Anti-Inflammatory Activity of a Cyclic Tetrapeptide in Mouse and Human Experimental Models.

A cyclic tetrapeptide Pro-Pro-Pheß3ho-Phe (4B8M) was tested for immunosuppressive activity and potential therapeutic utility in several in vitro and in vivo mouse and human models. The tetrapeptide was less toxic for mouse splenocytes in comparison to cyclosporine A (CsA) and a parent cyclolinopeptide (CLA). The tetrapeptide demonstrated potent anti-inflammatory properties in antigen-specific skin inflammatory reactions to oxazolone and toluene diisocyanate as well to nonspecific irritants such as salicylic acid. It also inhibited inflammatory processes in an air pouch induced by carrageenan. In addition, 4B8M proved effective in amelioration of animal models corresponding to human diseases, such as nonspecific colon inflammation induced by dextran sulfate and allergic pleurisy induced by ovalbumin (OVA) in sensitized mice. The tetrapeptide lowered expression of EP1 and EP3 but not EP2 and EP4 prostaglandin E2 (PGE2) receptors on lipopolysaccharide-stimulated Jurkat T cells and ICAM-1 expression on human peripheral blood mononuclear cells (PBMC). Its anti-inflammatory property in the carrageenan reaction was blocked by EP3 and EP4 antagonists. In addition, 4B8M induced an intracellular level of PGE2 in a human KERTr keratinocyte cell line. In conclusion, 4B8M is a low toxic and effective inhibitor of inflammatory disorders with potential therapeutic use, affecting the metabolism of prostanoid family molecules.

Three-Component Reaction of Diamines with Triethyl Orthoformate and Diethyl Phosphite and Anti-Proliferative and Antiosteoporotic Activities of the Products.

A three-component reaction between diamines (diaminobenzenes, diaminocyclohexanes, and piperazines), triethyl orthoformate, and diethyl phosphite was studied in some detail. In the case of 1,3- and 1,4-diamines and piperazines, products of the substitution of two amino moieties-the corresponding tetraphosphonic acids-were obtained. In the cases of 1,2-diaminobenzene, 1,2-diaminocyclohexanes and 1,2-diaminocyclohexenes, only one amino group reacted. This is most likely the result of the formation of hydrogen bonding between the phosphonate oxygen and a hydrogen of the adjacent amino group, which caused a decrease in the reactivity of the amino group. Most of the obtained compounds inhibited the proliferation of RAW 264.7 macrophages, PC-3 human prostate cancer cells, and MCF-7 human breast cancer cells, with 1, trans-7, and 16 showing broad nonspecific activity, which makes these compounds especially interesting in the context of anti-osteolytic treatment and the blocking of interactions and mutual activation of osteoclasts and tumor metastatic cells. These compounds exhibit similar activity to zoledronic acid and higher activity than incadronic acid, which were used as controls. However, studies of sheep with induced osteoporosis carried out with compound trans-7 did not support this assumption.

Micromorphological assessment of bone tissue remodeling in various hip degeneration conditions.

BACKGROUND: The reorganization of bone tissue is closely associated with its metabolism and changes in its internal structure. Metabolism of the bone, which results from the simultaneous processes of resorption and formation of new bone tissue, may depend on the presence and type of arthritis. OBJECTIVES: The objective of the study was to assess, based on the morphological features and mineral composition of bone tissue, changes in the femoral head in various types of hip joint degeneration. MATERIAL AND METHODS: The study group consisted of 21 patients surgically treated for hip joint degeneration. They included 17 women, aged 30-70 years (mean age 52.5 years), and 4 men, aged 38-51 (mean age 48.5 years). The assessment of the morphological condition of the bone and the mineral composition of bone tissue took into account quantitative and qualitative relationships among the mineral components and bone matrix. The structure of spongious bone tissue was analyzed in histological studies, with special attention paid to osteogenesis and osteoclastic processes and the advancement of degeneration. RESULTS: Three main types of degenerative changes in bone tissue of the examined femoral head were recognized: osteoporosis with a prevalence of coarse-fiber bone tissue and decreased osteogenic activity; osteolysis with few osteogenesis centers; and intensified reorganization of bone tissue. In more than half of the examined samples, coarse-fiber bone tissue was replaced by newly formed bone tissue. We observed bone resorption and osteogenesis, which indicate normal homeostasis of the bone tissue. Uneven saturation of spongious bone with mineral components was found. The content of organic and inorganic bone components measured with Ca : P and C : Ca + P ratios had similar values in all types of changes. Only the bone with intense osteolysis contained a smaller quantity of carbon (4.96-8.13%). CONCLUSIONS: Our observations indicate an intense adaptive reorganization of bone tissue depending on external and internal factors, including biomechanical condition.

