDP-glucose: flavonoid 3-O-glucosyltransferase (UFGT) were isolated from the pericarp of the fully red litchi cv. Nuomici, and their expression was analyzed in different cultivars and under the above mentioned treatments. Pericarp anthocyanin concentration varied from none to 734 mg m⁻² among the 12 litchi cultivars, which were divided into three coloration types, i.e. non-red (‘Kuixingqingpitian’, ‘Xingqiumili’, ‘Yamulong’and ‘Yongxing No. 2′), unevenly red (‘Feizixiao’ and ‘Sanyuehong’) and fully red (‘Meiguili’, ‘Baila’, Baitangying’ ’Guiwei’, ‘Nuomici’ and ‘Guinuo’). The fully red type cultivars had different levels of anthocyanin but with the same composition. The expression of the six genes, especially LcF3H, LcDFR, LcANS and LcUFGT, in the pericarp of non-red cultivars was much weaker as compared to those red cultivars. Their expression, LcDFR and LcUFGT in particular, was positively correlated with anthocyanin concentrations in the pericarp. These results suggest the late genes in the anthocyanin biosynthetic pathway were coordinately expressed during red coloration of litchi fruits. Low expression of these genes resulted in absence or extremely low anthocyanin accumulation in non-red cultivars. Zero-red pericarp from either immature or CPPU treated fruits appeared to be lacking in anthocyanins due to the absence of UFGT expression. Among these six genes, only the expression of UFGT was found significantly correlated with the pericarp anthocyanin concentration (r = 0.84). These results suggest that UFGT played a predominant role in the anthocyanin accumulation in litchi as well as pericarp coloration of a given cultivar.     

Interstitial Cajal-like cells in human gallbladder

We describe here an interstitial Cajal-like cell type (ICLC) in human gallbladder, resembling the archetypal enteric interstitial cells of Cajal. Gallbladder ICLC were demonstrated in fresh preparations (tissue cryosections) using methylene-blue, and fixed specimens in Epon semi-thin sections stained with toluidine blue or transmission electron microscopy (TEM). The positive diagnosis of gallbladder ICLC was further verified by immunohistochemistry: CD117/c-kit, CD34, and another 16 antigens: vimentin, desmin, nestin, α-smooth muscle actin, NK-1, S-100, PGP-9.5, tau protein, chromogranin A, NSE, GFAP, CD1a, CD62-P, CD68, estrogen and progesterone receptors. Double immunostaining was performed for CD117, CD34 and CD117 and nestin, respectively. In fresh specimens, the spatial density of gallbladder ICLC was 100–110 cells/mm². ICLC mainly appeared beneath the epithelium and in muscularis (about 7%, and ∼5%, respectively). In toto, ICLC represent in gallbladder ∼5.5% of subepithelial cells. TEM showed that diagnostic criteria were fulfilled by ICLC. Moreover, TEM indicated that the main ultrastructural distinctive feature for ICLC, the cell processes, develop into the characteristic shape at a relatively early stage of development. It remains to be established if, in humans, ICLC are involved in gallbladder (dis)functions (e.g. pace-making, secretion (auto-, juxta- and/or paracrine), intercellular signaling, or stone formation). M. E. Hinescu and C. Ardeleanu contributed equally to this study.     

Insights into the interstitium of ventricular myocardium: interstitial Cajal-like cells (ICLC)

We have previously described interstitial Cajal-like cells (ICLC) in human atrial myocardium. Several complementary approaches were used to verify the existence of ICLC in the interstitium of rat or human ventricular myocardium: primary cell cultures, vital stainings (e.g.: methylene blue), traditional stainings (including silver impregnation), phase contrast and non-conventional light microscopy (Epon-embedded semithin sections), transmission electron microscopy (TEM) (serial ultrathin sections), stereology, immunohistochemistry (IHC) and immunofluorescence (IF) with molecular probes. Cardiomyocytes occupy about 75% of rat ventricular myocardium volume. ICLC represent approximately 32% of the number of interstitial cells and the ratio cardiomyocytes/ICLC is about 70/1. In the interstitium, ICLC establish close contacts with nerve fibers, myocytes, blood capillaries and with immunoreactive cells (stromal synapses). ICLC show characteristic cytoplasmic processes, frequently two or three, which are very long (tens up to hundreds of microm), very thin (0.1-0.5 microm thick), with uneven caliber, having dilations, resulting in a moniliform aspect. Gap junctions between such processes can be found. Usually, the dilations are occupied by mitochondria (as revealed by Janus green B and MitoTracker Green FM) and elements of endoplasmic reticulum. Characteristically, some prolongations are flat, with a veil-like appearance, forming a labyrinthic system. ICLC display caveolae (about 1 caveola/ 1 microm cell membrane length, or 2-4% of the relative cytoplasmic volume). Mitochondria and endoplasmic reticulum (rough and smooth) occupy 5-10% and 1-2% of cytoplasmic volume, respectively. IHC revealed positive staining for CD34, EGFR and vimentin and, only in a few cases for CD117. IHC was negative for: desmin, CD57, tau, chymase, tryptase and CD13. IF showed that ventricular ICLC expressed connexin 43. We may speculate that possible ICLC roles might be: intercellular signaling (neurons, myocytes, capillaries etc.) and/or chemomechanical sensors. For pathology, it seems attractive to think that ICLC might participate in the process of cardiac repair/remodeling, arrhythmogenesis and, eventually, sudden death.     

