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The study of the embryonic development of the pancreas gives the opportunity to understand the mechanisms of organ regeneration in case of various pathologies. Worldwide research works, studying histo- and organogenesis of human pancreas, are based on data, received from model animals. Numerous processes of pancreatic development take several hours and remain unclear because white laboratory mouse has short gestation period. Spiny mouse (Acomys) has the prolonged prenatal period and can be a convenient model to study the stages of histo- and organogenesis of the pancreas. The review analyzed similarities and differences in the structure of human pancreas, white laboratory mouse and spiny mouse, the features of prenatal histo- and organogenesis of the pancreas, which should be considered in conducting and interpreting results of fundamental research, and possibility of using of spiny mice as a model animal to study embryonic development and pathology of the pancreas.

The article presents data on the regeneration of the auricle tissues of Acomys cahirinus mice (n=12) and Balb/c mice (n=12). Two experimental models were used: a full-thickness perforated defect with a diameter of 3 mm and subtotal removal of the auricle. Macrophotography and histological examination using general histological stains and detection of elastic fibers were performed after 2, 5, 15, 21, 30, 60, 90, and 120 days. It was found that regeneration in Acomys is more pronounced, which manifested itself in the complete elimination of the defect by 30-60 days in contrast to the control (Balb/c). A feature of this was less pronounced post-traumatic inflammation in Acomys mice. It was established the growth of a full-fledged auricle in case of its surgical removal. An important feature of regeneration in this case is the development of new cartilage tissue both from the edges of the original cartilage and developed in the form of separate islands of cartilage tissue. The newly formed cartilage was characterized by high cel-lularity, a smaller volume of the matrix, in the structure of which elastic fibers were formed. However, it should be noted that within 30-60 days. the recovery process, apparently, is not completed, the formed tissue regenerate enters the remodeling phase.

The study assessed reactivity of stromal-vascular skeletal muscle differons to acute chemical injury. Dysferlin-deficient Bla/J mice and the wild-type С57BL/6 mice were intramuscularly injected with 100 μl of 0.5% procaine solution. The middle segment of gastrocnemius muscle was taken on postsurgery days 2, 4, 10, and 14 for routine histological examination. To evaluate proliferation and vascularization, the paraffin sections were stained immunohistochemically with antibodies to α-smooth muscle actin and Ki-67. The connective tissue was stained according to Mallory. The study revealed diminished proliferative activity of stromal-vascular differons and decreased vascular density in muscles of Bla/J mice. Thus, mutations in the DYSF gene coding dysferlin down-regulate the reparation processes in all differons of skeletal muscle.

A number of sarcolemma proteins are responsible for muscle fiber repair. Dysferlin encoded by the

A tumor of the uterus, consisting of smooth muscles, is called myoma and fibroids, and in practice, the terms are used synonymously. Myoma of the uterus is a common disease in women of childbearing age. The frequency of its detection without regard to age is 2.45%. With age, its prevalence increases and reaches 8.31% by the age of 50. The estimate of the final probability of contracting uterine fibroids in the population throughout life is 9.7%. According to sectional data, uterine fibroids, including small nodes, occur in 20% of women. In 50% of cases, these tumors are manifested by clinically pronounced disorders that lead a woman to a doctor.

Different transcription factors, which are synthesized at different stages of cell differentiation, are often considered markers of p-cell precursors. One of these transcription factors is MafA, the role of which is not fully understood. According to one hypothesis, it activates insulin gene expression in the differentiating p-cells. According to another, this factor is only necessary for the regulation of insulin secretion by already differentiated p-cells. In favor of the latter hypothesis, we showed that MafA is not expressed in the immature p-cells during the prenatal development of the human pancreas. In order to finally determine whether MafA is a marker of differentiating cells or it is synthesized in already mature p-cells the aim of our investigation was the analysis of dynamical changes of MafA-positive cell population and C-kit-positive endocrinocyte precursors in Langerhans islets during experimental diabetes in rats. The study was performed on male Wistar rats (250-300g body weight) which were intraperitoneally injected with alloxan. Animals were sacrificed 1,2, 7, 14, 21 days of the experiment for the morphological analysis of pancreas. Paraffin sections of pancreas were stained immunohistochemically with antibodies against MafA, C-kit, insulin and glucagon. The maximum number of MafA-positive cells in the islets was found during normal prenatal development of pancreas. At all stages of the experimental diabetes the number of MafA-positive cells in the islets decreased, wherein the number of insulin-positive cells in the islets increased by the end of the first and third weeks of the experiment. It was also established that in experimental diabetes, changes in populations of MafA- and C-kit-positive cells occur in different ways. Thus, the results of our research showed that MafA cannot be considered as a marker of progenitor cells and is expressed only in the mature cells of the Langerhans islets, that confirms our previous data obtained during the study of prenatal development of human pancreas.

