Biochemical evidence that cytokeratins are present in smooth muscle.

Recent immunocytochemical studies have revealed that cytokeratin intermediate filaments, previously thought to be restricted to epithelial tissues, are present in muscle. In view of the implications of these reports for diagnostic pathology it is important to investigate by biochemical means whether these findings represent the presence of true cytokeratin proteins or an unexpected antigenic cross-reaction. In the present study intermediate filament proteins have been extracted from samples of human myometrium and identified by immunoblotting techniques using well characterized monoclonal antibodies, two against cytokeratins and two against desmin. The results show that the proteins of the appropriate molecular weight for cytokeratin were labelled by anti-cytokeratin antibodies and were quite distinct from those recognized by anti-desmin antibodies. This study therefore confirms previous immunocytochemical findings and emphasizes the need for caution when using anti-intermediate filament antibodies for diagnostic purposes.     

Antibodies to cytokeratins bind to epitopes in human uterine smooth muscle cells in normal and pathological pregnancies

Cytokeratin antibodies have been widely used for the identification of trophoblast cells in the placental bed, following their invasion from the developing conceptus. Their identification centres upon the expression of cytokeratin in epithelial cells, from which trophoblast cells are derived. Our recent observations indicate that this strict relationship may be more complex than was thought. Cryostat and paraffin sections of human decidua and myometrium, taken from the placental bed and the uterotomy cut, were examined immunocytochemically for cytokeratins using ten antibody clones selected to identify different cytokeratin proteins and antigenic epitopes. Biopsy specimens were obtained from normal and pathological pregnancies (pre-eclampsia, fetal retardation, amnioninfection, hysterorrhexis, placenta praevia) at the time of caesarean section (26–41 weeks of pregnancy). Antibodies against nine clones, CAM 5.2, MNF 116, AE1/AE3, CK5, KS-B17.2, CY-90, M20, E3, and 34βE12 identified, as expected, syncytial giant cells and mononuclear trophoblasts within the placental bed and glandular epithelial cells throughout the uterus. In addition, they stained numerous fusiform cells that were classified by established criteria to represent smooth muscle cells, both within blood vessels and myometrium. No staining differences were observed between normal and pathological disorders. These results indicate that cytokeratin antibodies CAM 5.2, MNF 116 and AE1/AE3, and other antibodies targeting proteins 8 and 18, cross-react with epitopes expressed in cells other than giant trophoblastic cells and mononuclear trophoblasts in the uterus and, thus, caution has to be used when such antibodies are used for the diagnostic characterization of tissues related to the placental bed.     

Both smooth and skeletal muscle precursors are present in foetal mouse oesophagus and they follow different differentiation pathways.

Muscularis externa of mouse oesophagus is composed of two skeletal muscle layers in the adult. Unlike rest of skeletal muscle in the body, the oesophageal skeletal muscle in the mouse has been proposed to be derived from fully differentiated smooth muscle cells by transdifferentiation during later foetal and early postnatal development (Patapoutian et al. [1995] Science 270:1818-1821). Here we characterised the nature of cells in muscularis externa of the mouse oesophagus by ultrastructural and immunoctyochemical analyses. The presence of differentiated skeletal muscle cells identified by positive staining for skeletal muscle specific myosin heavy chain became first apparent in the outer layer of cranial oesophagus at 14 days gestation. The transient expression of smooth muscle type alpha-actin in mouse oesophageal muscle was also apparent during foetal development. This isoform, however, was not smooth muscle specific during early development as it was also detected in foetal skeletal muscles. Compared with oesophagus, the suppression of this smooth muscle type alpha-actin during foetal development was faster in non-oesophageal skeletal muscle cells. The development of skeletal muscle in oesophagus showed a cranial to caudal and an outer layer to inner layer progression. During early foetal development, mouse oesophagus is composed of undifferentiated mesenchymal cells that formed cell clusters. Two types of cells with different staining densities could be distinguished within these cell clusters by electron microscopy. The centrally located pale staining cells gave rise to skeletal muscle cells while the peripherally positioned dense staining cells gave rise to smooth muscle cells, indicating the existence of both skeletal and smooth muscle cell precursors in mouse oesophagus during early foetal development. Further development showed an increase in the proportion of skeletal muscle cells and a decrease in size and number of the smooth muscle type cells. Apart from decrease in cell size, some other morphological features of smooth muscle cell degeneration were also observed during later foetal and early neonatal development. No smooth muscle cells undergoing transdifferentiation were observed. Both immunochemical and ultrastructural observations, thus, demonstrated the presence of skeletal muscle cells in early foetal oesophagus. It is concluded that the transient appearance of smooth muscle cells may provide a scaffold for the laying down of skeletal muscle layers in mouse oesophagus, the final disappearance of which may be triggered by lack of smooth muscle innervation.     