Therapeutic effects of an azaphenothiazine derivative in mouse experimental colitis.

Phenothiazines represent a class of compounds of potential therapeutic utility. In this report we evaluated therapeutic value of an azaphenothiazine derivative, 6-acetylaminobutyl-9-chloroquino[3,2-b]benzo[1,4]thiazine (QBT), given intragastrically, in the model of dextran sodium sulfate-induced colitis in C57BL/6 mice using 5-aminosalicylic acid (5-ASA) as a reference drug. Colitis symptoms such as body weight loss, diarrhea and hematochezia (blood in stool) were observed and registered and disease activity index (DAI) was calculated. In addition, weight and cell numbers in the lymphatic organs and histological parameters of the colon wall were analyzed. The effects of QBT on viability of colon epithelial cell lines were also determined. We showed that weight and cell number of draining mesenteric lymph nodes were lower in mice treated with QBT in comparison to their control counterparts. The number of thymocytes, drastically reduced in control mice, was elevated in mice treated with the compounds with a significant effect of 5-ASA. In addition, an abnormal composition of blood cell types was partially corrected in these groups. Histological analysis of the colon revealed that the pathological changes were partially normalized by QBT and even to a higher degree by 5-ASA. In conclusion we demonstrated a therapeutic efficacy of the compound in amelioration of local and systemic pathological changes associated with chemically-induced colitis in mice. A possible mechanism of action of the compound is discussed.

A Case of Syphilis with High Bone Arsenic Concentration from Early Modern Cemetery (Wroclaw, Poland).

Venereal syphilis is a sexually transmitted disease caused by Treponema pallidum - Gram-negative, slowly growing bacteria. The spread of the disease in the Old World was due to increased birth rate, urban population growth, migration and lack of knowledge concerning the epidemiology. In the past, the treatment was mainly symptomatic and included application of mercury compounds. The goal of the study was to present the case of advanced venereal syphilis found in early modern (16th-18thc) graveyard localized in Wroclaw, Poland. The object of the study is a cranium of a male whose age at death has been estimated to be over 55. In order to observe the morphological and paleopathological characteristics of the examined material, anthropometrics, computed tomography, spectrometry and microscopic methods were incorporated. Microscopic analysis revealed the presence of the extensive inflammatory lesions. Analyses indicate tertiary stage of venereal syphilis as the most probable cause of the observed lesions. Concentration of arsenic (16.17±0.58 µg/g) in examined bone samples was about hundred times bigger than average arsenic concentration in bones reported in other studies. Advanced stage of observed lesions along with high arsenic level may suggest long-lasting palliative care and usage of arsenic compound in therapeutic treatment of this chronic disease.

Topically applied azaphenothiazines inhibit experimental psoriasis in mice.

The therapeutic efficacy of topically applied azaphenothiazine derivatives: 9-chloro-6-acetylaminobutylquinobenzo[3,2-b][1,4]thiazine (compound 4) and 6-chloroethylureidoethyldiquino[3,2-b;2';3'-e][1,4]thiazine (compound 5) in the amelioration of inflammatory symptoms of imiquimod-induced psoriasis in mice was investigated. Clobederm®, containing clobetasol propioniate, served as a reference drug. The application of the compounds led to thinning of the epidermis and reduction of the cell layers. The suppressive actions of the compounds were even stronger with regard to pathological changes of the dermis. The compounds also exerted generalized, anti-inflammatory effects by decreasing the number of circulating leukocytes, lowering subiliac lymph node weight and partially normalizing an altered blood cell composition. The changes in the composition of main cell types in the epidermis and dermis were less affected by the compounds. In addition, both compounds inhibited to a similar degree production of tumor necrosis factor α (TNF α) in human whole blood cell culture. Whereas compound 5 strongly inhibited IL-8 and CXCL10 chemokines in human keratinocytes - KERTr cell line, transfected with poly(I:C), the suppressive action of compound 4 in this model was weak. In addition, compound 5, but not compound 4, exhibited at low doses proapoptotic properties with regard to colonic cell lines. In summary, we demonstrated the therapeutic potential of two selected azaphenotiazines in the amelioration of the skin pathology elicited in a mouse experimental model of psoriasis.

