Long-term ultrastructural indices of lead intoxication in pulmonary tissue of the rat.

In the present research long-term pulmonary toxicity of lead was investigated in rats treated by intraperitoneal administration of lead acetate for three consecutive days (25 mg/kg per day). Five weeks after treatment average lead content in the whole blood was 0.41 µg/dL ± 0.05, in the lung homogenates it measured 3.35 µg/g ± 0.54, as compared to the control values of 0.13 ± 0.07 µg/dL and 1.03 µg/g ± 0.59, respectively. X-ray microanalysis of lung specimens displayed lead localized mainly within type II pneumocytes and macrophages. At the ultrastructural level the effects of lead toxicity were found in lung capillaries, interstitium, epithelial cells, and alveolar lining. Alveolar septa showed intense fibrosis, consisting of collagen, elastin, and fibroblasts. Thinned alveolar septa had emphysematous tissue with some revealing signs of angiogenesis. Type II pneumocytes contained lamellar bodies with features of laminar destruction. Fragments of the surfactant layer were often detached from the alveolar epithelium. These findings indicate that 5 weeks after exposure, lead provokes reconstruction of the alveolar septa including fibrosis and emphysematous changes in the lung tissue.

Functional alterations in endothelial NO, PGI2 and EDHF pathways in aorta in ApoE/LDLR-/- mice.

Adequate endothelial production of nitric oxide (NO), endothelium-derived hyperpolarizing factor (EDHF), and prostacyclin (PGI2) is critical to the maintenance of vascular homeostasis. However, it is not clear whether alterations in each of these vasodilatory pathways contribute to the impaired endothelial function in murine atherosclerosis. In the present study, we analyze the alterations in NO-, EDHF- and PGI2-dependent endothelial function in the thoracic aorta in relation to the development of atherosclerotic plaques in apoE/LDLR⁻/⁻ mice. We found that in the aorta of 2-month-old apoE/LDLR⁻/⁻ mice there was no lipid deposition, subendothelial macrophage accumulation; and matrix metalloproteinase (MMP) activity was low, consistent with the absence of atherosclerotic plaques. Interestingly, at this stage the endothelium was already activated and hypertrophic as evidenced by electron microscopy, while acetylcholine-induced NO-dependent relaxation in the thoracic aorta was impaired, with concomitant upregulation of cyclooxygenase-2 (COX-2)/PGI2 and EDHF (epoxyeicosatrienoic acids, EETs) pathways. In the aorta of 3-6-month-old apoE/LDLR⁻/⁻ mice, lipid deposition, macrophage accumulation and MMP activity in the intima were gradually increased, while impairment of NO-dependent function and compensatory upregulation of COX-2/PGI2 and EDHF pathways were more accentuated. These results suggest that impairment of NO-dependent relaxation precedes the development of atherosclerosis in the aorta and early upregulation of COX-2/PGI2 and EDHF pathways may compensate for the loss of the biological activity of NO.

Ultrastructural changes in lung tissue after acute lead intoxication in the rat.

Pulmonary toxicity of lead was studied in rats after an intraperitoneal administration of lead acetate at a dose of 25 mg/kg. Three consecutive days of treatment increased lead content in the whole blood to 2.1 µg/dl and in lung homogenate it attained 9.62 µg/g w.w. versus control values of 0.17 µg/dl and 0.78 µg/g w.w., respectively. At the ultrastructural level, the effects of lead toxicity were observed in lung capillaries, interstitium, epithelial cells and alveolar lining layer. Accumulation of aggregated platelets, leucocytic elements and monocytes was found within capillaries. Interstitium comprised a substantial number of collagen, elastin filaments and lipofibroblasts. Lamellar bodies of type II pneumocytes contained phospolipid lamellae, which stratified into an irregular arrangement. Pulmonary alveoli were filled with macrophages. The extracellular lining layer of lung alveoli was partially destroyed. This study provided evidence that acute lead intoxication affects the whole lung parenchyma and by impairing production of the surfactant might disturb the regular respiratory function.

