Malondialdehyde Epitopes as Targets of Immunity and the Implications for Atherosclerosis.

Accumulating evidence suggests that oxidation-specific epitopes (OSEs) constitute a novel class of damage-associated molecular patterns (DAMPs) generated during high oxidative stress but also in the physiological process of apoptosis. To deal with the potentially harmful consequences of such epitopes, the immune system has developed several mechanisms to protect from OSEs and to orchestrate their clearance, including IgM natural antibodies and both cellular- and membrane-bound receptors. Here, we focus on malondialdehyde (MDA) epitopes as prominent examples of OSEs that trigger both innate and adaptive immune responses. First, we review the mechanisms of MDA generation, the different types of adducts on various biomolecules and provide relevant examples for physiological carriers of MDA such as apoptotic cells, microvesicles, or oxidized low-density lipoproteins. Based on recent insights, we argue that MDA epitopes contribute to the maintenance of homeostatic functions by acting as markers of elevated oxidative stress and tissue damage. We discuss multiple lines of evidence that MDA epitopes are proinflammatory and thus important targets of innate and adaptive immune responses. Finally, we illustrate the relevance of MDA epitopes in human pathologies by describing their capacity to drive inflammatory processes in atherosclerosis and highlighting protective mechanisms of immunity that could be exploited for therapeutic purposes.

Activation of cortical and inhibited differentiation of medullary epithelial cells in the thymus of lymphotoxin-beta receptor-deficient mice: an ultrastructural study.

The reciprocal influences of thymic lymphocyte and nonlymphocyte populations, i.e. thymic cross-talk, are necessary for the proper maturation of thymocytes and the development/maintenance of thymic stromal microenvironments. Although the molecular influences exerted by thymic stromal cells on maturing thymocytes have been extensively studied, the identity of signalling molecules used by thymocytes to influence the thymic stromal cells is still largely unknown. Our study provides the first ultrastructural evidence that the functional lymphotoxin-beta receptor (LTbetaR) signalling pathway is engaged in the cross-talk between thymocytes and the thymic stromal cell population. We show that LTbetaR signalling is of the utmost significance for the preservation of the subcellular integrity of all thymic epithelial cells. In the absence of LTbetaR there is (1) hypertrophy and activation of cortical thymic epithelial cells, (2) the complete loss of fully differentiated medullary thymic epithelial cells, and (3) the inhibited differentiation of remaining medullary thymic epithelial cells with the appearance of prominent intercellular cysts in the thymic medulla.

A quantitative morphometric study of rectal mucosa in adult and aged healthy subjects.

Rectal mucosa is relatively susceptible to pathological processes and frequently it is affected by various diseases. However, there is a notable lack of quantitative data regarding normal rectal mucosa, which would provide a reference for histoquantitative studies of the pathologically changed tissue. Therefore, we obtained the tissue from 27 healthy patients subjected to diagnostic rectoscopy during active screening for asymptomatic cancer of the large intestine, in which no disease was found. Using computer-aided morphometric analysis, we studied all structural elements of the rectal mucosa. The patients were divided into four groups according to the age and sex: adult males, elderly males, adult females and elderly females. The patients under 60 years of age were grouped as adult and those older than 60 years as aged subjects. A decreased height of surface epithelium was registered in both elderly male and female groups. This finding, however, was significant only when adult and elderly male groups were compared. The tendency towards reduction of the mucosal height was also registered comparing male adult and elderly groups. The number of crypts per 0.1 mm2 of tissue increased with aging in both males and females, whereby the crypts were always more numerous in males than in females. The increase in number of crypts in male subjects was accompanied by a decrease in their diameter and perimeter. The changes associated with ageing were discrete and affected only the male subjects.

Comparison of the efficacy of two different dosage dacarbazine-based regimens and two regimens without dacarbazine in metastatic melanoma: a single-centre randomized four-arm study.

