Contractile Microfilaments Research Articles

Cell Migration Guided by Cell-Cell Contacts in Innate Immunity.

The directed migration of leukocytes to sites of damage or infection is necessary for a productive immune response. There is substantial evidence supporting a key role for chemoattractants in directed migration, however, less is known about how cell-cell contacts affect the migratory behavior of leukocytes in innate immunity. Here, we explore how cell-cell contacts can affect the directed migration of innate immune cells, including their role in attracting, repelling, or stopping cell motility. Further investigation of cell contact dynamics as guidance cues may yield new insights into the regulation of innate immunity.

Cytochalasin B induces apoptosis through the mitochondrial apoptotic pathway in HeLa human cervical carcinoma cells

Cytochalasin B (CB) is a cell-permeable mycotoxin. It inhibits cytoplasmic division by blocking the formation of contractile microfilaments, it inhibits cell movement and induces nuclear extrusion. In the present study, we investigated the anticancer activity of CB in HeLa human cervical carcinoma cells. CB showed significant cytotoxicity, with an IC50 of 7.9 µM, in a WST-8 assay and significantly inhibited cell proliferation. Furthermore, results from Annexin V-FITC/propidium iodide double-staining indicated that CB induced early apoptosis of HeLa cells in a time-dependent manner. The cells exhibited apoptotic morphology, including cell shrinkage and nuclear condensation. CB induced cell cycle arrest at the S phase. We also observed inhibition of DNA replication in a [3H]-thymidine incorporation assay. Furthermore, CB induced a time-dependent increase in reactive oxygen species and a decrease in mitochondrial membrane potential. Western blot analysis showed an increase in levels of mitochondrial factors Bax and Bcl-2, which was followed by activation of caspase-9 and -3. These results suggested that CB induced apoptosis via a mitochondrial-dependent pathway in HeLa cells.

Antioxidants counteract lipopolysaccharide-triggered alterations of human colonic smooth muscle cells

Gut dysmotility develops in individuals during and after recovering from infective acute gastroenteritis and it is apparently due to a direct effect of circulating lipopolysaccharides (LPS). This is an endotoxin with a prooxidant activity derived from gram-negative bacteria. Due to the lack of human models available so far, the mechanisms underlying LPS-induced gut dysmotility are, however, poorly investigated. In the present work long-term effects of LPS and their reversibility have been assessed by means of different analytical cytology methods on pure primary cultures of human colonic smooth muscle cells. We found that LPS triggered the following alterations: (i) a redox imbalance with profound changes of contractile microfilament network, and (ii) the induction of cell cycle progression with dedifferentiation from a contractile to a synthetic phenotype. These alterations persisted also after LPS removal. Importantly, two unrelated antioxidants, alpha-tocopherol and N-acetylcysteine, were able to reverse the cytopathic effects of LPS and to restore normal muscle cell function. The present data indicate that LPS is capable of triggering a persistent and long-term response that could contribute to muscle dysfunction occurring after an infective and related inflammatory burst and suggest a reappraisal of antioxidants in the management of postinfective motor disorders of the gut.

The Differential Action of Cytocalasin D in T-tubular Remodelling of Ventricular Myocytes

Cytocalasin D (cytoD) is a fungal metabolite that inhibits cytokinesis by blocking formation of contractile microfilament structures resulting in multi-nucleated cells, reversible inhibition of cell movement, and the induction of cellular extrusion. However, in muscle cells reports are controversial and vary from actin stabilization to actin disruption at concentrations of 40 µM. The aim of this study was to investigate the putative cytoD effects on adult cardiomyocytes in culture and how it can be used as a pharmacological tool in single cell models. This investigation is based on our optimized serum-free culture method for adult rat ventricular myocytes.Initially, for global calcium transients we performed repetitive dose response relationships over the time course of 6 days to identify the optimal cytoD concentration. At this identified concentration we analyzed action potentials, calcium handling (incl. post rest behavior and caffein induced calcium release) and cellular contractility. In addition, we investigated cytoD-mediated changes in the T-tubular system and actin cytoskeleton by confocal and STED microscopy, respectively.In contrast to previous reports we were able to identify a cytoD concentration that resulted in a high degree of morphological and functional conservation of properties found in freshly isolated cells over a time course of one week.In conclusion our findings represent a further significant improvement for single cell models of cardiomyocytes, enabling chronic stimulation and an extended time period for adenoviral protein expression. The identified relationship between actin filament modulation and prevention of T-tubular loss might shed new light on the loss of T-tubules in cardiac diseases.This work was supported by the DFG (KFO196) and the Medical Faculty (HOMFOR).