Peritubular myoid cells of human and rat testis are smooth muscle cells that contain desmin-type intermediate filaments

We studied the cytoskeletal composition of human and rat testicular myoid cells by using immunofluorescence microscopy with polyclonal and monoclonal antibodies. In adult human and rat testis, the peritubular myoid cell layer was brightly positive for desmin, the muscle type of intermediate filament protein, and a faint reaction was also seen with antibodies to vimentin, the intermediate filament protein of fibroblasts and diverse other mesenchymal cells. The desmin-positive myoid cell layer could already be identified in newborn rat testis but was more compact in appearance 23 days after birth. Both squash preparations and cultured cells from adult rat seminiferous tubules revealed distinct cell populations positive for desmin. The adult myoid cells of both species also showed a strong reaction with antibodies to myosin and p230, a nonerythroid avian alpha-spectrin analogue. The immunostaining results could be confirmed by the western blotting technique: Experiments with isolated seminiferous tubules showed a specific reaction with a 55,000-dalton and a 58,000-dalton polypeptide when desmin and vimentin antibodies were used, respectively. The present results show that the peritubular myoid cells are genuine smooth muscle cells with desmin-type intermediate filament cytoskeleton and suggest that these cells can be identified by this feature before their ultrastructural maturation.     

Hyperpolarization-activated cationic channels in smooth muscle cells are stretch sensitive

The properties of hyperpolarization-activated channels were studied in single smooth muscle cells from the stomach of the toad, Bufo marinus, using the patchclamp technique. In cell-attached patches, inward channel currents were activated by hyperpolarizing pulses from a holding potential of –20 mV to potentials more negative than –60 mV. The activity of the channels increased and their latency of activation decreased as the hyperpolarization was increased. The slope conductance of the channels with standard high sodium concentration pipette solution was 64.2±9.1 pS (SD, n=17). Stretching the patch, by suction applied to the back of the patch pipette, also increased the activity and shortened the latency of activation. We designate these channels as HA-SACs (hyperpolarization- and stretch-activated channels). HA-SACs were observed in 83% (175/210) of the patches studied. HA-SAC currents were carried by sodium and potassium ions, but their amplitude was increased by replacing extracellular sodium with potassium. Extracellular magnesium and calcium ions significantly reduced the single-channel conductance of HA-SACs. These permeation characteristics and the single-channel conductance of HA-SACs were indistinguishable from those of stretch-activated channels (SACs) previously described in these cells. The following observations are consistent with HA-SACs being a subset of SACs. First, SACs were at times found in cell-attached patches which lacked HA-SACs. Second, the number of channels in a cell-attached patch simultaneously activated by stretch (usually 5–10 and often more) exceeded by far the number simultaneously activated by hyperpolarization (usually one or two). Third, in excised insideout patches, the ability of hyperpolarization to activat HA-SACs was lost within 3–5 min, whereas stretch continued to activate a large number of channels. The last observation also suggests that activation by hyperpolarization requires an intracellular agent, whereas stretch activation does not.     

Autoantibodies to vascular smooth muscle are pathogenic for vasculitis

We have previously shown that microvascular smooth muscle activates CD4+ T lymphocytes in sterile co-culture, presents antigen, and produces inflammatory cytokines. Adoptive transfer of lymphocytes co-cultured with syngeneic smooth muscle cells to healthy recipient mice results in vasculitic lesions predominantly in postcapillary venules. The present study assessed the pathogenic role of immunoglobulin and B cells in a murine model of vasculitis. Here, we show that transferred B cells, including plasmablast cells, accumulated, persisted, and proliferated in lung and secondary lymphoid organs of recipient mice. The induction of vasculitis was accompanied by production of IgM and IgG2a autoantibodies specific for vascular smooth muscle intracellular antigens. Circulating immunoglobulin had a pathogenic role in this vasculitis model, because the disease could be induced by transfer of serum from vasculitic mice to untreated animals but not by transfer of serum depleted of anti-smooth muscle autoantibodies. Additionally, the pathogenic mechanisms triggered by the transfer of vasculitogenic serum were dependent on T lymphocytes because both wild-type and B cell-deficient mice developed the disease after serum transfer, whereas RAG2-deficient mice did not. Thus, immunoglobulin and cell-mediated pathways work in concert to produce vasculitis in this model.     