Dysferlinopathies are a group of muscular dystrophies with autosomal-recessive inheritance caused by mutations in DYSF gene. Dysferlin is a 237 kDa transmembrane protein responsible for reparation of the sarcolemma. It has calcium-sensitive C2 domains and after dysferlin binding with calcium ions the first one activates vesicles fusion and patch mechanism repair. There are number of knockout animal strains with dysferlin gene mutations. Bla/J mice have the ETn retrotransposon inserted in intron 4 of the DYSF gene of wild-type mice - C57Bl/6. The pathogenesis ascertainment of dysferlinopathies is important not only for revealing of physiological function of dysferlin, but its deficiency influence on reparative regeneration of skeletal muscles. In this paper the description of main pathohistological processes in skeletal muscle that take place in mice with dysferlinopathy after acute myotoxic injury is present. This article reviews quantitative evaluation of main pathomorphological processes in reparative regeneration: alteration (necrotized muscle fibers ratio), proliferation (Ki-67-positive myonuclei ratio), differentiation (mean cros-sectional area, percentage of centrinucleated muscle fibers, myo-genin-positive nuclei ratio, slow/fast muscle fibers ratio). It was identified that dysferlin-deficient mice have increased alteration level with more necrotized muscle fibers (35,1% (29,4%; 42,9%) in Bla/J vs. 25,8% (17,9%; 37,4%) in C57Bl/6 on 2 day after alteration, p

Perineuronal net (PNN) is a highly structured portion of the CNS extracellular matrix (ECM) regulating synaptic plasticity and a range of pathologic conditions including posttraumatic regeneration and epilepsy. Here we studied Wisteria floribunda agglutinin-stained histological sections to quantify the PNN size and enrichment of chondroitin sulfates in mouse brain and spinal cord. Somatosensory cortex sections were examined during the period of PNN establishment at postnatal days 14, 21 and 28. The single cell PNN size and the chondroitin sulfate intensity were quantified for all cortex layers and specifically for the cortical layer IV which has the highest density of PNN-positive neurons. We demonstrate that the chondroitin sulfate proteoglycan staining intensity is increased between P14 and P28 while the PNN size remains unchanged. We then addressed posttraumatic changes of the PNN expression in laminae 6 and 7 of cervical spinal cord following hemisection injury. We demonstrate increase of the chondroitin sulfate content at 1.6–1.8 mm rostrally from the injury site and increase of the density of PNN-bearing cells at 0.4–1.2 mm caudally from the injury site. We further demonstrate decrease of the single cell PNN area at 0.2 mm caudally from the injury site suggesting that the PNN ECM takes part in the posttraumatic tissue rearrangement in the spinal cord. Our results demonstrate new insights on the PNN structure dynamics in the developing and posttraumatic CNS.

The study assessed reactivity of stromal-vascular skeletal muscle differons to acute chemical injury. Dysferlin-deficient Bla/J mice and the wild-type С57BL/6 mice were intramuscularly injected with 100 μl of 0.5% procaine solution. The middle segment of gastrocnemius muscle was taken on postsurgery days 2, 4, 10, and 14 for routine histological examination. To evaluate proliferation and vascularization, the paraffin sections were stained immunohistochemically with antibodies to α-smooth muscle actin and Ki-67. The connective tissue was stained according to Mallory. The study revealed diminished proliferative activity of stromal-vascular differons and decreased vascular density in muscles of Bla/J mice. Thus, mutations in the DYSF gene coding dysferlin down-regulate the reparation processes in all differons of skeletal muscle.

Herein we present a clinical case of the Caroli syndrome caused by the compound heterozygous mutation in the PKHD1 gene. Histopathological assessment of liver detected biliary cirrhosis, numerous dilated bile ducts of various sizes, hyperplastic cholangiocytes containing a large amount of acid mucopolysaccharides, decreased s-tubulin expression and increased proliferation of cholangiocytes. A significant proportion of hepatic tissue was composed of giant cysts lined with a single layer of cholangiocytes, containing pus and bile in its lumen and surrounded by granulation tissue. An accumulation of neutrophils in the lumen of the bile ducts was observed, as well as an infiltration of the ducts and cysts surrounding connective tissue by CD4+ and to a lesser extent CD8+ lymphocytes. This may be caused by the expression of HLA-DR by cholangiocytes. Atrophy and desquamation of the epithelium of collecting tubules with the formation of microcysts were detected in the kidneys without a clinically significant loss of renal function. Morphopathogenetic mechanisms of the Caroli syndrome can be targets for a potential pathogenetic therapy and prevention of its manifestations and complications.