Androgen activity on the peripheral female genital target: ultrastructural study

There are still many areas in the field of female sexual physiopathologies that remain to be fully studied, among which are disturbances of the excitation phase, particularly the hemodynamics of the clitoral corpora cavernosa. Low levels of estrogens and vascular risks lie at the base of female sexual arousal disorders. The aim of our study was to investigate the ultrastructure of the clitoral cavernosal tissue in healthy women and those affected by metabolic and hormonal pathologies. Recent data show the presence of nitric oxide synthase in the clitoral tissue, suggesting that nitric oxide could be involved in the mechanisms of clitoral tumescence. Moreover, the presence of type 5 phosphodiesterase activity in clitoral tissue has been hypothesized. Informed consent was obtained from the 24 women enrolled in the study. Five were diabetic premenopausal women, and 8 were naturally post menopausal and 6 surgically post menopausal women affected by HSDD. They all underwent a micro-biopsy of the clitoral body by means of fine needle aspiration under total anesthesia during gynaecological operation for stress urinary incontinence. Five health women undergoing surgically procedures for benign gynaecological pathology were considered as control group. The tissue removed was fixed in Karnowski's liquid and underwent ultrastructural analysis with standard procedures. Histomorphometric studies have shown that there is a correlation between age and smooth muscle fibers of the clitoral corpora cavernosa due to an increase of connective tissue. In the clitoris there is also a correlation between large diameter thin-walled vessels and smooth muscle fibrocells(SMF), which make up the majority of the cellular elements of the concavity. In health premenopausal women there are extensive bundles of smooth muscle fibrocells SMF, seen both transversely and longitudinally; the SMF have regular borders, regular nuclei with dense chromatin and evident nucleoli. The cytoplasm includes contractile microfilaments and dense bodies. The cell borders do not have continuous basal membranes(BM) and electro-ionic junctions of the “tight” type. The SMF, in all pre menopause subjects, are isometric with regular borders, having a homogeneous distribution of the microfilaments and dense bodies. The basal membranes are regular with a constant thickness, rarely reduplicate. The blood vessels are thin-walled, made up of a flattened endothelial layer and by an incomplete layer of pericytes, a close association with the interstitial SMF is observed, which, with the pericytes, form tight junctions. The vascular lacunae are particularly interesting, having a single line of flattened epithelial cells with a cytoplasm relatively poor in organules, resting on continuous BM. The SMF have a direct relationship with the vascular lacunae, giving rise to an anatomical-functional complex comparable to that of the corpora cavernosa of the penis. In diabetic women, the SMF of the BM and the vascular lacunae are notably thickened. In natural post menopausal subjects the general arrangement is maintained, and only rarely is there a discrete thinning of the SMF, which can, in places, have an irregular border. The concavity, made up of isometric collagen fibers that can be poorly cohesive and moderately thickened in post menopausal subjects. In surgically postmenopausal women, clitoral tissue is reduced; the SMF have irregular distribution of the microfilaments and borders. The blood vessels are riduced, and the vascular lacunae have not relationship the SMF. Transdermic testosterone therapy of surgically postmenopausal women affected by HSDD seems to also act at the level of the clitoral tissue. In fact, the ultrastructure of the clitoral corpora cavernosa has features almost identical to those found in pre menopausal women. We hope the electronic microscopic study of the clitoral cavernosal tissue can give similar information to that recorded above, and can be used as an additional tool for the diagnosis, prognosis and therapeutic orientation of female sexual dysfunctions.