A cytokeratin immunohistochemical study of cholestatic liver disease: evidence that hepatocytes can express 'bile duct-type' cytokeratins

A cytokeratin immunohistochemical study was performed on 38 liver biopsies from cases of primary biliary cirrhosis, primary sclerosing cholangitis, extrahepatic biliary obstruction or drug-induced liver disease in order to analyse the cytoskeletal changes in detail. On paraffin sections of 27 cases, a variable number of hepatocytes were reactive with a polyclonal anti-cytokeratin antiserum that, in the normal liver, stains bile duct cells only. On cryostat sections of 23 cases, a variable number of hepatocytes were immunoreactive with a monoclonal antibody specifically directed against cytokeratin no. 7 and were most numerous in cases of long-standing cholestasis irrespective of the aetiology. In three cases of primary sclerosing cholangitis and two cases of primary biliary cirrhosis a few hepatocytes were also weakly positive with a monoclonal antibody specific for cytokeratin no. 19. Since cytokeratins no. 7 and no. 19 are, in the normal liver, restricted to bile duct cells, these results further support the concept of 'ductular metaplasia' of hepatocytes, the mechanism of which remains unclear.     

A cytokeratin immunohistochemical study of alcoholic liver disease: evidence that hepatocytes can express ''bile duct-type'' cytokeratins

A cytokeratin immunohistochemical study was performed on 40 liver biopsies diagnosed as alcoholic liver disease to further investigate the cytoskeletal changes occurring in this disease. On paraffin sections of 29 cases, a variable number of hepatocytes were reactive with a polyclonal antiserum that normally stains only bile ducts. Using monoclonal antibodies specific for a single cytokeratin polypeptide on cryostat sections, a variable number of hepatocytes were immunoreactive for cytokeratin no. 7 in 23 cases and also for cytokeratin no. 19 in seven cases. Both these polypeptides are restricted to bile duct cells in the normal liver. The number of hepatocytes positive for bile duct-type cytokeratins increased and their location changed with the severity of the disease. Mallory bodies were reactive with monoclonal antibodies CAM 5.2 and anti-cytokeratin no. 18 but unreactive with anti-cytokeratin no. 8. except in one case. In two cases, Mallory bodies reactive with both monoclonal antibodies anti-cytokeratin no. 7 and anti-cytokeratin no. 19 were found. These results clearly indicate that hepatocytes in alcoholic liver disease can express immunoreactivity for bile duct-type cytokeratins. Our data also demonstrate heterogeneity in the composition of Mallory bodies. Whether hepatocytes expressing bile duct-type cytokeratins are the precursors of Mallory body-containing cells is not clear at present.     

Cytokeratins in smooth muscle cells and smooth muscle tumours

A keratin positive metastatic leiomyosarcoma in the lung, which resulted in diagnostic error, is reported. The results of additional studies of 17 benign and malignant leiomyogenic tumours with various keratin antibodies are presented and discussed in the light of recent bibliographical data.     

Cytokeratin expression in smooth muscle and smooth muscle tumours

The expression of cytokeratin intermediate filaments by a tumour has been accepted as evidence of an epithelial origin. Although there have been anecdotal reports of cytokeratin expression within tissues and neoplasms of non-epithelial origin, particularly muscle, there have been no comprehensive studies of its frequency and distribution. In order to investigate this we have studied 51 cases of normal smooth muscle and benign and malignant smooth muscle tumours using a panel of monoclonal antibodies against a range of intermediate filaments (cytokeratins, desmin and vimentin). Cytokeratin expression was noted overall in 50% of normal, benign and malignant smooth muscle tissues. Such expression tended to have a focal or patchy distribution. No case expressed cytokeratins in the absence of both desmin and vimentin. The implication of these findings for diagnostic immunocytochemistry is that intermediate filaments alone are not completely reliable markers of tumour histogenesis and should be used as part of a larger panel of monoclonal antibodies.     