Since the 1990s, when it was demonstrated by Hammel and others that liver fibrosis is reversible, researchers and physicians actively search for new antifibrotic therapies. In recent years, knowledge of liver fibrosis pathophysiology has greatly advanced and new cellular and molecular mechanisms were described. The cells that determine extracellular matrix components distribution are myofibroblasts, but their origin is diverse. They can be activated hepatic stellate cells (HSCs), portal fibroblasts (PF), or circulating mesenchymal stem cells of the bone marrow. Among large number of substrates to inhibit activation, to inhibit proliferation of myofibroblasts, and to induce their apoptosis we, chose curcumin and gliotoxin. Primarily, in the current work, we optimized the explantation culture method for isolation of hepatic myofibroblasts and received two different cultures—myofibroblasts of HSC and PF origin. Exposition of 50 μM curcumin and 0.1 μM gliotoxin was the most optimal; we observed suppression of hepatic myofibroblast activation and inhibition of their proliferation. These results extend the current knowledge of the cells within the liver fibrogenic populations and prove inhibitory influence of biologically active substances (curcumin and gliotoxin) on portal myofibroblasts.

Global changes in recent decades, caused by scientific and technological breakthroughs, which are characterized as a genomic revolution and the fourth industrial revolution, raise new pressing issues both for society as a whole and for public health. The latter can not exist and develop without a qualitative, and most importantly, modern medical education, in which significant changes are also taking place. In particular, the transition to standards by the World Federation of Medical Education (WFME) and the Association of Medical Education in Europe (AMEE) - «International Standards of Medical Education for Improving the Quality of Health» (Copenhagen, March 15-19, 2003). In this article, based on the analysis of trends in the development of health care and medical education, possible options for correcting approaches in the teaching of morphological disciplines (normal anatomy, histology, pathology) in medical universities are considered.

Aim. Development of a model of hindlimb ischemia in rats to assess the effectiveness of gene and cell therapy. Methods. In the first stage of this approach the external iliac artery was ligated immediately after bifurcation of the common iliac artery. Then the femoral artery was ligated before its branching into the descending genicular, popliteal and saphenous arteries with following dissection of arteries between ligatures. The second stage of this approach was performed seven days after the completion of the first stage: branches of popliteal artery to gastrocnemius muscle and formed collaterals were ligated and dissected. Hindlimb blood flow was assessed by laser Doppler flowmetry. Animals were euthanazied 14, 17, 21, 28, 35, 42 days after the first stage. The paraffin sections of gastrocnemius muscles were stained with Mallory trichrome and with antibodies to CD34. Results. Histopathological analysis showed a continuous hindlimb ischemia of operated limb without any native skeletal muscle structure restoration and with significant interstitial fibrosis (11.895.53% vs 2.552.13% in intact limb by day 42, p 0.05). Polymorphic muscle fibers in operated limb with significantly smaller diameter and reduced capillary density (0.820.03 vs 1.910.06 in intact limb by day 42, p 0.05) were revealed. Laser Doppler flowmetry revealed reduction of blood flow in the operated limb (0.670.22 of intact limb blood flow by day 42). Conclusion. Our developed two-stage model of hindlimb ischemia in rats leads to histologic changes characteristic for the same pathology in human.

Perineuronal nets (PNN) ensheath GABAergic and glutamatergic synapses on neuronal cell surface in the central nervous system (CNS), have neuroprotective effect in animal models of Alzheimer disease and regulate synaptic plasticity during development and regeneration. Crucial insights were obtained recently concerning molecular composition and physiological importance of PNN but the microstructure of the network remains largely unstudied. Here we used histochemistry, fluorescent microscopy and quantitative image analysis to study the PNN structure in adult mouse and rat neurons from layers IV and VI of the somatosensory cortex. Vast majority of meshes have quadrangle, pentagon or hexagon shape with mean mesh area of 1.29µm(2) in mouse and 1.44µm(2) in rat neurons. We demonstrate two distinct patterns of chondroitin sulfate distribution within a single mesh - with uniform (nonpolar) and node-enriched (polar) distribution of the Wisteria floribunda agglutinin-positive signal. Vertices of the node-enriched pattern match better with local maxima of chondroitin sulfate density as compared to the uniform pattern. PNN is organized into clusters of meshes with distinct morphologies on the neuronal cell surface. Our findings suggest the role for the PNN microstructure in the synaptic transduction and plasticity.

C-kit—a stem cell receptor, or CD117, is one of the markers of human pancreatic endocrinocyte progenitor cells. Its important role in the proliferation and differentiation of pancreatic endocrine cells during human prenatal development has already been proven. However, the presence and role of c-kit-positive cells in adult pancreatic islets remains unclear. The purpose of our study was to establish the presence of c-kit-positive cells in the adult pancreatic islets and to assess whether they retain the ability to proliferate during a lifetime. We studied pancreatic autopsies of adults aged 50 to 70 years, which were obtained in pathology departments of Kazan hospitals. Samples of the pancreas were embedded in paraffin using standard techniques. Paraffin sections of the pancreas were stained immunohistochemically with commercial antibodies against c-kit, insulin, glucagon, and Ki-67. Few c-kit-positive cells simultaneously secreting insulin or glucagon were detected in all samples studied. These data suggest that such cells are in a state of differentiation into endocrinocytes, but no proliferation of c-kit-positive cells was observed.