The infection processes of Sclerotinia sclerotiorum in cotyledon tissue of a resistant and a susceptible genotype of Brassica napus

Background and Aims

Sclerotinia sclerotiorum can attack >400 plant species worldwide. Very few studies have investigated host–pathogen interactions at the plant surface and cellular level in resistant genotypes of oilseed rape/canola (Brassica napus).

Methods

Infection processes of S. sclerotiorum were examined on two B. napus genotypes, one resistant cultivar ‘Charlton’ and one susceptible ‘RQ001-02M2’ by light and scanning electron microscopy from 2 h to 8 d post-inoculation (dpi).

Key Results

The resistant ‘Charlton’ impeded fungal growth at 1, 2 and 3 dpi, suppressed formation of appresoria and infection cushions, caused extrusion of protoplast from hyphal cells and produced a hypersensitive reaction. At 8 dpi, whilst in ‘Charlton’ pathogen invasion was mainly confined to the upper epidermis, in the susceptible ‘RQ001-02M2’, colonization up to the spongy mesophyll cells was evident. Calcium oxalate crystals were found in the upper epidermis and in palisade cells in susceptible ‘RQ001-02M2’ at 6 dpi, and throughout leaf tissues at 8 dpi. In resistant ‘Charlton’, crystals were not observed at 6 dpi, whereas at 8 dpi they were mainly confined to the upper epidermis. Starch deposits were also more prevalent in ‘RQ001-02M2’.

Conclusions

This study demonstrates for the first time at the cellular level that resistance to S. sclerotiorum in B. napus is a result of retardation of pathogen development, both on the plant surface and within host tissues. The resistance mechanisms identified in this study will be useful for engineering disease-resistant genotypes and for developing markers for screening for resistance against this pathogen.     

Telocytes and putative stem cells in ageing human heart

Tradition considers that mammalian heart consists of about 70% non‐myocytes (interstitial cells) and 30% cardiomyocytes (CMs). Anyway, the presence of telocytes (TCs) has been overlooked, since they were described in 2010 (visit www.telocytes.com ). Also, the number of cardiac stem cells (CSCs) has not accurately estimated in humans during ageing. We used electron microscopy to identify and estimate the number of cells in human atrial myocardium (appendages). Three age‐related groups were studied: newborns (17 days–1 year), children (6–17 years) and adults (34–60 years). Morphometry was performed on low‐magnification electron microscope images using computer‐assisted technology. We found that interstitial area gradually increases with age from 31.3 ± 4.9% in newborns to 41 ± 5.2% in adults. Also, the number of blood capillaries (per mm²) increased with several hundreds in children and adults versus newborns. CMs are the most numerous cells, representing 76% in newborns, 88% in children and 86% in adults. Images of CMs mitoses were seen in the 17‐day newborns. Interestingly, no lipofuscin granules were found in CMs of human newborns and children. The percentage of cells that occupy interstitium were (depending on age): endothelial cells 52–62%; vascular smooth muscle cells and pericytes 22–28%, Schwann cells with nerve endings 6–7%, fibroblasts 3–10%, macrophages 1–8%, TCs about 1% and stem cells less than 1%. We cannot confirm the popular belief that cardiac fibroblasts are the most prevalent cell type in the heart and account for about 20% of myocardial volume. Numerically, TCs represent a small fraction of human cardiac interstitial cells, but because of their extensive telopodes, they achieve a 3D network that, for instance, supports CSCs. The myocardial (very) low capability to regenerate may be explained by the number of CSCs, which decreases fivefold by age (from 0.5% to 0.1% in newborns versus adults).     