Genetic and ultrastructural studies in dilated cardiomyopathy patients: a large deletion in the lamin A/C gene is associated with cardiomyocyte nuclear envelope disruption.

Major nuclear envelope abnormalities, such as disruption and/or presence of intranuclear organelles, have rarely been described in cardiomyocytes from dilated cardiomyopathy (DCM) patients. In this study, we screened a series of 25 unrelated DCM patient samples for (a) cardiomyocyte nuclear abnormalities and (b) mutations in LMNA and TMPO as they are two DCM-causing genes that encode proteins involved in maintaining nuclear envelope architecture. Among the 25 heart samples investigated, we identified major cardiomyocyte nuclear abnormalities in 8 patients. Direct sequencing allowed the detection of three heterozygous LMNA mutations (p.D192G, p.Q353K and p.R541S) in three patients. By multiplex ligation-dependant probe amplification (MLPA)/quantitative real-time PCR, we found a heterozygous deletion encompassing exons 3-12 of the LMNA gene in one patient. Immunostaining demonstrated that this deletion led to a decrease in lamin A/C expression in cardiomyocytes from this patient. This LMNA deletion as well as the p.D192G mutation was found in patients displaying major cardiomyocyte nuclear envelope abnormalities, while the p.Q353K and p.R541S mutations were found in patients without specific nuclear envelope abnormalities. None of the DCM patients included in the study carried a mutation in the TMPO gene. Taken together, we found no evidence of a genotype-phenotype relationship between the onset and the severity of DCM, the presence of nuclear abnormalities and the presence or absence of LMNA mutations. We demonstrated that a large deletion in LMNA associated with reduced levels of the protein in the nuclear envelope suggesting a haploinsufficiency mechanism can lead to cardiomyocyte nuclear envelope disruption and thus underlie the pathogenesis of DCM.

Ultrastructural and immunochemical studies of glial scar formation in diabetic rats.

We explored the rebuilding of the brain parenchyma after surgical injury due to reactive astrogliosis. In the present study, we investigated the initial stages of rebuilding in the perilesional cortex of streptozotocin-induced diabetic rats. Our methods utilized ultrastructural and immunohistochemical studies as well as Western blot analysis of glial fibrillary acidic protein (GFAP) and vimentin. Data was collected at 2 days, 7 days and 2 months following a unilateral sensorimotor cortex lesion. Electron-microscopic studies revealed not only formation of glial scar tissue but also ultrastructural features of death in the elements of neurovascular unit. Immunohistochemical studies, confirmed by Western blot analysis, demonstrated the enhancement of vimentin and GFAP immunoreactivity (IR) in astrocytes located in the perilesion cortical area of the diabetic rats that were operated upon. We suggest that the process of rebuilding brain parenchyma following surgical injury may be disturbed by the induction of astrocytes and the degeneration of astrocytes, as well as by morphological changes within capillaries that are accompanied by the presence of macrophages.

Staphylococcal cysteine protease staphopain B (SspB) induces rapid engulfment of human neutrophils and monocytes by macrophages.

Abstract Circulating neutrophils and monocytes constitute the first line of antibacterial defence, which is responsible for the phagocytosis and killing of microorganisms. Previously, we have described that the staphylococcal cysteine proteinase staphopain B (SspB) cleaves CD11b on peripheral blood phagocytes, inducing the rapid development of features of atypical cell death in protease-treated cells. Here, we report that exposure of phagocytes to SspB critically impairs their antibacterial functions. Specifically, SspB blocks phagocytosis of Staphylococcus aureus by both neutrophils and monocytes, represses their chemotactic activity and induces extensive, nonphlogistic clearance of SspB-treated cells by macrophages. The proteinase also cleaves CD31, a major repulsion ('do not-eat-me') signal, on the surface of neutrophils. We suggest that both proteolytic degradation of repulsion signals and induction of 'eat-me' signals on the surface of leukocytes are responsible for the observed intensive phagocytosis of SspB-treated neutrophils by human monocyte-derived macrophages. Collectively, this may lead to the depletion of functional neutrophils at the site of infection, thus facilitating staphylococcal colonisation and spreading.