This clinical study describes the intralesional application of the plasmid DNA encoding two therapeutic species-specific growth factors: vascular endothelial growth factor (VEGF164) and fibroblast growth factor 2 (FGF2) in seven horses to restore naturally occurring injuries of the superficial digital flexor tendon (SDFT) (tendinitis) and in three horses with suspensory ligament branch desmitis. Following application all horses were able to commence a more rapid exercise program in comparison to standardized exercise programs. Clinical observation and ultrasonic imaging was used to evaluate the regeneration rate of the tendon and ligament injury recovery and to confirm the safety of this gene therapy in horses, throughout a 12 month period. Follow-up data of the horses revealed a positive outcome including significant ultrasonographic and clinical improvements in 8 out of 10 horses with SDFT and suspensory ligament branch lesions, with return to their pre-injury level of performance by 2–6 months after the completion of treatment. The ninth horse initially presenting with severe suspensory ligament branch desmopathy, showed no significant ultrasonographic improvements in the first 2 months after treatment, however, it improved clinically and became less lame. The final horse, presenting with severe tendinitis of the SDFT returned to their pre-injury level of performance, but experienced re-injury 6 months after treatment. This data is highly promising, however, further research in experimental models, with the histopathological, immunohistochemical and gene expression evaluation of the equine tendon/ligament after gene therapy application is required in order to fully understand the mechanisms of action. This treatment and the significant clinical impacts observed represents an important advancement in the field of medicine.

Experimental study. Several neuro-degenerative disorders such as Alzheimer’s dementia, Parkinson’s disease and amyotrophic lateral sclerosis (ALS) are associated with genetic mutations, and replacing or disrupting defective sequences might offer therapeutic benefits. Single gene delivery has so far failed to achieve significant clinical improvements in humans, leading to the advent of co-expression of multiple therapeutic genes. Co-transfection using two or more individual constructs might inadvertently result in disproportionate delivery of the products into the cells. To prevent this, and in order to rule out interference among the many promoters with varying strength, expressing multiple proteins in equimolar amounts can be achieved by linking open reading frames under the control of only one promoter. Kazan, Russian Federation. Here we describe a strategy for adeno-viral co-expression of vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF2) interconnected through picorna-viral 2A-amino-acid sequence in transfected human umbilical cord blood mono-nuclear cells (hUCB-MCs). Presence of both growth factors, as well as absence of immune response to 2A-antigen, was demonstrated after 28–52 days. Following injection of hUCB-MCs into ALS transgenic mice, co-expression of VEGF and FGF2, as well as viable xeno-transplanted cells, were observed in the spinal cord after 1 month. These results suggest that recombinant adeno-virus containing 2A-sequences could serve as a promising alternative in regenerative medicine for the delivery of therapeutic molecules to treat neurodegenerative diseases, such as ALS.

Peripheral arterial diseases are characterized by a progressing tissue ischemia which results in the invalidization of patients. The aim of our research was to study the morphological effects of autologous peripheral blood stem cells intramuscular injection into patients with peripheral arterial disease. Peripheral blood stem cells were transplanted intramuscularly into a 48-year-old male patient with peripheral arterial disease stage IIb by Fontaine. The biopsies of his gastrocnemius muscle were taken before the stem cells were transplanted and 3 months after transplantation. These biopsies were stained with H&E and also with antibodies against CD34, myogenin, caspase 3, and bcl-2. Immunohistochemical study results showed an increase of capillary density of 32.7 % (P = 0.005). In muscular biopsies obtained before therapy, we identified single myogenin+ myosatellite cells, while 3 months after transplantation we detected the presence of cells with myogenin-positive nuclei and multinucleated myotubes. We also observed the formation of young myogenin+ muscle fibers with central nuclei. There was no significant difference in the expression of caspase-3 before transplantation and 3 months after transplantation. An increased number of bcl-2+ myosatellite cells, myotubes, and muscle fibers were detected after transplantation. The patient’s ankle-brachial index increased by 13.56 % (0.59 before and 0.67 3 months post transplantation). The patient’s pain-free walking distance by 89.97 % (from 59.56 to 113.77 m). Control arteriograms showed the formation of new collaterals. Transplanted autologous peripheral blood stem cells stimulated the formation of new capillaries, the activation of myosatellite cells and bcl-2 expression in muscle fibers.

In this clinical study, for the first time we used the direct gene therapy to restore severe injuries of the suspensory ligament branch and superficial digital flexor tendon in horses (Equus caballus). We injected the plasmid DNA encoding two therapeutic species-specific growth factors: vascular endothelial growth factor (VEGF164) and fibroblast growth factor 2 (FGF2) at the site of injury in the suspensory ligament branch and tendon. Treatment effects were evaluated with the use of clinical observation and ultrasound imaging during a period of a few months. We showed that gene therapy used within a period of 2-3 months after the injury resulted in the complete recovery of functions and full restoration of the severely damaged suspensory ligament and superficial digital flexor tendon.