4NQO-Induced Rat Tongue Carcinoma: An Ultrastructural Study

The 4-nitroquinoline 1-oxide (4NQO)-induced rat tongue carcinoma, in which the carcinogen is administered systemically in drinking water, is the most comparable animal model to the development of human oral carcinoma. This is the first study to report the ultrastructural changes in this model. The most significant changes were observed in the carcinoma cells at the invasion front and included unique modifications in the basal lamina, presence of micropinocytotic vesicles (plasmalemmal caveolae), and emergence of cytoplasmic microfilaments featuring a parallel arrangement. The microfilaments, in both appearance and organization, were consistent with contractile microfilaments. These observations may be the morphological reflection of the phenotypic modifications occurring within the carcinoma cells, approaching smooth muscle differentiation.

A stromal myoid cell line provokes thymic erythropoiesis between 16th to 20th weeks of intrauterine life.

The thymus provides an optimal cellular and humoral microenvironment for cell line committed differentiation of haematopoietic stem cells. The immigration process requires the secretion of at least one peptide called thymotaxine by cells of the reticulo-epithelial (RE) network of the thymic stromal cellular microenvironment. The thymic RE cells are functionally specialised based on their intrathymic location and this differentiation is modulated by various interaction signals of differentiating thymocytes and other non lymphatic haematopoietic stem cells. To study the role of another cell line in fetal thymic haematopoietic proliferation and differentiation in different stages of development: the stromal myoid cells. Fifteen cases of fetal thymic specimens (4th to 8th weeks: five cases 16th to 20th weeks: five cases and 28th to 32nd weeks: five cases respectively) were studied. Tissue paraffin samples were stained immunohistochemically using (i) a monoclonal antibody recognising alpha-smooth muscle actin, a contractile microfilament expressed exclusively by smooth muscle cells, myofibroblasts and related cells, (ii) a monoclonal antibody glycophorin C recognising the erythropoietic cells. Histology-Embryology Department of Democritus University of Thrace (Alexandroupolis) over ten year period (1991-2001). The number of alpha-smooth muscle actin-positive cells significantly increased during the late second and third trimester of gestation. In the above period a relevant increase in the number of glycophorin C positive cells were observed. Our data suggest that a myoid cell line is involved in the formation of an appropriate microenvironment for homing and proliferation of erythropoietic cells.

The role of contractile microfilaments in the morphogenesis of the developing foregut of chick embryos

The role of contractile microfilaments in the morphogenesis of the developing foregut of chick embryos

The role of contractile microfilaments in the morphogenesis of the developing foregut of chick embryos.

The cellular processes that lead to changes in shape (morphogenesis) and organ formation (organogenesis) are poorly understood. Local contraction of microfilaments can change cell shape and lead to changes of tissue shape. To clarify the role of contractile microfilaments in the foregut morphogenesis of chick embryos, 2- to 4-day-old embryos were exposed to cytochalasin D (CD), which is known to disrupt microfilaments. Untreated age-matched embryos were used as controls. Sections of treated embryos and controls were stained with phalloidin, which binds to actin, and examined with a fluorescence microscope. Microdissected specimens were examined using a scanning electron microscope (SEM). Immunofluorescent staining showed a bright signal belt toward the apical cell region of the foregut epithelium in controls. This signal was not evident in CD-exposed embryos. SEM micrographs of the controls showed the cranial foregut as a smooth, even, cylindrical structure in all stages studied. The lumen was narrow and perfectly straight, the ventral and dorsal walls were in close apposition. The foregut of CD-exposed specimens, however, showed a wide lumen and the walls were separated from each other. The structure seemed atonic and appeared conical, curved, or tilted. We observed a dense microfilament network toward the apical cell pole of the epithelial foregut cells of controls that was no longer evident after CD exposure. This network seems to play an important role in foregut morphogenesis, since actin-filament disruption by CD causes loss of the normal shape.

Vascular Smooth Muscle α-Actin Gene Transcription during Myofibroblast Differentiation Requires Sp1/3 Protein Binding Proximal to the MCAT Enhancer