The aim of this randomized four-arm phase III study was to evaluate whether there is a difference in activity between regimens containing dacarbazine and regimens without dacarbazine in metastatic melanoma, whether there is a dose-effect relationship for dacarbazine, and whether non-dacarbazine-containing aggressive regimens are in any way superior to non-aggressive ones. A total of 219 patients with metastatic cutaneous melanoma were included in this study; 196 of them were evaluable for activity. The patients were randomized into four treatment arms: arm A (standard dose dacarbazine arm), vincristine 1.4 mg/m2 on day 1, carmustine (BCNU) 60 mg/m2 on day 1, and dacarbazine 300 mg/m2 per 24 h on days 2-5; arm B (high-dose dacarbazine arm), vincristine and BCNU as in arm A and dacarbazine 600 mg/m2 per 24 h on days 2-5; arm C ('aggressive' regimen without dacarbazine), vindesine 3 mg/m2 on day 1, bleomycin 7 mg/m2 per 24 h on days 1-4, and cisplatin 30 mg/m2 per 24 h on days 5-8; arm D ('non-aggressive' regimen without dacarbazine), BCNU 100 mg/m2 on day 1 and procarbazine 90 mg/m2 per 24 h on days 1-10. The four arms were well balanced with regard to patient- and disease-related characteristics. On an intend-to-treat basis, the response rate was 11 out of 49 (22%) in arm A, nine out of 47 (19%) in arm B, 16 out of 63 (25%) in arm C and nine out of 60 (15%) in arm D. There was a large overlap between the 95% confidence intervals and no significant differences in the response rates between the four arms. Median survival in the four treatment arms was 4, 5, 6 and 4 months, respectively, again with no significant differences. Median survival for responders (8, 11, 10 and 13 months, respectively) in all four arms was significantly longer than in non-responders (4, 3, 5 and 4 months, respectively). Arms A, B and C were significantly more toxic compared with arm D, which was for all practical purposes devoid of toxicities. The efficacy of all four regimens thus appeared comparable both in terms of response rate and survival. Responders in all four arms achieved a survival benefit. There does not seem to be a dose-effect relationship for dacarbazine in metastatic melanoma. Chemotherapy from arm D, might be well suited for 'fragile' or elderly patients due to the lack of toxicity.

Involution of bursa cloacalis (Fabricii) and thymus in Cyclosporin A-treated chickens.

Fifty-day-old Arbor Acres chickens received a daily oral dose of 30 mg/kg Cyclosporin A for 21 consecutive days. This treatment induced the striking morphological changes in the bursa cloacalis (Fabricii). The number of bursal follicles did not change, but they were markedly decreased in size. The pars lymphoreticularis was collapsed and contained smaller accumulations of lymphocytes. The pars lymphoepithelialis was almost totally devoid of lymphocytes and it was composed of an empty network of epithelial cells. Interfollicular epithelium, follicle-associated epithelium, basement-membrane-associated epithelium and bursal epithelial cytoreticulum were not changed. The thymus also displayed substantial structural changes, whereby the thymic medulla was strongly reduced in size.

Splenectomy of rats selectively reduces lymphocyte function-associated antigen 1 and intercellular adhesion molecule 1 expression on B-cell subsets in blood and lymph nodes.

Splenectomy increases the number of B cells in the blood of humans and animals. It is unknown whether this is due to changes in migration, proliferation, or both. The numbers of naïve (IgD(+)IgM(+)), memory (IgD(-)IgM(high)), newly formed (IgM(high)CD90(high)), early recirculating follicular (IgM(low)CD90(high)), recirculating follicular (IgM(low)CD90(-)), and marginal zone (IgM(high)CD90(-)) phenotype B cells were determined in control and splenectomized rats by flow cytometry. All subsets increased significantly in the blood after splenectomy. Because surface molecules are involved in the regulation of migration and proliferation, their expression (lymphocyte function-associated antigen 1 [LFA-1], intercellular adhesion molecule 1 (ICAM-1), L-selectin, alpha4-integrins, CD44, major histocompatability complex class II, interleukin 2 receptor-alpha chain) was determined on B- and T-cell subsets of both groups. B cells, but not T cells, showed a significantly reduced LFA-1 and ICAM-1 expression in blood and lymph nodes, whereas the expression of the other surface molecules analyzed remained unchanged. The down-regulation of these molecules did not influence the adherence of B cells to high endothelial venules in vitro. In vivo, however, ICAM-1(low)-expressing B cells migrated significantly faster through lymph nodes (ICAM-1(low) 41 +/- 5 hours versus ICAM-1(high) 58 +/- 3 hours), whereas proliferation of B cells in bone marrow, lymph node, and blood remained unchanged. Thus, the presence of one organ is necessary for appropriate expression of LFA-1 and ICAM-1 on B cells in other, distant organs. The more rapid transit of ICAM-1(low) B cells through lymph nodes may be responsible for the increased B-cell number in the blood after splenectomy.