Immunocytochemical evidence that endometrial stromal granulocytes are granulated lymphocytes

Endometrial stromal granulocytes (EGs) are prominent in late luteal phase human endometrium and in early pregnancy decidua. They have been believed to develop from endometrial stromal cells and to secrete relaxin. Recent immunohistochemical studies have suggested that EGs are derived from bone marrow but this has been difficult to prove, mainly because the characteristic cytoplasmic granules are not preserved in frozen tissues. Two separate approaches have now been employed to investigate the cellular lineage of EGs. Formalin-fixed paraffin-embedded sections of first trimester decidua were labelled by an immunoperoxidase method with four monoclonal antibodies (mAbs) reactive with routinely fixed and processed tissues. In addition, acetone-fixed smears of decidual cell suspensions were labelled with a panel of mAbs. Sections and smears were counterstained to demonstrate the characteristic cytoplasmic granules of EGs. Endometrial granulocytes were LCA+, CD2+, MT1+, and UCHL1+, which provides evidence that they are leucocytes. EGs are probably members of the large granular lymphocytes series and may have an essential role in normal implantation and placentation.     

Myosin filaments isolated from skinned amphibian smooth muscle cells are side-polar

Summary The structure of myosin filaments isolated from skinned toad stomach smooth muscle cells has been examined by electron microscopy as a step toward identifying thein vivo structure. When negatively stained following exposure to relaxing conditions, the filaments exhibited a continuous 14-nm axial repeat of crossbridge projections with no central bare zone. The filaments thus differed from the bipolar filaments found in striated muscle and displayed instead features resembling side-polar and mixed-polarity filament models. By rotation of isolated filaments around their longitudinal axes it was found that cross bridges occurred only along two sides of the filament, an arrangement consistent with the side-polar but not the mixed-polarity model. The polarity is thus similar to that proposed for ribbons (Small & Squire,J. molec. Biol. 67, (1972) 17–149) and for synthetic smooth muscle myosin filaments (Craig and Megerman,J. Cell Biol. 75, (1977) 990–996); their appearance in cross-section, however, shows that these structures are filaments (i.e. with two axes of similar dimensions) and not broad ribbons. As the filaments were derived directly from skinned cells which contracted and relaxed in response to physiological levels of MgATP and Ca²⁺ at rates comparable to those of native, isolated cells, this unusual arrangement of cross bridges appears to be an effective, functional form of myosin in the contractile apparatus. Side-polar filaments therefore merit consideration as plausible candidates for the native organization of myosin in vertebrate smooth muscle cells.     

Genetic evidence that metacyclic forms of Trypanosoma brucei are diploid

The hypothesis that metacyclic trypanosomes are haploid has been tested genetically. Five cloned stocks of Trypanosoma brucei (each having four known isoenzyme markers and six known restriction fragment length polymorphisms) have been independently transmitted through tsetse flies. Fifteen individual metacyclic organisms were taken from flies with mature cyclical infections and used to establish fresh clones. All the sub-clones from all the flies proved to be identical to the starting (parental) stocks, with respect to all the markers examined, including those markers which were heterozygous in the parental stocks. We conclude that metacyclic trypanosomes are diploid, and are not the product of an obligatory meiosis.     

Microtubule-associated protein tau epitopes are present in fiber lesions in diverse muscle disorders.

The microtubule-associated protein tau is a major cytoskeletal protein involved in the neurofibrillary tangles of Alzheimer's disease. Although tau is predominantly a neuronal protein, it has been demonstrated in glia and other nonneuronal cells. We describe the presence of microtubule-associated protein tau epitopes in various muscle fiber lesions in oculopharyngeal and Becker muscular dystrophy, dermatomyositis, central core disease, neurogenic atrophy, and in the recovery phase of an attack of malignant hyperthermia. Western blot demonstrated a 100- to 110-kd tau-immunoreactive protein probably corresponding to 'big tau' as described in peripheral nerves. Tau immunoreactivity in muscle fiber lesions usually co-localized with tubulin, although electron microscopy failed to show an increase in microtubules. Tau and tubulin reactivity also correlated with the presence of desmin and vimentin epitopes. Possible explanations for the presence of tau are briefly discussed.     