The conversion of stromal fibroblasts into contractile myofibroblasts is an essential feature of the wound-healing response that is mediated by transforming growth factor beta1 (TGF-beta1) and accompanied by transient activation of the vascular smooth muscle alpha-actin (SmalphaA) gene. Multiple positive-regulatory elements were identified as essential mediators of basal SmalphaA enhancer activity in mouse AKR-2B stromal fibroblasts. Three of these elements bind transcriptional activating proteins of known identity in fibroblasts. A fourth site, shown previously to be susceptible to single-strand modifying agents in myofibroblasts, was additionally required for enhancer response to TGF-beta1. However, TGF-beta1 activation was not accompanied by a stoichiometric increase in protein binding to any known positive element in the SmalphaA enhancer. By using oligonucleotide affinity isolation, DNA-binding site competition, gel mobility shift assays, and protein overexpression in SL2 and COS7 cells, we demonstrate that the transcription factors Sp1 and Sp3 can stimulate SmalphaA enhancer activity. One of the sites that bind Sp1/3 corresponds to the region of the SmalphaA enhancer required for TGF-beta1 amplification. Additionally, the TGF-beta1 receptor-regulated Smad proteins, in particular Smad3, are rate-limiting for SmalphaA enhancer activation. Whereas Smad proteins collaborate with Sp1 in activating several stromal cell-associated promoters, they appear to operate independently from the Sp1/3 proteins in activating the SmalphaA enhancer. The identification of Sp and Smad proteins as essential, independent activators of the SmalphaA enhancer provides new insight into the poorly understood process of myofibroblast differentiation.

Contraction-mediated pinocytosis of RGD-peptide by dermal fibroblasts: inhibition of matrix attachment blocks contraction and disrupts microfilament organisation.

Force generation in collagen and matrix contraction are basic functions of fibroblasts and important elements of tissue repair. Cell-matrix attachment is critical to this contraction, involving RGD-binding integrins. We have investigated how this process operates, in terms of force generation (in the Culture Force Monitor) and cytoskeletal structure, using a synthetic RGD-decapeptide. The RGD-peptide blocked force generation over the first 6 h, followed by near complete recovery by 20 h. However, dose response was complex indicating multiple processes were operating. Analysis of cytoskeletal structure after treatment with RGD-peptide indicated major disruption with condensed aggregates of actin and microtubular fragmentation. Fluorescent labeling and tracking of the RGD-peptide demonstrated intracellular uptake into discrete cytoplasmic aggregates. Critically, these RGD-peptide pools co-localised with the condensed actin microfilament aggregates. It is concluded that RGD-peptide uptake was by a form of contraction-mediated pinocytosis, resulting from mechanical tension applied to the untethered RGD-peptide-integrin, as contractile microfilament were assembled. These findings emphasize the importance of sound mechanical attachment of ligand-occupied integrins (e.g., to extracellular matrix) for normal cytoskeletal function. Conversely, this aspect of unrestrained cytoskeletal contraction may have important pathogenic and therapeutic applications.

Cytoskeletal control of cell length regulation

It was shown that mouse embryo fibroblasts and human foreskin diploid fibroblasts of AGO 1523 line cultivated on specially prepared substrates with narrow (15 +/- 3 microns) linear adhesive strips were elongated and oriented along the strips, but the mean lengths of the fibroblasts of each type on the strips differed from those on the standard culture substrates. In contrast to the normal fibroblasts, the length of mouse embryonic fibroblasts with inactivated gene-suppresser Rb responsible for negative control of cell proliferation (MEF Rb-/-), ras-transformed mouse embryonic fibroblasts (MEF Rb-/-ras), or normal rat epitheliocytes of IAR2 line significantly exceeded those of the same cells on the standard culture substrates. The results of experiments with the drugs specifically affecting the cytoskeleton (colcemid and cytochalasin D) suggest that the constant mean length of normal fibroblasts is controlled by a dynamic equilibrium between two forces: centripetal tension of contractile actin-myosin microfilaments and centrifugal force generated by growing microtubules. This cytoskeletal mechanism is disturbed in MEF Rb-/- or MEF Rb-/-ras, probably, because of an impaired actin cytoskeleton and also in IAR2 epitheliocytes due to the different organization of the actin-myosin system in these cells, as compared to that in the fibroblasts.

Evidence of tubulin in the scolex gland ducts of Gymnorhynchus gigas plerocercoid (Cestoda: Trypanorhyncha).

Evidence of tubulin in the scolex gland ducts of Gymnorhynchus gigas plerocercoid (Cestoda: Trypanorhyncha).

Opposing views on tensegrity as a structural framework for understanding cell mechanics.