Ultrastructural identification of specialized endocytic compartments in macrophages of the thymic cortico-medullary zone and germinal centers of peripheral lymphatic organs of the rat.

In this study transmission electron microscopy was used to investigate the ultrastructural features of macrophages which are strategically positioned in the thymic cortico-medullary zone and the dark zone of germinal centers of peripheral lymphatic organs in adult Wistar rats. We show that this, morphologically distinct, type of macrophage displays the entire range of cytoplasmic inclusions, which structurally closely correspond to those of endosomal/MHC-II-enriched compartments of antigen presenting cells. The macrophages of the cortico-medullary zone and germinal centers contain numerous multivesicular bodies, as well as various kinds of cytoplasmic inclusions ranging from single to aggregated multivesicular/multilamellar bodies to large vacuoles. These multilamellar inclusions are composed of elongated, irregularly shaped cisternae, with abundance of internal membrane profiles and dense bodies. Often, cortico-medullary zone and germinal center macrophages contain the typical multilamellar bodies. Polysaccharides are detected by the thiocarbohydrazide-silver proteinate method within the dense bodies of these macrophages. The functional significance of cortico-medullary zone and germinal center macrophages is briefly discussed.

Cyclosporin A-induced changes of the thymic microenvironment. A review of morphological studies.

Cyclosporin A is an immunosuppressive drug, which disrupts the activation of peripheral T-lymphocyte pool and blocks the maturation of thymocytes within the thymus. Normally, thymic nonlymphoid cells provide the optimal inductive microenvironment for development of T-lymphocytes. After application of cyclosporin A the complex alterations of the thymic microenvironment occur, affecting all types of nonlymphoid cells. All subsets of thymic epithelial cells are thoroughly changed. The subcapsular epithelial cells show the prominent enlargement of cytokeratin contents. In electron microscopy, however, these cells present the morpho-functional aspect of resting cells. The epithelial cells in deeper cortex become enlarged and stockier, whereby their cell processes appear more ramified and thicker. Thus, the cytoreticulum they create seems much denser. These cells strongly express MHC antigens. Their subcellular organization is suggestive of increased synthetic and secretory activity. The number of medullary epithelial cells is decreased. The cells with the most mature phenotype are the most prominently depleted and the ones with phenotypically and morphologically immature appearance predominate. The number of Hassall's bodies is also decreased. The number of cortical macrophages does not increase. However, these cells become enlarged showing the prominent changes in enzyme capacity, histochemical features and ultrastructural organization. Thus, they become similar to macrophages located in the cortico-medullary zone of the normal rat thymus. Cortical macrophages increase the activity of hydrolytic enzymes, acid phosphatase and nonspecific esterase, develop the strong activity of chloroacetate esterase, the strong activity of respiratory enzyme succinic dehydrogenase and begin to show the marked presence of prostaglandin synthase. Moreover, the cytoplasmic inclusions, which are aldehyde fuchsin- and PAS-positive and show sudanophilia, appear within cortical macrophages. In electron microscopy these cells show an abundant cytoplasm a very active appearance and the variety of vacuolar cytoplasmic inclusions. The mitoses of neighboring thymocytes are often seen. The number of interdigitating cells is decreased due to reduced size of thymic medulla, but these cells do not show the substantial phenotype changes. The description and classification of all types of nonlymphoid cells, which constitute the normal thymic microenvironment, is also presented. The functional significance and possible mechanisms of CSA-induced changes of the thymic microenvironment are discussed.

[Alloarthroplasty of war injuries of the elbow joint]. / Aloartroplastika lakatnog zgloba kod ratnih povreda.

War injuries require specific approach in order to be optimally managed. In case of penetrating or explosive joint injuries, the management should be done with regard of the war surgery doctrine. Besides, it is very hard to prognose the disability degree after the completed treatment. If we point out that the average age of our patients with implanted elbow endoprosthesis was between 23 to 35 years, our efforts to reduce the disability are understandable. For all the reasons cited above, in the last two years we started with the total elbow arthroplasty. In all the patients with implanted elbow endoprosthesis, the improvement in the amplitude of elbow movements was observed. In 20% cases the pain was significantly decreased, and in 80% cases, the elbow became painless.

Stereological study of splenic tissue compartments in FK506-treated rats.