Topically applied azaphenothiazines inhibit contact sensitivity to oxazolone in mice.

In this work we investigated the efficacy of two topically applied azaphenothiazine derivatives, 9-chloro-6-acetylaminobutylquinobenzo[3,2-b][1,4]thiazine (compound 4) and 6-chloroethylureidoethyldiquino[3,2-b;2';3'-e][1,4]thiazine (compound 5), in the amelioration of inflammatory symptoms of contact sensitivity (CS) to oxazolone in mice, in relation to the commercial ointment Protopic® (tacrolimus), the reference drug. The compounds were administered 24 h following elicitation of CS and, 24 h later, the parameters of inflammation, such as ear edema, blood composition, leukocyte level, numbers of cells in the draining lymph nodes, histological picture of the inflamed tissue, and the morphometric analysis, were analyzed. The study showed that the effectiveness of the studied azaphenothiazines applied as a 0.1% ointment was comparable to the reference drug regarding suppression of the inflammatory process, when all the investigated histological parameters are taken into account.

Effect of Glucocorticoids on Ultrastructure of Myocardial Muscle in the Course of Experimentally Induced Acute Myocardial Ischemia.

The search for effective methods of myocardial cytoprotection against ischemia is the most significant issue in modern cardiology and cardiac surgery. Glucocorticoids are deemed very strong modulators of inflammatory response and thus can potentially protect heart muscle from postreperfusion injury and myocardial ischemia during cardiac surgery. Ultrastructural examination of the left ventricle heart samples revealed that the intravenous application of dexamethasone and hydrocortisone proved to exert cytoprotective effect on cardiomyocytes during experimentally induced acute ischemia in rats.

The rabbit brachial plexus as a model for nerve repair surgery--histomorphometric analysis.

One of the most devastating injuries to the upper limb is trauma caused by the avulsion. The anatomical structure of the rabbit's brachial plexus is similar to the human brachial plexus. The aim of our study was to analyze the microanatomy and provide a detailed investigation of the rabbit's brachial plexus. The purpose of our research project was to evaluate the possibility of utilizing rabbit's plexus as a research model in studying brachial plexus injury. Studies included histomorphometric analysis of sampled ventral branches of spinal nerves C5, C6, C7, C8, and Th1, the cranial trunk, the medial part of the caudal trunk, the lateral part of the caudal trunk and peripheral nerve. Horizontal and vertical analysis was done considering following features: the axon diameter, fiber diameter and myelin sheath. The number of axons, nerve area, myelin fiber density and minimal diameter of myelin fiber, minimal axon diameter and myelin area was marked for each element. The changes between ventral branches of spinal nerves C5-Th1, trunks and peripheral nerve in which the myelin sheath, axon diameter and fiber diameter was assessed were statistically significant. It was found that the g-ratio has close value in the brachial plexus as in the peripheral nerve. The peak of these parameters was found in nerve trunks, and then decreased coherently with the nerves travelling peripherally.

Collateral sprouting axons of end-to-side nerve coaptation in the avulsion of ventral branches of the C5-C6 spinal nerves in the brachial plexus.