Interstitial Cajal-like cells (ICLC) in atrial myocardium: ultrastructural and immunohistochemical characterization

We have previously reported (Hinescu & Popescu, 2005) the existence of interstitial Cajal-like cells (ICLC), by transmission electron microscopy, in human atrial myocardium. In the present study, ICLC were identified with non-conventional light microscopy (NCLM) on semi-thin sections stained with toluidine blue and immunohistochemistry (IHC) for CD117/c-kit, CD34, vimentin and other additional antigens for differential diagnosis. Quantitatively, on semi-thin sections, ICLC represent about 1-1.5% of the atrial myocardial volume (vs. approximately 45% working myocytes, approximately 2% endothelial cells, 3-4% for other interstitial cells, and the remaining percentage: extracellular matrix). Roughly, there is one ICLC for 8-10 working atrial myocytes in the intercellular space, beneath the epicardium, with a characteristic (pyriform, spindle or triangular) shape. These ICLC usually have 2-3 definitory processes, emerging from cell body, which usually embrace atrial myocytes (260 nm average distance plasmalemma/sarcolemma) or establish close contact with nerve fibers or capillaries (approximately 420 nm average distance to endothelial cells). Cell prolongations are characteristic: very thin (mean thickness = 0.15+/-0.1 microm), very long for a non-nervous cell (several tens of microm) and moniliform (uneven caliber). Stromal synapses between ICLC and other interstitial cells (macrophages) were found (e.g. in a multicontact type synapse, the average synaptic cleft was approximately 65 nm). Naturally, the usual cell organelles (mitochondria, smooth and rough endoplasmic reticulum, intermediate filaments) are relatively well developed. Caveolae were also visible on cell prolongations. No thick filaments were detected. IHC showed that ICLC were slightly and inconsistently positive for CD117/c-kit, variously co-expressed CD34 and EGF receptor, but appeared strongly positive for vimentin, along their prolongations. Some ICLC seemed positive for a-smooth muscle actin and tau protein, but were negative for nestin, desmin, CD13 and S-100. In conclusion, we provide further evidence of the existence of ICLC in human atrial myocardium, supporting the possible ICLC role in pacemaking, secretion (juxta- and/or paracrine), intercellular signaling (neurons and myocytes). For pathology, ICLC might as well be 'players' in arrhythmogenesis and atrial remodeling.     

Entomopathogenic fungus as a biological control for an important vector of livestock disease: the Culicoides biting midge

Background

The recent outbreak of bluetongue virus in northern Europe has led to an urgent need to identify control measures for the Culicoides (Diptera: Ceratopogonidae) biting midges that transmit it. Following successful use of the entomopathogenic fungus Metarhizium anisopliae against larval stages of biting midge Culicoides nubeculosus Meigen, we investigated the efficacy of this strain and other fungi (Beauveria bassiana, Isaria fumosorosea and Lecanicillium longisporum) as biocontrol agents against adult C. nubeculosus in laboratory and greenhouse studies.

Methodology/Findings

Exposure of midges to ‘dry’ conidia of all fungal isolates caused significant reductions in survival compared to untreated controls. Metarhizium anisopliae strain V275 was the most virulent, causing a significantly decrease in midge survival compared to all other fungal strains tested. The LT₅₀ value for strain V275 was 1.42 days compared to 2.21–3.22 days for the other isolates. The virulence of this strain was then further evaluated by exposing C. nubeculosus to varying doses (10⁸–10¹¹ conidia m⁻²) using different substrates (horse manure, damp peat, leaf litter) as a resting site. All exposed adults were found to be infected with the strain V275 four days after exposure. A further study exposed C. nubeculosus adults to ‘dry’ conidia and ‘wet’ conidia (conidia suspended in 0.03% aq. Tween 80) of strain V275 applied to damp peat and leaf litter in cages within a greenhouse. ‘Dry’ conidia were more effective than ‘wet’ conidia, causing 100% mortality after 5 days.

Conclusion/Significance

This is the first study to demonstrate that entomopathogenic fungi are potential biocontrol agents against adult Culicoides, through the application of ‘dry’ conidia on surfaces (e.g., manure, leaf litter, livestock) where the midges tend to rest. Subsequent conidial transmission between males and females may cause an increased level of fungi-induced mortality in midges thus reducing the incidence of disease.