Model-based stereology was used to study the changes of splenic structure and proportions of splenic tissue compartments in FK506-treated male Wistar rats (0.1 mg/kg three times a week for 14 days). The point-counting method was utilized to study the volume densities of the following tissue compartments: red pulp; white pulp (this compartment was divided in two subcompartments: follicles and periarteriolar lymphocyte sheath); marginal zone; and connective tissue. The following stereological parameters of lymphoid follicles were also determined: areal numerical density (the number of follicles per mm2 of tissue section); the numerical density (number of follicles per mm3 of tissue); and the mean follicle diameter. After FK506 treatment a significant reduction of volume density of white pulp was observed, which was due to the very prominent decline in the amount of periarteriolar lymphocyte sheath, whereby the cellular density of this tissue compartment was decreased. In the marginal zone of FK506-treated spleens the presence of numerous, large, densely packed lymphoblastic cells of very uniform appearance was registered. These observations are in very good keeping with the changes of splenic morphology registered in cyclosporin A-treated rats, which further documents the parallelism in actions between these immunosuppressive agents.

Ultrastructure of different types of thymic epithelial cells in normal and cyclosporin-A-treated rats.

Transmission electron microscopy was used to study the ultrastructural features of thymic epithelial cells of intact and cyclosporin-A-treated adult male Wistar rats. The animals received a peroral daily dose of 30 mg of cyclosporin A per kg body weight for 21 consecutive days. On the basis of ultrastructural features, seven subsets of epithelial cells were distinguished within the intact rat thymus. Four subsets were predominantly situated in the cortex (type 4 - "dark" cells - occasionally penetrated into the medullar region) and three were positioned within the thymic medulla. All subsets of thymic epithelial cells were markedly changed after the application of cyclosporin A. The prominent enlargement of cytokeratin contents was registered in type 1 - "subcapsular/paraseptal/perivascular" - epithelial cells. At the same time these cells acquired the morpho-functional aspect of resting cells. All other subsets of cortical epithelial cells were enlarged (though in different ways) in comparison to the corresponding subsets of the control thymus, whereby their subcellular organization was suggestive of increased synthetic and secretory activity. The decrease in the number of epithelial cells, with the prevalence of phenotypically immature subsets, was evident within the residual islands of thymic medullary tissue.

Stereological study of tissue compartments of the human spleen.

Morphometric reports on animal and human spleen are very few and no studies have been carried out using stereological methods to investigate all of the tissue compartments of the human spleen. Eighteen samples of spleens, which were either surgically removed after traumatic injury or during treatment for early stage carcinoma or gastric ulcer, were investigated. The point-counting method was used to study the volume densities of the following tissue compartments: red pulp, perifollicular zone, white pulp (this tissue compartment was divided in two subcompartments: follicles and periarteriolar lymphatic sheath), marginal zone and connective tissue of trabeculas. The following stereological parameters of the follicles were investigated: the number of follicles per mm2 of spleen section, numerical density, volume density, and the mean follicular diameter. The identity of spleen tissue compartments was verified using immunohistochemical staining for B- and T-lymphocytes. The volume densities of tissue compartments, as well as stereological parameters of lymphoid follicles, were similar in both groups of splenic samples, except for the volume densities of perifollicular zone and periarteriolar lymphatic sheath, where a statistically significant difference was registered.

Stereologic study of splenic tissue compartments from traumatically injured and cancer patients.

Carefully age-matched groups of patients, surgically treated for either traumatic injury (n = 5; mean = 71.2 years) or for early stage carcinoma (n = 5; mean = 74.2 years), were used to investigate all the splenic tissue compartments applying model-based stereology. The point-counting method was utilized to study the volume densities of following tissue compartments: red pulp, perifollicular zone, white pulp (this compartment was divided into two subcompartments: follicles and periarteriolar lymphatic sheath), marginal zone and connective tissue. The following stereologic parameters of lymphoid follicles were determined: areal numerical density (the number of follicles per mm2 of tissue section), the numerical density (number of follicles per mm3 of tissue), and the mean follicle diameter. The identity of tissue compartments was verified using immunohistochemical staining for B- and T- lymphocytes. Significantly increased volume densities of perifollicular zone and periarteriolar lymphatic sheath were registered in patients treated for traumatic spleen injury. On the other hand, the significantly increased number of lymphoid follicles per mm2 and per mm3 of splenic tissue were registered in the group of cancer patients. The differences observed, whether attributable to the immune stimulation of the ruptured spleen or the alterations of the immune system due to malignant disease, suggest that this issue deserves further attention.