Amyotrophic lateral sclerosis is a neurodegenerative disease characterized by progressive death of cerebral and spinal motorneurons. Using behavioral tests we studied the efficiency of gene-cell therapy in SOD1 G93A transgenic mice receiving xenotransplantation of human umbilical cord blood mononuclear cells genetically modified with adenoviral vectors encoding vascular endothelial growth factor (VEGF) and reporter green fluorescent protein (EGFP) genes. The cells were transplanted to mice on week 27 of life (preclinical stage of the disease). Behavioral tests (open field, grip strength test) showed that transplantation of umbilical cord blood mononuclear cells expressing VEGF significantly improved the parameters of motor and explorative activity, grip strength, and animal survival. Thus, gene-cell therapy based on genetically modified mononuclear cells expressing VEGF can be efficient for the treatment of amyotrophic lateral sclerosis.

Alzheimer's disease (AD) is a devastating and progressive form of dementia that is typically associated with a build-up of amyloid-β plaques and hyperphosphorylated and misfolded tau protein in the brain. Presently, there is no single test that confirms AD; therefore, a definitive diagnosis is only made after a comprehensive medical evaluation, which includes medical history, cognitive tests, and a neurological examination and/or brain imaging. Additionally, the protracted prodromal phase of the disease makes selection of control subjects for clinical trials challenging. In this study we have utilized a gene-expression array to screen blood and skin punch biopsy (fibroblasts, keratinocytes, and endothelial cells) for transcriptional differences that may lead to a greater understanding of AD as well as identify potential biomarkers. Our analysis identified 129 differentially expressed genes from blood of dementia cases when compared to healthy individuals, and four differentially expressed punch biopsy genes between AD subjects and controls. Additionally, we identified a set of genes in both tissue compartments that showed transcriptional variation in AD but were largely stable in controls. The translational products of these variable genes are involved in the maintenance of the Golgi structure, regulation of lipid metabolism, DNA repair, and chromatin remodeling. Our analysis potentially identifies specific genes in both tissue compartments that may ultimately lead to useful biomarkers and may provide new insight into the pathophysiology of AD.

Alzheimer's dementia (AD) is a degenerative brain disorder characterized mainly by cholinergic failure, but other neuro-transmitters are also deficient especially at late stages of the disease. Misfolded β-amyloid peptide has been identified as a causative agent, however inflammatory changes also play a pivotal role. Even though the most prominent pathology is seen in the cognitive functions, specific abnormalities of the central nervous system (CNS) are also reflected in the periphery, particularly in the immune responses of the body. The aim of this study was to characterize the dopaminergic and serotonergic systems in AD, which are also markedly disrupted along with the hallmark acetyl-choline dysfunction. Peripheral blood mono-nuclear cells (PBMCs) from demented patients were judged against comparison groups including individuals with late-onset depression (LOD), as well as non-demented and non-depressed subjects. Cellular sub-populations were evaluated by mono-clonal antibodies against various cell surface receptors: CD4/CD8 (T-lymphocytes), CD19 (B-lymphocytes), CD14 (monocytes), and CD56 (natural-killer (NK)-cells). The expressions of dopamine D(3) and D(4), as well as serotonin 5-HT(1A), 5-HT(2A), 5-HT(2B) and 5-HT(2C) were also assessed. There were no significant differences among the study groups with respect to the frequency of the cellular sub-types, however a unique profound increase in 5-HT(2C) receptor exclusively in NK-cells was observed in AD. The disease-specific expression of 5-HT(2C), as well as the NK-cell cyto-toxicity, has been linked with cognitive derangement in dementia. These changes not only corroborate the existence of bi-directional communication between the immune system and the CNS, but also elucidate the role of inflammatory activity in AD pathology, and may serve as potential biomarkers for less invasive and early diagnostic purposes as well.

Functionalized living cells are regarded as effective tools in directed cell delivery and tissue engineering. Here we report the facile functionalization of viable isolated HeLa cells with superparamagnetic cationic nanoparticles via a single-step biocompatible process. Nanoparticles are localized on the cellular membranes and do not penetrate into the cytoplasm. The magnetically responsive cells are viable and able to colonize and grow on substrates. Magnetically facilitated microorganization of functionalized cells into viable living clusters is demonstrated. We believe that the technique described here may find a number of potential applications in cell-based therapies and in development of whole-cell biosensors.

The presence of several subtypes of P2X receptors on early hemopoietic precursors (CD34+) from human umbilical blood was detected by flow cytometry. The expression of P2X receptors on umbilical blood lymphocytes was an order of magnitude higher than that on adult human blood cells. Our results attest to early involvement of P2X receptors in differentiation of human hemopoietic cells.