INTRODUCTION: This study assessed the collateral sprouting in avulsion of the ventral branches of the C5 and C6 spinal nerves treated by coaptation of these nerves to the C7 spinal nerve on the brachial plexus of rabbits. MATERIAL AND METHODS: Thirty-six New Zealand rabbits were randomly divided into four groups: end-to-side coaptation (ESN) (n = 12), side-to-side coaptation (SSN) (n = 12), direct neurorrhaphy (end-to-end) (EEN) (n = 6) and no coaptation (n = 6). The operations were performed on the left brachial plexus. The contralateral, non-operated right brachial plexi were used as the control group. The groups were compared using morphological, electrophysiological and behavioral methods. The follow-up duration was 20 weeks. RESULTS: Significant differences were observed in all parameters when the experimental groups were compared with the control group and the no coaptation group. The histology of axonal regeneration after ESN, but not after SSN, was comparable to that after EEN. There were no significant differences in the electrophysiological, behavioral assessment or G-ratio parameters between the ESN and EEN groups. There were significant differences in the behavioral assessment, G-ratio and the histomorphometric parameters between the SSN and EEN groups, which disagreed with the electrophysiological results. Sensory axon collateral sprouting was more rapid than motor axon collateral sprouting. CONCLUSIONS: The electrophysiological, histomorphometric and behavioral results obtained using end-to-side coaptation of ventral branches of the C5 and C6 spinal nerves to the C7 spinal nerve in the brachial plexi of rabbits confirm the occurrence of collateral sprouting at this level. After further research is performed to confirm the results of this study, end-to-side coaptation might emerge as an alternative method in the treatment of brachial plexus avulsion.

Homologous lactoferrin triggers mobilization of the myelocytic lineage of bone marrow in experimental mice.

The effects of lactoferrin (LF), an iron binding protein, on myelopoiesis have been studied extensively in vitro and in vivo in human and murine models over the past three decades. Due to the lack of high-quality homologous LFs, however, the conclusions are still unequivocal. Recently, both human and murine LFs have become available as recombinant products expressed in Chinese hamster ovary (CHO) cell lines showing mammalian type of glycosylation, thus apparently species compatible. In this study, we present the effects of homologous recombinant mouse LF (rmLF) on myelopoiesis in CBA mice. The myelocytic lineage has been assessed by their appearance in circulating blood and bone marrow, and induction of relevant mediators of inflammation. Intravenous injection of rmLF (100 µg/mouse) resulted in a significantly increased number of myelocytic cells in the circulating blood after 24 h. Mouse serum transferrin, used as a control protein, showed no stimulatory effect. The increase in output of neutrophil precursors, neutrophils, and eosinophils was correlated with a twofold increase of leukocyte concentrations. The analysis of the bone marrow sections confirmed increased myelopoiesis. The alterations in the bone marrow cell composition were statistically significant regarding mature neutrophils (10.8% vs. 27.7%), metamyelocytes (11.4% vs. 16.0%), and myelocytes (2.4% vs. 4.0%). The mobilization of the myelocytic cells in the bone marrow and the increased output of these cells into circulation were accompanied by elevated serum concentrations of interleukin-6 at 6 h and haptoglobin at 24 h following administration of rmLF. In conclusion, the homologous LF elicits significant and transient myelopoiesis in experimental mice.

Experimental xenoimplantation of antlerogenic cells into mandibular bone lesions in rabbits: two-year follow-up.

Different types of cells require activation, and take part in annual, dynamic growth of deer antlers. Stem cells play the most important role in this process. This report shows the results of a two-year long observation of xenogenic implant of antlerogenic stem cells (cell line MIC-1). The cells were derived from growing antler of a deer (Cervus elaphus), seeded onto Spongostan and placed in postoperative lesions of mandibular bones of 15 experimental rabbits. The healing process observed in the implantation sites in all rabbits was normal, and no local inflammatory response was ever observed. Histological and immunohistochemical evaluations were performed after 1, 2, 6, 12 and 24 months, and confirmed the participation of xenogenic cells in the regeneration processes, as well as a lack of rejection of the implants. The deficiencies in the bones were replaced by newly formed, thick fibrous bone tissue that underwent mineralization and was later remodelled into lamellar bone. The results of the experiment with rabbits allow us to believe that antlerogenic cells could be used in reconstruction of bone tissues in other species as well.

Calf thymus extract attenuates severity of experimental encephalomyelitis in Lewis rats.