Effects of cis-diamminedichloroplatinum II (cisplatin) on the splenic tissue of rats: a histoquantitative study.

Histoquantitative methods were applied to study the changes of splenic structure and proportions of splenic tissue compartments in cisplatin-treated (6 mg/kg body wt) male Wistar rats. Six days after treatment the significant reduction of volume density of white pulp was observed, which was due to the very prominent decrease of volume density of follicles. The volume density of periarteriolar lymphatic sheaths did not change substantially. Volume density of the marginal zone was also significantly reduced. A significant increase of volume density of connective tissue was observed. Morphometrical parameters of the follicles were also markedly altered: the number of follicles per mm2 of spleen section area, the numerical density of the follicles in the spleen, and the total number of the follicles were significantly reduced.

Immunocytochemical demonstration of prostaglandin synthase (cyclooxygenase) in thymic macrophages of normal and cyclosporin-treated rats.

As revealed with ED1 and ED2 monoclonal antibodies, macrophages are scattered throughout the thymic tissue. However, in contrast to the cortex and medulla, in the cortico-medullary zone macrophages are large and show strong reactivity with rabbit polyclonal antisera to cyclooxygenase. Only few smaller cortical macrophages also show weaker presence of prostaglandin synthase. After cyclosporin treatment cortical macrophages become strikingly similar to the macrophages of the cortico-medullary zone of the normal thymus. Cortical macrophages become enlarged and develop the strong expression of prostaglandin synthase. Our results show that a specific type of macrophages (with distinct histochemical characteristics, enzyme profile and ultrastructural organization, which is strategically positioned within the thymic tissue--as we demonstrated earlier) possesses the enzyme capacity required for prostaglandin synthesis. After cyclosporin treatment, which interferes with the maturation of thymocytes, cortical macrophages thoroughly change and develop the strong prostaglandin synthase expression, similar to that of normal cortico-medullary zone macrophages.

Relationship between naphthol AS-D chloroacetate esterase and prostaglandin synthase.

The close topographical correlation between naphthol AS-D chloroacetate esterase-positive macrophages and prostaglandin synthase-rich macrophages in the thymus of normal and cyclosporin-treated rats was observed. It seems possible that chloroacetate esterase is an enzyme related to metabolism of arachidonic acid and/or production of its active metabolites.

Ultrastructural study of macrophages of the rat thymus after cyclosporin treatment.

Young adult male Wistar rats were given 30 mg per kg of cyclosporin (CS) for 21 consecutive days. After CS treatment thymic medulla virtually disappears and the thymus is almost entirely composed of cortical tissue. Macrophages are scattered throughout the thymic cortex. These cells are very large, rounded, with inconspicuous prolongations and euchromatic nucleus with prominent nucleoli. These cells are loaded with lipid bodies and vacuolar cytoplasmic inclusions of different size and diverse content, but very rarely contain phagocytosed lymphocyte remnants. The cytoplasm between inclusions has very active aspect with abundance of polyribosomes, granular endoplasmic reticulum and vesicles. Mitoses of lymphocytes in the vicinity of macrophages are frequently seen. We discuss the morphological similarity between cortical macrophages of CS-treated thymus and macrophages of cortico-medullary zone (CMZ) of the normal rat thymus, as well as functional significance of described morphological characteristics of this type of thymic macrophages, which probably reflect the metabolism of arachidonic acid.

Structure of the bursa of fabricius and immune response to Brucella abortus in the chicken after neonatal gonadectomy.

The structure of the bursa of Fabricius and the immune response to Brucella abortus in Prelux chickens were studied three and six months after neonatal gonadectomy. The different structural changes and immune response were revealed in male and female chickens. In six-months-old gonadectomized males an increased volume density of lymphoid follicles and follicular medulla was found. The follicular number and the follicular diameter were increased. In three- and six-months-old orchidectomized animals, the immune response exceeded that of the nonoperated males of the same age. In ovariectomized chickens, antibody levels were lower than (three months) or equivalent to (six months) to those of nonoperated females. Histoquantitative analysis showed that the volume density of lymphoid follicles and follicular cortex decreased. The follicular number and the mean follicular diameter also decreased. Our results demonstrate that sex steroids have an immunomodulatory role and influence the size and structure of the bursa.