Cognitive dysfunction following surgery is a common complication, which increases the incidence of other co-morbid conditions, hospital and health-care costs. The reported rate of the occurrence of post-operative cognitive decline varies with different studies, depending on population profile, type of surgery, definition of cognitive disorder and detection methods, design of study, etc. It remains unclear whether these psychiatric signs and symptoms are direct results of the effects of surgery or general anesthesia. Nonetheless they are more frequent after cardiac surgery and are likely to be multi-factorial, but the patho-mechanisms are not yet fully characterized. This communication provides a synopsis of proteomics tools and delineates novel SELDI-TOF results to evaluate biomarkers in this regard. Presented for the first time is a classification of the clinically relevant forms of post-operative cognitive decline with the advent of a novel subclass.

In regenerative medicine, stem cell therapy can be used to replace, restore or enhance the biological function of damaged tissues and organs. Mesenchymal stem cells (MSCs) isolated from various sources are inexhaustible sources of therapeutic products for cell-based therapies. Various tissues in an adult organism can serve as sources of MSCs. MSCs have multipotent, regenerative and immunosuppressive properties. The main advantage of MSC is the safety of their use, but the effectiveness of stem cell therapy might be limited by low survivability and insufficient expression of various biologically active factors by the transplanted cells. To enhance the viability of MSC and increase their therapeutic potential, scientists perform genetic modification of such cells. For this purpose, recombinant genetic material can be delivered using various viral and non-viral methods. This chapter describes the advantages and disadvantages of commonly used viral, physical and chemical gene delivery vector systems in respect to MSC genetic modification. It should be noted that the MSCs derived from different tissues of the tooth have higher pro-angiogenic, neurogenic and regenerative potential compared to the stem cells of the bone marrow and adipose tissue. The main research areas for genetic engineering of MSCs derived from dental tissues are modulation of phenotype, immortalization, controlling the processes of differentiation and apoptosis, as well as increasing secretion of therapeutic growth factors. In this chapter, we summarize prospective studies of genetically modified MSCs from different tissues of the tooth in the context of their application in regenerative medicine for treatment of dental, ischemic and neurodegenerative diseases.

Mental illnesses are frequent co-morbid conditions in chronic systemic diseases. High incidences of depression, anxiety and cognitive impairment complicate cardiovascular and metabolic disorders such as hypertension and diabetes mellitus. Lifestyle changes including regular exercise have been advocated to reduce blood pressure and improve glycaemic control. The purpose of this project was to evaluate the effect of physical training on the most prevalent corollary psychiatric problems in patients with chronic organic ailments. This longitudinal study assessed the mental health of hypertensive (age: 57 ± 8 years) and/or diabetic (age: 53 ± 8 years) patients using mini-mental state examination, Beck’s depression inventory, Beck’s anxiety inventory and self-reporting questionnaire-20 before and after a 3-month supervised resistance and aerobic exercise programme comprising structured physical activity three times a week. Clinically relevant improvement was observed in the Beck’s depression inventory and Beck’s anxiety inventory scores following the 12-week training (61%, p = 0.001, and 53%, p = 0.02, respectively). Even though statistically not significant ( p = 0.398), the cognitive performance of this relatively young patient population also benefited from the programme. These results demonstrate positive effects of active lifestyle on non-psychotic mental disorders in patients with chronic systemic diseases, recommending exercise as an alternative treatment option.

Chronic disorders such as hypertension and diabetes mellitus are often associated with depressive and anxiety symptoms, as well as cognitive decline. Once developed, psychological support is essential for improving the quality of life. This study is aimed at identifying impaired mental health in connection with these systemic metabolic disorders. A total of 34 patients were included in this cross-sectional study: 17 hypertensive individuals with a mean age of 59 ± 10 years, and 17 diabetic patients aged 54 ± 10 years. The following psychometric tests were used: Mini-Mental State Examination (MMSE), Beck Depression Inventory (BDI), Beck Anxiety Inventory (BAI), and self-reporting questionnaire (SRQ-20). A large number of patients with high blood pressure or diabetes was associated with mental health problems (82% or 65%, respectively; p = 0.246). Affective disorder, especially moderate to severe depression, was seen mainly in diabetic patients (76%), whereas hypertensive individuals had higher prevalence of anxiety (64%). There was no cognitive impairment in this middle-aged population. This study shows a high proportion of depression and anxiety symptoms in patients with hypertension or diabetes mellitus, reinforcing the importance of psychiatric support for appropriate control of these metabolic disorders.