The aim of this study was to evaluate the efficacy of treatment of Lewis rats with calf thymus extract (TFX®) and its six-peptide fraction on the course of experimental allergic encephalomyelitis (EAE). Interferon- ß served as a reference drug. We found that intramuscular administration of the thymus extract fraction significantly reduced clinical, immunological, histological, and ultrastructural alterations inherent in the disease. We suggest that TFX® or TFX®-derived fractions have potential as therapeutics in treatment of neurodegenerative diseases such as multiple sclerosis.

The evaluation of the influence of a high injury to brachial plexus elements on the condition of neurons of the anterior horns of the spinal cord--experimental research.

The aim of the experimental research was to assess the impact of a high damage to brachial plexus elements on the condition of neurons of spinal cord anterior horns. The research was conducted on 12 rabbits, in which the ventral branches of spinal nerves C5-Th1 were severed. During dissections carried out 7, 30, 60, 180 days after the operation the cervicothoracic segment of the spinal cord was collected. The material was subjected to microscopic histological and ultrastructural examinations, which showed that where brachial plexus elements had been severed some of the neurons of spinal cord anterior horns had died and that the process intensity depended on the time that had passed after the injury.

Immunoregulatory function of lactoferrin in immunosuppressed and autoimmune animals.

In this article we review our recent results on the effects of lactoferrin (LF), given orally, on the immune status of mice subjected either to chemotherapy or immobilization stress as well as on rats with experimentally induced autoimmune encephalomyelitis (EAE). We demonstrated that LF accelerated reconstitution of the immune system function after administration of a sublethal dose cyclophosphamide (CP) and normalized the ratio of major blood cell types in that model. Also, after application of methotrexate (MTX) LF was effective to speed up reconstitution of the cellular and humoral immune response. Mice treated with lethal dose of busulfan (Bu) and CP and reconstituted with bone marrow cells (BMC) were able to quicker develop optimal immune responses when administered LF. In addition LF was shown to accelerate engraftment of bone marrow cells from syngeneic donors in that model. Using immobilization stress model was shown that LF accelerates reconstitution of the cellular and humoral immune response. In rats with EAE lactoferrin lowered the clinical score of the disease and diminished pathohistological changes in the spinal cord. In summary, in a series of studies we demonstrated a benefit of orally administered LF in immunocompromised animals.

Experimental studies on the effect of osteotomy technique on the bone regeneration in distraction osteogenesis.

The objective of the experiment was to study the effect of osteotomy technique on the course of bone regeneration in distraction osteogenesis in sheep. Thirty five merino sheep were divided into 5 groups of 7 sheep each, depending on the osteotomy technique: subcutaneous corticotomy, open corticotomy, Cattaneo corticotomy, open osteotomy and osteoclasia. Formation of regenerate and its remodeling was assessed, based on roentgenograms and morphological examination (bone sections, histological evaluation, SEM, superficial X-ray analysis). Irrespective from the osteotomy technique applied, in all experimental groups, regenerates filling the distraction gap were obtained. Analysis of bone sections and histological slides indicated the most advanced osteogenesis processes in the osteoclasia group. SEM examination and X-ray microanalysis confirmed not only the presence of mature bone tissue structures in this group, but also showed a mineralization close to that of a healthy bone. Osteogenesis was the slowest in the group of subcutaneous corticotomy. The observed histological picture and analysis of elemental composition indicate a normal course of distraction osteogenesis in this group, but its consecutive phases proceed with a certain delay, compared to the remaining groups. With a good deal of caution, it can be assumed that the delay of osteogenesis in the group of subcutaneous corticotomy in the 14th post-operative week was ca. 3 weeks compared to the osteoclasia group. In all experimental groups, few cartilaginous nodules were observed in regenerates 5 weeks and, in few cases, 9 weeks old. Ossification in such places was indirect, cartilage-based. In the remaining regions direct, membrane-based ossification prevailed. Direct apposition bone lamellae on islands of fibrous bone tissue was observed in regenerates aged 9 and 14 weeks, especially in periosteal regions. The intensity and rate of formation of new bone tissue varied between the experimental groups, based on roentgenological analysis and histological evaluation. Complex morphological studies on the course of regeneration in sheep showed the most advanced remodeling and mineralization of bone tissue in animals subject to osteoclasia.