Donald E. Ingber: Important new theories in science often ignite heated debates. If they do not, they are probably of little significance. Thus a strong argument in support of the importance of the tensegrity model of cell and tissue architecture first proposed almost 20 years ago ([23][1], [24][2

Cytokinesis in Tetrahymena: determination of division plane and organization of contractile ring.

A protein, Tetrahymena p85, is localized to the presumptive division plane before the formation of the contractile ring. p85 directly interacts with Tetrahymena calmodulin (CaM) in a Ca(2+)-dependent manner, and p85 and CaM colocalize in the division furrow. A Ca(2+)/CaM inhibitor N-(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide HCl (W7) inhibits the direct interaction between p85 and Ca(2+)/CaM. W7 also inhibits the localization of p85 and CaM to the division plane, and the formation of the contractile ring and division furrow. Tetrahymena fimbrin and elongation factor-1a (EF-1alpha), which induce bundling of Tetrahymena F-actin, are also localized to the division furrow during cytokinesis. The Tetrahymena fimbrin has two actin-binding domains, but lacks the EF-hand Ca(2+)-binding motif, suggesting that Tetrahymena fimbrin probably cross-links actin filaments in a Ca(2+)- insensitive manner during cytokinesis. The evidence also indicates that Ca(2+)/CaM inhibits the F-actin-bundling activity of EF-1alpha; and EF-1alpha and CaM colocalize in the division furrow. In this review, we propose that the Ca(2+)/CaM signal and its target protein p85 cooperatively regulate the determination of the division plane, and that a Ca(2+)-insensitive actin-bundling protein, Tetrahymena fimbrin, and a Ca(2+)/CaM-sensitive actin-bundling protein, EF-1alpha, play pivotal roles in regulating the organization of the contractile ring microfilaments.

Neutralizing antibody to TGFβ modulates stromal fibrosis but not regression of photoablative effect following PRK

Previous studies have suggested that corneal fibrosis controlled by a TGFbeta-mediated cytocrine pathway underlies the development of clinical corneal haze and associated regression of photoablative effect following excimer laser PRK. Using a unique blocking antibody, we evaluated the role of TGFbeta in post-PRK corneal wound healing as measured by in vivo Confocal Microscopy Through Focusing (CMTF). Twelve rabbits received a monocular, 6-mm diameter, 9.0 D PRK myopic correction. Six animals received 50 microg of anti-TGFbeta blocking antibodies applied topically 3x/day for three days post-PRK, while six animals received vehicle alone. An additional six animals served as unoperated controls. At various times during a four-month-period, animals were evaluated using CMTF, which generates a quantitative image intensity depth profile of the cornea. The location and reflectivity of corneal structures were identified from CMTF-profiles and used to determine epithelial and stromal thickness and corneal light reflectivity as an objective estimate of corneal haze. To correlate in vivo and ex vivo morphology, an additional six rabbits were analyzed at differing temporal intervals post-PRK for the expression and cytoskeletal organization of contractile microfilaments: f-actin (stress fibers) and alpha-smooth muscle actin (a molecular marker for myofibroblast transformation). Anti-TGFbeta treated corneas showed significantly less CMTF measured light reflectivity (ANOVA, p < 0.02) following PRK compared to vehicle treated corneas with a 34% decrease at two weeks (2513 +/- 758 U compared to 3810 +/- 1262 U) and a 61% reduction in reflectivity at four months (447 +/- 208 U compared to 1154 +/- 585 U). The reduction in early development of light reflecting structures and the more rapid decline appeared related to anti-TGFbeta-mediated inhibition of keratocyte activation and proliferation, myofibroblast transformation, and stromal fibrosis. Between anti-TGFbeta and vehicle treated corneas, no significant differences were detected in either photoablation depth (126 +/- 9 microm versus 126 +/- 7 microm) or regression of photoablative effect (postoperative stromal thickening at four months: 95 +/- 16 microm versus 95 +/- 10 microm). Histologic examination demonstrated that regression of photoablative effect in anti-TGFbeta treated corneas was related entirely to regeneration by corneal growth underlying the photoablated stromal surface. In vehicle treated corneas, fibrosis or deposition of new fibrotic tissue above the photoablated stromal surface was observed but contributed only about 25% of the total postoperative stromal thickening. No epithelial hyperplasia was detected. In unoperated control animals, a physiologic stromal thickening of 5 +/- 2 microm per month (p < 0.001) was observed. This study confirms our earlier observations that increased corneal light reflectivity following PRK is predominantly due to: (1) distortion of the photoablated stromal surface leading to prominent reflections; and (2) increased reflections from activated and transformed keratocytes. Anti-TGFbeta reduced keratocyte activation and transformation and inhibited stromal fibrosis, leading to a reduction in early light reflectivity as well as to a more rapid decline. Of greatest interest is the unexpected finding that anti-TGFbeta treatment inhibited stromal fibrosis without reducing or delaying post-PRK stromal re-thickening. Based on these findings we propose that corneal thickness may be tightly and dynamically regulated by an unknown, non-TGFbeta mediated pathway. We propose that anti-TGFbeta treatment may be useful in reducing post-PRK corneal haze development in patients by: (1) inhibiting the recruitment of highly reflective, activated keratocytes, (2) inhibiting myofibroblast transformation, and 3) reducing stromal fibrosis.