Amyotrophic lateral sclerosis (ALS) is an incurable, chronic, fatal neuro-degenerative disease characterized by progressive loss of moto-neurons and paralysis of skeletal muscles. Reactivating dysfunctional areas is under earnest investigation utilizing various approaches. Here we present an innovative gene-cell construct aimed at reviving inert structure and function. Human umbilical cord blood cells (hUCBCs) transduced with adeno-viral vectors encoding human VEGF, GDNF and/or NCAM genes were transplanted into transgenic ALS mice models. Significant improvement in behavioral performance (open-field and grip-strength tests), as well as increased life-span was observed in rodents treated with NCAM-VEGF or NCAM-GDNF co-transfected cells. Active trans-gene expression was found in the spinal cord of ALS mice 10 weeks after delivering genetically modified hUCBCs, and cells were detectable even 5 months following transplantation. Our gene-cell therapy model yielded prominent symptomatic control and prolonged life-time in ALS. Incredible survivability of xeno-transpanted cells was also observed without any immune-suppression. These results suggest that engineered hUCBCs may offer effective gene-cell therapy in ALS.

We evaluated the relationship between pathological changes in the liver and the state of intestinal microflora in rats with experimental dysbiosis. Changes in the intestinal microflora were accompanied by alteration of the morphological structure in the liver. Enhanced proliferation of Ito cells served as an indirect evidence of damage to the liver. Ito cells did not undergo transformation into myofibroblasts that excluded the possibility of fibrosis.

Loss of function may ensue when internal bodily mechanisms fall short of healing a damaged tissue. Regenerative medicine has recently been established as an innovative discipline to restore such failing systems by either replacing impaired parts and/or by stimulating the body’s own repair processes. Gross transplantation of entire organs, where possible, is now being changed with more sophisticated methods that involve cells or even genes only. Stem cells have the ability to self-renew and to differentiate into various tissues and therefore can be used to substitute lost cells. When genetically modified to express growth and trophic factors, they are also able to fulfill the task of promoting self-healing. Neuro-science is one of the fastest emerging fields of biology and medicine, however neuro-degenerative disorders still pose an insurmountable clinical challenge despite massive pioneering studies. This chapter presents the latest developments in gene-cell therapy and dissects advances in the use of human umbilical cord blood for the treatment of various diseases of the brain.

We estimated the count of pancreatic alpha- and beta-cells and blood glucose level at various stages of alloxan-induced diabetes in rats. Alloxan decreased the count of insulin-producing beta-cells, but increased the number of glucagon-secreting alpha-cells in the pancreas (week 1 of diabetes). These changes were accompanied by hyperglycemia. The decrease in blood glucose level in diabetic rats was associated with an increase in beta-cell count against the background of high density of pancreatic alpha-cells.

Several biomarkers are used in confirming the diagnosis of cognitive disorders. This study evaluates whether the level of these markers after heart surgery correlates with the development of cognitive dysfunction, which is a frequent complication of cardiac interventions. Concentrations of amyloid-β peptide, tau, and S100β in the cerebro-spinal fluid were assessed, as well as cognitive functions were evaluated before and after coronary artery bypass grafting, utilizing immuno-assays and psychometric tests, respectively. A drastic rise in the level of S100β was observed one week after the surgery, a mark of a severe generalized cerebral injury. The level of amyloid-β peptide significantly decreased, whereas the concentration of tau markedly increased six months postoperatively. Gradual cognitive decline was also present. These findings clearly demonstrate post-surgical cognitive impairment associated with changes in biomarkers similar to that seen in Alzheimer's disease, suggesting a unifying pathognomic factor between the two disorders. A holistic approach to coronary heart disease and Alzheimer's type dementia is proposed.

Current therapy of a number of neuropsychiatric maladies has only symptomatic modality. Effective treatment of these neuro-degenerative diseases, including amyotrophic lateral sclerosis (ALS), may benefit from combined gene/stem-cell approaches. In this report, mononuclear fraction of human umbilical cord blood cells (hUCBCs) were transfected by electroporation with dual plasmid constructs, simultaneously expressing vascular endothelial growth factor 165 (VEGF165) and human fibroblast growth factor 2 (FGF2) (pBud-VEGF-FGF2). These genetically modified hUCBCs were injected retro-orbitally into presymptomatic ALS transgenic animal models (G93A mice). Lumbar spinal cords of rodents were processed for immunofluoresent staining with antibodies against human nuclear antigen (HNA), oligodendrocyte-specific protein, S100, iba1, neuronal β3-tubulin and CD34. Co-localization of HNA and S100 was found in the spinal cord of mice after transplantation of genetically modified hUCBCs over-expressing VEGF-FGF2. Double staining in control animals treated with unmodified hUCBCs, however, revealed HNA+ cells expressing iba1 and CD34. Neuron-specific β3-tubulin or oligodendrocyte-specific protein were not expressed in hUCBCs in either control or experimental mice. These results demonstrate that genetically naïve hUCBCs may differentiate into endothelial (CD34+) and microglial (iba1+) cells; however when over-expressing VEGF-FGF2, hUCBCs transform into astrocytes (S100+). Autocrine regulation of VEGF and FGF2 on hUCBCs, signal molecules from dying motor neurons in spinal cord, as well as self-differentiating potential may provide a unique microenvironment for the transformation of hUCBCs into astrocytes that eventually serve as a source of growth factors to enhance the survive potential of surrounding cells in the diseased regions.