Immunosuppressory activity of an isoxazolo[5,4-e]triazepine-compound RM-33 II. Effects on the carrageenan-induced inflammation.

The aim of this investigation was to evaluate effectiveness of RM-33, a new isoxazolotriazepine, in the model of carrageenan-induced inflammation in rats. Wistar rats were pretreated with intraperitoneal (ip) or oral (po) doses of RM-33, at daily doses ranging from 250 to 1000 microg, administered 1-3 days before elicitation of the carrageenan reaction. We showed that both routes of RM-33 administration were effective in significantly diminishing the footpad edema. The effects were dose-dependent and better pronounced at the ip administration of the compound. We found a lower production of tumor necrosis factor alpha (TNF-alpha) by mitogen-stimulated splenocytes isolated from rats pretreated with RM-33 and injected with carrageenan, as well as lower serum TNF-alpha levels in these rats, as compared to the respective control. Histological analysis of the skin reaction site revealed that in the rats pretreated with RM-33, the carrageenan-induced inflammation was reduced, as reflected by a lesser damage of mast cells, smaller infiltration by macrophages and a diminished edema of the connective tissue. Together with our previous data, indicating the antagonistic action of RM-33 in the adjuvant-induced footpad inflammation in mice, the present results confirm the anti-inflammatory activity of RM-33 compound.

[Application of elemental microanalysis for estimation of osteoinduction and osteoconduction of hydroxyapatite bone implants]. / Zastosowanie mikroanalizy pierwiastkowej do oceny osteoindukcji i osteokondukcji dokostnych implantów hydroksyapatytowych.

Hydroxyapatite (HAp) ceramics based on calcium phosphates, chemical compounds being natural constructive element of bones is now regarded to be one of better implantation materials in osseous surgery and stomatology. HAp is poorly soluble and slowly resorbing in tissues material. Easiness of connecting of hydroxyapatite ceramics with other materials creates possibilities to produce new composites of chemical compounds containing calcium and easier resorbing. An important and till now not fully known problem is influence of hydrodroxyapatite grafts on the processes leading to production of calcium and phosphorus ions responsive for mineralization of bone tissue around the graft. Proliferation of osteoblasts and rate of osseous trabeculas production depends, among others, concentration of calcium and phosphorus ions. The main target of this study was the comparative analysis of the bone mineralisation rate after implantation of hydroxyapatite (HAp) and composites hydroxyapatite + beta tricalcium phosphate (HAp + TCP) and hydroxyapatite + calcium hydrosulfate (HAp + gypsum), on the basis of elemental microanalysis. Implantation studies were carried out on 24 rabbits. Sections were carried out 5, 9, 11 and 14 weeks after implantation of the tested materials. The carried out punctual analysis of the occurring elements and their topographic location (mapping) in bones after implantation showed essential differences among the tested grafts. Those differences were most significant after 5 and 14 weeks and depended on the rate of the tested materials resorption. On the basis of conducted macroscopic evaluation and scanning microscope assessment we can stated that the composite of HAp+TCP showed the best osteocunductive properties, while the best osteoinductive influence was shown by the composite HAp + gypsum.

[Macrophages and the expression of endothelin in sulphur dioxide induced bronchitis in rats]]. / Makrofagi i endotelina w zapaleniu oskrzeli wywolanym inhalacjami SO2 u szczurów.

The aim of the study was to evaluate the influence of SO2 on bronchial mucosa in rats. Our interest was focused on macrophages function and expression of endothelin. 20 animals were used in the study; 5 of them constituted the control group, 15 of them were submitted to long term SO2 irritation. In animals exposed to SO2 the BALF evaluation revealed the increase of inflammatory cells concentration: neutrophils, macrophages, lymphocytes. Electron microscope examination demonstrated that macrophages from study group were activated in comparison with healthy rats. Immunoreactive endothelin was found in bronchial specimens and mainly in alveolar macrophages (we used a computer-assisted system of image analysis). The expression of endothelin was significantly increased in study group in comparison with control group.