Quickly tapping targets that are flashed during smooth pursuit reveals perceptual mislocalisations

In various studies subjects have been shown to misperceive the positions of targets that are flashed during pursuit eye movements. They mislocalise them in the direction of pursuit. Nevertheless, Hansen (1979) found that subjects accurately hit targets that are flashed during pursuit with a quick hammer blow. We examined whether this is because there is a fundamental difference between the information that determines our perceptual judgements of a targets position and the information that is used to guide our hand to a similar target. Subjects were asked to quickly tap targets that were flashed during pursuit with their index finger. They systematically tapped ahead of the position of the flash, in accordance with the above-mentioned perceptual mislocalisations. Thus the lack of systematic errors in Hansens study is not a general property of fast motor responses.This work was financed by the Research Council for Earth and Life Sciences (ALW, grant number 809–37.006) of the Netherlands Organisation for Scientific Research (NWO)     

Guthrie card methylomics identifies temporally stable epialleles that are present at birth in humans

A major concern in common disease epigenomics is distinguishing causal from consequential epigenetic variation. One means of addressing this issue is to identify the temporal origins of epigenetic variants via longitudinal analyses. However, prospective birth-cohort studies are expensive and time consuming. Here, we report DNA methylomics of archived Guthrie cards for the retrospective longitudinal analyses of in-utero-derived DNA methylation variation. We first validate two methodologies for generating comprehensive DNA methylomes from Guthrie cards. Then, using an integrated epigenomic/genomic analysis of Guthrie cards and follow-up samplings, we identify interindividual DNA methylation variation that is present both at birth and 3 yr later. These findings suggest that disease-relevant epigenetic variation could be detected at birth, i.e., before overt clinical disease. Guthrie card methylomics offers a potentially powerful and cost-effective strategy for studying the dynamics of interindividual epigenomic variation in a range of common human diseases.There is increasing interest in exploring the possibility that interindividual epigenetic variation plays a role in common human disease etiology (for review, see Petronis 2010; Rakyan et al. 2011). Consequently, epigenome-wide association studies (EWASs), analogous to genome-wide association studies (GWASs) for disease-associated genetic variants (Rakyan et al. 2011), are currently being conducted to systematically uncover epigenetic variants for a range of different diseases (www.roadmapepigenomics.org/participants lists some of the current NIH-funded EWASs). However, the disease process itself can also induce epigenetic changes. Therefore, an EWAS design that just compares post-disease affected with unaffected individuals will not distinguish causal from consequential disease-associated epigenetic variation. The inability to make this distinction will ultimately prove to be a significant barrier to establishing the etiological role or medical value for any disease-associated epigenetic variants.One way of addressing this issue is to determine whether the epigenetic variants are detectable before overt disease. Although such variants could arise at any point during an individual''s lifespan (Fraga et al. 2005), in-utero development is considered to be a period during which the epigenome is particularly susceptible to stochastic and/or environmental factors such as infection, poor diet, or stress (for review, see Warner and Ozanne 2010). A variety of common diseases have been proposed to have a component of in-utero origins including asthma, metabolic conditions including type 2 diabetes, and autoimmune disorders like multiple sclerosis (Heijmans et al. 2008; Warner and Ozanne 2010). Hence, being able to determine epigenomic profiles at birth, and by implication before overt disease, would be invaluable for elucidating the origins and potentially the etiological role of disease-associated epigenetic variants. In this regard, prospective birth-cohort studies are very useful but, unfortunately, are expensive, time-consuming, and difficult to establish.In 1963, Robert Guthrie proposed spotting a small amount of neonatal blood onto filter paper for the large-scale biochemical screening of various diseases such as phenylketonuria (Guthrie and Susi 1963). “Guthrie cards” are now routinely created by major national health authorities; e.g., since 2000, over 90% of all neonates in the United States had Guthrie cards made shortly after birth (Pollitt 2009). Remarkably, in many cases Guthrie cards are being stored indefinitely. This led us to consider genome-wide DNA methylation profiling—DNA methylomics—of Guthrie cards as a means of investigating the origins of disease-associated epigenetic variation. Crucially, this longitudinal investigation could be done retrospectively at a fraction of the cost and time investment required for prospective studies.