MMP2 and MMP9 in immature endothelial cells following surgical injury of rat cerebral cortex--a preliminary study.

Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases capable of extracellular matrix remodeling. They have been implicated in various physiological and pathological phenomena involving neurovascular unit elements (neurons, astrocytes and capillary vessels). Surgical injury of the fronto-temporal region of rat cerebral cortex induces massive neurodegeneration and cell death accompanied by astrogliosis. In the area adjacent to the damage, the induction of angiogenesis is observed, in which process immature endothelial cells are important players. The aim of this study was to examine the expression of MMP2 and MMP9 in cortical blood vessels following the surgical injury of cerebral cortex in the rat. Our data demonstrated absence of immature endothelial cells and lack of or only weak MMP2 and MMP9 immunoreactivities in some cortical capillaries in intact rats. Following the surgery, we found high MMPs' immunoreactivities in walls of the vessels located in the cortex adjacent to the lesion site, and particularly in the immature endothelial cells. These results show for the first time the presence of MMPs in the immature (progenitor?) endothelial cells following surgical brain injury..

Apoptotic death of cortical neurons following surgical brain injury.

Unilateral surgical brain injury of the rat is a model of surgery-related brain damage of humans. Our preliminary experiments showed two phenomena within the damaged cortical region of rat brain. Those were: degeneration and death of neurons and massive gliosis. In the present study we aimed to investigate the mechanisms of neuronal death following brain injury and to characterize responses of glial cells to the damage. We analyzed the morphological changes and alterations of immunochemical profile of cells localized in the brain areas adjacent to the lesion. Our data show the massive neuronal death following the lesion. Neurons undergo necrosis and apoptosis, but on the 4th day following the operation apoptosis prevails. Apoptotic cells showed heavy immunostaining for proapoptotic Bax and caspase 3. This result suggests the involvement of these proteins in neuronal apoptosis in our experimental model. Neuronal death is accompanied by the induction of astrogliosis in the perilesional cortical area. Astrocytes became hypertrophic. We did not detect any dying astrocytes at the investigated time point, but there is a possibility that apoptosis may occur in astroglia during another time period following the damage. This question requires further studies in our experimental model.

Obliteration of cardiomyocyte nuclear architecture in a patient with LMNA gene mutation.

OBJECTIVE: The aim of our study was to perform an immunohistochemical and ultrastructural analysis of the nuclear architecture of cardiomyocytes from an end-stage DCM patient with a missense point mutation in the exon 3 of the LMNA gene which is predicted to result in a D192G substitution. METHODS: We studied endomyocardial biopsy samples taken from the right ventricle by immunostaining using antibodies against the lamins A and C and by electron microscopy. The cardiomyocyte ultrastructure was analysed, with particular attention to the nuclear architecture. RESULTS: Thirty percent of cardiomyocyte nuclei from the D192G carrier showed chromatin disorganization and a changed nuclear shape. The most surprising finding was the appearance of sarcoplasmic organelles within the nuclear matrix of well enveloped nuclei. To our knowledge, this intriguing phenomenon was observed for the first time in cardiomyocytes. CONCLUSION: The study documents that D192G mutation in LMNA gene may lead to the disruption of the nuclear wall in cardiomyocytes, thus supporting the mechanical hypothesis of dilated cardiomyopathy development in humans, which might be mutation-specific.

Detection of mitochondrial dysfunction by EPR technique in mouse model of dilated cardiomyopathy.