Neutralizing antibody to TGFβ modulates stromal fibrosis but not regression of photoablative effect following PRK

Purpose. Previous studies have suggested that corneal fibrosis controlled by a TGFβ-mediated cytocrine pathway underlies the development of clinical corneal haze and associated regression of photoablative effect following excimer laser PRK. Using a unique blocking antibody, we evaluated the role of TGFβ in post-PRK corneal wound healing as measured by in vivo Con-focal Microscopy Through Focusing (CMTF).Methods. Twelve rabbits received a monocular, 6-mm diameter, 9.0 D PRK myopic correction. Six animals received 50 μg of anti-TGFβ blocking antibodies applied topically 3×/day for three days post-PRK, while six animals received vehicle alone. An additional six animals served as unoperated controls. At various times during a four-month-period, animals were evaluated using CMTF, which generates a quantitative image intensity depth profile of the cornea. The location and reflectivity of corneal structures were identified from CMTF-profiles and used to determine epithelial and stromal thickness and corneal light reflectivity as an objective estimate of corneal haze. To correlate in vivo and ex vivo morphology, an additional six rabbits were analyzed at differing temporal intervals post-PRK for the expression and cytoskeletal organization of contractile micro-filaments: f-actin (stress fibers) and α-smooth muscle actin (a molecular marker for myofibroblast transformation).Results. Anti-TGFβ treated corneas showed significantly less CMTF measured light reflectivity (ANOVA, p < 0.02) following PRK compared to vehicle treated corneas with a 34% decrease at two weeks (2513 ± 758 U compared to 3810 ± 1262 U) and a 61% reduction in reflectivity at four months (447 ± 208 U compared to 1154 ± 585 U). The reduction in early development of light reflecting structures and the more rapid decline appeared related to anti-TGFβ-mediated inhibition of keratocyte activation and proliferation, myofibroblast transformation, and stromal fibrosis. Between anti-TGFβ and vehicle treated corneas, no significant differences were detected in either photoablation depth (126 ± 9 μm versus 126 ± 7 μm) or regression of photoablative effect (postoperative stromal thickening at four months: 95 ± 16 μm versus 95 ± 10 μm). Histologic examination demonstrated that regression of photoablative effect in anti-TGFβ treated corneas was related entirely to regeneration by corneal growth underlying the photoablated stromal surface. In vehicle treated corneas, fibrosis or deposition of new fibrotic tissue above the photoablated stromal surface was observed but contributed only about 25% of the total postoperative stromal thickening. No epithelial hyperplasia was detected. In unoperated control animals, a physiologic stromal thickening of 5 ± 2 μm per month (p > 0.001) was observed.Conclusions. This study confirms our earlier observations that increased corneal light reflectivity following PRK is predominantly due to: (1) distortion of the photoablated stromal surface leading to prominent reflections; and (2) increased reflections from activated and transformed keratocytes. Anti-TGFβ reduced keratocyte activation and transformation and inhibited stromal fibrosis, leading to a reduction in early light reflectivity as well as to a more rapid decline. Of greatest interest is the unexpected finding that anti-TGFβ treatment inhibited stromal fibrosis without reducing or delaying post-PRK stromal re-thickening. Based on these findings we propose that corneal thickness may be tightly and dynamically regulated by an unknown, non-TGFβ mediated pathway. We propose that anti-TGFβ treatment may be useful in reducing post-PRK corneal haze development in patients by: (1) inhibiting the recruitment of highly reflective, activated keratocytes, (2) inhibiting myofibroblast transformation, and 3) reducing stromal fibrosis. Curr. Eye Res. 17:736–747, 1998.