Tgalphaq44 mice with targeted overexpression of activated Galphaq protein in cardiomyocytes mimic many of the phenotypic characteristics of dilated cardiomyopathy in humans. However, it is not known whether the phenotype of Tgalphaq44 mice would also involve dysfunction of cardiac mitochondria. The aim of the present work was to examine changes in EPR signals of semiquinones and iron in Fe-S clusters, as compared to classical biochemical indices of mitochondrial function in hearts from Tgalphaq44 mice in relation to the progression of heart failure. Tgalphaq44 mice at the age of 14 months displayed pulmonary congestion, increased heart/body ratio and impairment of cardiac function as measured in vivo by MRI. However, in hearts from Tgalphaq44 mice already at the age of 10 months EPR signals of semiquinones, as well as cyt c oxidase activity were decreased, suggesting alterations in mitochondrial electron flow. Furthermore, in 14-months old Tgalphaq44 mice loss of iron in Fe-S clusters, impaired citrate synthase activity, and altered mitochondrial ultrastructure were observed, supporting mitochondrial dysfunction in Tgalphaq44 mice. In conclusion, the assessment of semiquinones content and Fe(III) analysis by EPR represents a rational approach to detect dysfunction of cardiac mitochondria. Decreased contents of semiquinones detected by EPR and a parallel decrease in cyt c oxidase activity occurs before hemodynamic decompensation of heart failure in Tgalphaq44 mice suggesting that alterations in function of cardiac mitochondria contribute to the development of the overt heart failure in this model.

[Morphological and clinical aspects of Danon disease]. / Morfologiczne i kliniczne aspekty choroby Danona.

BACKGROUND: Danon disease, an X-linked hypertrophic cardiomyopathy, is caused by primary deficiency of lysosome-associated membrane protein (LAMP-2). The pathological hallmark of the disease is the appearance of intracytoplasmic vacuoles containing autophagic material and the absence of LAMP-2 activity in the muscle. AIM: To define the LAMP-2 protein deficiency we investigated cardiac and skeletal muscle of a 19-year-old man with hypertrophic cardiomyopathy without clinically apparent skeletal myopathy or mental impairment, whose mother died suddenly at 46 years of age. METHODS: Clinical, morphological, immunohistochemical and ultrastructural analysis was performed. Paraffin sections of cardiac muscle were stained using routine histochemical methods. Frozen sections of skeletal muscle were stained using histochemical methods as well as using monoclonal antisera against N-terminal of dystrophin and antisera against LAMP-2. Ultrastructural examination of both cardiac and skeletal muscle specimens were performed. RESULTS: Cardiac and skeletal muscle revealed an excessive accumulation of early and late autophagic vacuoles containing various cytoplasmic debris. In immunohistochemical analysis the vacuolar membrane seen in skeletal muscle was decorated with antibody against dystrophin and such vacuoles were negative for LAMP-2. CONCLUSION: Ultrastructural and immunohistochemical analysis of skeletal muscle (less invasive than myocardial biopsy) may be used in diagnosis of Danon disease. Early diagnosis of Danon disease is important for timely cardiac transplantation, the only effective therapeutic option.

The immature endothelial cell in human glioma. Ultrastructural features of blood capillary vessels.

New vessel formation is a prerequisite for the growth of a tumour mass. Growing evidence suggests that endothelial progenitor cells circulate in the blood and participate in that process. The purpose of the present study was ultrastructural and electron microscopic immunocytochemical examination of capillary blood vessels in human glioma. The results showed striking morphological changes in these vessels. Our observations indicate that tumours build vessels by cooption of pre-existing vasculature and de novo recruitment of endothelial progenitor cells. Immature endothelial cells characterized by fibrils in the cytoplasm and Flk-1 positive immunoreactivity were observed as small clusters or luminally localized individual endothelial progenitor cells that participate in intussusceptive vessel growth. This observation indicates that the tumour microenvironment determines biological and functional attributes of endothelial progenitor cells.

Abnormal chaperone-mediated autophagy (CMA) in cardiomyocytes of a boy with Danon disease.

Ultrastructural analysis of the cardiomyocyte structure in Danon disease reveals dramatic accumulation of abnormal late autophagic vacuoles (AVd) suggestive of primary lysosomal defect. Moreover, the accumulation of AVd in cardiomyocytes is consistent with a decreased rate of autophagic to lysosomal trafficking. These results suggest that the loss of the LAMP-2 protein strongly inhibits uptake of proteins into lysosomes for degeneration. The significant reduction of chaperone-mediated autophagy (CMA) activity in the affected cardiomyocytes induces a dramatic increase in the number and size of AVd and a severe reduction of myocardial contractility.

[Ultrastructural features of normal and dysfunctional endothelium of the blood vessels]. / Cechy ultrastrukturalne prawidlowego i dysfunkcyjnego sródblonka naczyn krwionosnych.

Endothelium of the blood vessels plays a very important role in blood circulation, inflammation, atherosclerosis and cancer progression. We describe the ultrastructural morphology, function of endothelium and its participation in vessel formation during regenerative or inflammatory processes in adults. Transmission and scanning electron microscopic studies indicate on endothelial cell function in atherosclerotic plaque formation.

Dilated cardiomyopathy caused by LMNA mutations. Clinical and morphological studies.

BACKGROUND: Dilated cardiomyopathy (DCM) is familial in about 20-35% of patients. The most frequently encountered mutations associated with DCM are found in LMNA. AIM: To define the frequency of LMNA mutations in a series of consecutive DCM patients and to evaluate the phenotype of mutation carriers. METHODS: We screened the 12 exons of LMNA in a series of 61 Polish patients with DCM diagnosed angiographically, as well as in two DCM families. RESULTS: Two mutations were detected in 5 mutation carriers (D192G in one proband and Y481Stop in one proband and 3 of his offspring), which represents 3.3% (2/61) of the DCM patients. These mutations were absent from 100 controls. The D192G mutation was found in a 26-year-old patient with mild DCM and heart failure leading to death within two years after onset of symptoms. Mild conduction disease was also present. Ultrastructural analysis of the endomyocardial biopsy showed a striking alteration of nuclear morphology. This finding can explain nuclear fragility and is in agreement with the pathophysiological mechanical hypothesis of LMNA mutations. All four Y481Stop mutation-carriers were affected. Three phenotypes were found: in the proband, cardiac dysrhythmia and pacemaker requirement preceded DCM leading to heart transplantation; the proband's 13-year old daughter had conduction disease (2nd degree A-V block) with subtle skeletal muscle involvement documented by immunofluorescence study; ventricular arrhythmia was detected in the proband's son at the age of 11 and in the proband's daughter at the age of 18. Serum creatine kinase was normal in all mutation carriers.

The immature endothelial cell in new vessel formation following surgical injury in rat brain.

OBJECTIVES: We investigated neovasculatization in the cerebral cortex of the adult rat after surgical brain injury by ultrastructural, immunocytochemical and immunochemical means. Previously we described endothelial-like cell that participates in new vessel formation on plasma proteins that served as a provisional matrix in the region immediately adjacent to the traumatic injury. In the present study we describe new vessel formation in the multistep process with the alterations in endothelial-like cell immunophenotype. METHOD: The observations were conducted from 2 to 7 days after induction of cortical trauma. Traumatic injury was induced in the frontotemporal region of cerebral cortex. RESULTS: We show that endothelial-like cell could not successfully terminate its development without the presence of pericyte and astrocyte. New formed blood vessels were accompanied by fibroblast and lipofibroblast cells differentiated probably from common progenitor. MAIN FINDINGS: Our result suggests that endothelial-like cell is committed endothelial cell between progenitor endothelial cell and terminally differentiated endothelium stage. Therefore, we propose that our of endothelial stem cells present in blood at different stages of morphogenetic differentiation and specifically arrested in "check points" of development trauma mobilize the group of the most differentiated progenitors. These may contribute to new vessel formation. CONCLUSION: Our model should be useful for the characterization of endothelial commitment and endothelial cell differentiation after brain injury.

Application of the lithium and magnesium initiators for the synthesis of glycolide, lactide, and epsilon-caprolactone copolymers biocompatible with brain tissue.