Balanced mechanical forces and microtubule contribution to fibroblast contraction.

Fibroblast locomotion is thought to generate tractional forces which lead to contraction and reorganisation of collagen in tissue development and repair. A culture force monitor device (CFM) was used to measure changes in force in fibroblast populated collagen lattices, which resulted from cytoskeletal reorganisation by cytochalasin B, colchicine, vinblastine, and taxol. Microfilament disruption abolished contraction forces, microtubule disruption elicited a new peak of contraction, while taxol stabilisation of microtubules produced a gradual fall in measured force across the collagen gel. Based on these measurements, it is suggested that the cell can be viewed as an engineering structure in which residual intracellular forces, from contractile microfilaments, exert compressive loading on microtubular elements. This microtubular structure appears to act as a "balanced space frame" (analogous to an aeroplane chassis), maintaining cell shape and consequently storing a residual internal tension (RIT). In dermal fibroblasts this hidden RIT was up to 33% of the measurable force exerted on the collagen gel. Phenotypic differences between space frame organisation and RIT levels could explain site and pathological variations in fibroblast contraction.

A novel method for monitoring Mycobacterium bovis BCG trafficking with recombinant BCG expressing green fluorescent protein.

To better understand intracellular and extracellular trafficking of Mycobacterium bovis bacillus Calmette-Guérin (BCG) when used as an intravesical agent in the treatment of transitional cell carcinoma (TCC) of the bladder, recombinant BCG (rBCG) expressing the jellyfish green fluorescent protein (GFP) was created. When the MB49.1 murine TCC cell line was incubated with GFP-expressing rBCG, internalization of the pathogen could be directly visualized by UV microscopy and quantitated by flow cytometry. The in vitro internalization of the GFP rBCG by the bladder tumor cells was temperature dependent, occurring most readily at 37 degrees C and being severely inhibited at 4 degrees C. Optimum internalization was achieved in vitro at a 10:1 BCG-to-tumor cell ratio over 24 h during which approximately 16% of the tumor cells became infected. Cytochalasin B, a phagocytosis inhibitor, abrogated the ingestion by almost 100% at a concentration of 200 micrograms/ml, indicating that contractile microfilaments likely played an important role in this process. By using mitomycin, a DNA cross-linking reagent, to inhibit proliferation of MB49.1 cells, clearance of about 40% of the green rBCG was achieved by 3 days postinfection. No significant difference between the GFP rBCG and wild-type BCG was observed in the ability to induce the expression of cell membrane proteins of major histocompatibility classes I and II, ICAM-I and -II, B7-1 and -2, of Fas from MB49.1 cells or cytokine production from mouse spleen cells. These results indicate that GFP rBCG may serve as a useful substitute for wild-type BCG in future studies of in vivo trafficking experimental and clinical immunotherapy.

Investigation into the histogenesis of congenital epulis of the newborn

Five previously unreported cases of congenital epulis of the newborn are presented. All five cases were on the anterior maxillary alveolar ridge. Four were removed at 2 days of age and one at 7 weeks. Light microscopy demonstrated large eosinophilic granular cells within vascular fibrous connective tissue. Immunohistochemical studies revealed a positivity for vimentin and neuron specific enolase. Cytogenetic evaluation performed on one case was normal. Estrogen and progesterone receptors were absent in the one case so studied. Electron microscopy demonstrated tumor cells that were filled with autophagosomes. Cellular organelles were significantly reduced and inversely related to the number of cytoplasmic autophagosomes. Many of the autophagosomes contained collagen precursors. Poorly formed junctional complexes were seen. Occasional tumor cells demonstrated long processes that contained contractile microfilaments, pinocytosis, and areas of exocytosis. These studies suggest the tumor cells represent early mesodermal cells that express pericytic and myofibroblastic features that undergo cytoplasmic autophagocytosis.