The subject of this work is new method of the synthesis of biodegradable copolymers compatible with brain tissue. Copolymerization of glycolide with lactide was conducted in solution or in bulk in the presence of LiBu, LiAcac, MgBu(2), Mg(acac)(2) as initiators. In all cases, copolymers with molecular weight of 20000-40000 were obtained, which enables to use them as drug carriers. During the reactions of copolymer chain growth, the intermolecular transesterification occurs, changing the distribution of comonomeric units in copolymer chain. Magnesium initiators showed a lower contribution to transesterification in comparison with lithium and calcium compounds. The copolymerization of glycolide with epsilon-caprolactone using magnesium compounds as initiators was also described. The random glycolide/epsilon-caprolactone copolymer (10/90) obtained with MgBu(2) was used in in vivo study in the forms of microspheres and foils. Complete degradation of microspheres during 6 weeks was observed after the implantation to brain tissue. All implanted copolymers are compatible with brain tissue.

Involvement of immature endothelial cells in vascular alterations in Alzheimer's disease.

The aim of the present study was to investigate ultrastructural features of cerebral capillaries and the pattern of new vessel formation in a patient with Alzheimer's disease (AD). Recent neuropathological studies have demonstrated that patients with AD have cerebrovascular pathology. Using electron microscopy, we showed that alterations of the capillaries are a common finding both in vascular disease and in AD, suggesting that vascular factors may also play a role in the pathogenesis of AD. We also found regionally increased capillary density, and in many sections immature endothelial cells lying on the preexisting endothelium were present in the lumen of capillaries. These cells might thus contribute to the pathological pattern of capillaries. The cytoplasm of immature endothelial cells in the patient with AD was characterized by accumulation of amyloid fibrils. We suggest that immature endothelial cells may be an important source of circulation-derived amyloid in the brain.

Inhibition of poly(ADP-ribose) polymerase activity protects hippocampal cells against morphological and ultrastructural alteration evoked by ischemia-reperfusion injury.

Poly(ADP-ribose) polymerase 1 (PARP-1 EC 2.4.2.30) is a nuclear enzyme that plays an important role in cell survival and death. PARP is involved in DNA repair machinery, however, massive DNA damage leads to over-activation of PARP-1 and to depletion of its substrate bNAD+ which causes cell death. Our previous study indicated that the PARP activity was significantly activated during ischemia-reperfusion injury. In this study we investigated the effect of PARP inhibitor, 3-aminobenzamide (3-AB) on intracellular organelles alteration. Gerbils were submitted to 3 and 10 min transient global ischemia followed by recirculation and survival for 1 till 7 days. The histological and electron microscopic examination indicated a pronounced protective effect of 3-AB on the swelling of astrocytes and neurons 1 day after 3 and 10 min ischemic insult. It decreased also the swelling of pericytes. 3-AB decreases evoked by ischemia swelling of mitochondria and Golgi apparatus. The significant ameliorating effect of 3-AB was also observed on the 7th day of reperfusion after 3 min ischemia and was also visible on the 1st day after 10 min ischemia. However, 7 days after prolonged 10 min ischemia almost all neurons in the CA1 hippocampal layer died and 3-AB was not able to protect these cells. In spite of that, 3-AB markedly decreased immunostaining of glial fibrillary acidic protein (GFAP), which was enhanced in the stratum: oriens, radiatum and lacunosum-moleculare at the 7th day after 10 min ischemia. These data indicated that inhibition of PARP may have a protective effect on neuronal cells affected by ischemia-reperfusion injury.

Calcification of Trichinella spiralis larval capsule.

Trichinella spiralis larvae were examined in TEM to identify calcareous corpuscles in the outer part of parasite capsule. The microroentgenographic analysis of calcareous corpuscles mainly demonstrated the presence of phosphorus and calcium. The physiological importance of calcareous corpuscles, as well as their significance in decay of T. spiralis larvae were discussed.