Objectives Among several other factors, the neuro-toxic β-amyloid peptide (βAP)-induced inflammatory mechanisms have also been implicated in the pathogenesis of Alzheimer's dementia (AD). Cytokines have recently emerged as prime candidates underlying this immune reaction. The purpose of this study was to evaluate the inflammatory response of peripheral blood mono-nuclear cells (PBMC) in AD. Design Cross-sectional (observational) study. Setting Behavioral and cognitive neurology clinic of the Universidade Federal de Minas Gerais in Belo Horizonte, Brazil. Participants AD patients (n = 19), healthy elderly (n = 19) and young (n = 14) individuals. Measurements Cytokine levels were assessed by enzyme-linked immuno-sorbent assay (ELISA) after exposing cells to a broad range of βAP concentrations (10 − 4 –10 − 10 M) as a stimulus. AD samples were weighed against leukocytes harvested from non-demented young and elderly subjects. Results Cytokine production of PBMCs in the youth was characterized by low baseline levels when compared to cells from the older generation. In the aging population, AD cells were distinguished from the healthy elderly sub-group by an even higher basal cytokine secretion. The low resting concentration in young individuals was markedly increased after treatment with βAP, however cells from the elderly, irrespective of their disease status, showed unchanged cytokine release following βAP administration. Non-specific activation of PBMCs with anti-CD3/CD28 antibodies resulted in elevated interleukin (IL)-1β concentrations in AD. Conclusions These results demonstrate a general over-production of cytokines and resistance to βAP in the old comparison group, with a more pronounced disruption/boosted pattern in AD. Our findings are in line with the hypothesis of “inflammaging”, i.e. an enhanced inflammatory profile with normal aging and a further perturbed environment in AD. The observed cytokine profiles may serve as diagnostic biomarkers in dementia.

The common drawback of many in vitro cell culture systems is the absence of appropriate micro-environment, which is formed by the combination of factors such as cell-cell contacts, extracellular matrix and paracrine regulation. Micro-environmental factors in a tumor tissue can influence physiological status of the cancer cells and their susceptibility to anticancer therapies. Interaction of cancer cells with their micro-environment and regional stem cells, therefore, is of particular interest. Development of in vitro systems which allow more accurate modeling of complex relations occurring in real tumor environments can increase efficiency of preclinical assays for screening anticancer drugs. The aim of this work was to study interactions between human mesenchymal stem cells (MSCs) and neuro-blastoma cancer SH-SY5Y cells under co-culture conditions on different coated surfaces to determine the effect of co-existence of cancer and stem cells on each cellular population under various stress conditions. We developed an efficient in vitro system for studying individual cancer and stem cell populations during co-culture using differential live fluorescent membrane labeling, and demonstrated self-organization of cancer and stem cells during co-culture on various coated surfaces. Our findings support the evidence that cancer and stem cell interactions play important roles in cellular behavior of cancer cells. These properties can be used in different fields of cancer research, tissue engineering and biotechnology.

The use of mesenchymal stem cells (MSCs) has been shown to be promising in chronic disorders such as diabetes, Alzheimer's dementia, Parkinson's disease, spinal cord injury and brain ischemia. Recent studies revealed that human tooth germs (hTG) contain MSCs which can be easily isolated, expanded and cryo-preserved. In this report, we isolated human tooth germ stem cells (hTGSCs) with MSC characteristics from third molar tooth germs, cryo-preserved them at -80( degrees )C for 6 months, and evaluated for their surface antigens, expression of pluri-potency associated genes, differentiation capacity, karyotype, and proliferation rate. These characteristics were compared to their non-frozen counterparts. In addition, neuro-protective effects of cryo-preserved cells on neuro-blastoma SH-SY5Y cells were also assessed after exposure to stress conditions induced by hydrogen-peroxide (oxidative stress) and paclitaxel (microtubule stabilizing mitotic inhibitor). After long term cryo-preservation hTGSCs expressed surface antigens CD29, CD73, CD90, CD105, and CD166, but not CD34, CD45 or CD133, which was typical for non-frozen hTGSCs. Cryo-preserved hTGSCs were able to differentiate into osteo-, adipo- and neuro-genic cells. They also showed normal karyotype after high number of population doublings and unchanged proliferation rate. On the other hand, cryo-preserved cells demonstrated a tendency for lower level of pluri-potency associated gene expression (nanog, oct4, sox2, klf4, c-myc) than non-frozen hTGSCs. hTGSCs conditioned media increased survival of SH-SY5Y cells exposed to oxidative stress or paclitaxel. These findings confirm that hTGSCs preserve their major characteristics and exert neuro-protection after long-term cryo-preservation, suggesting that hTGSCs, harvested from young individuals and stored for possible use later as they grow old, might be employed in cellular therapy of age-related degenerative disorders.

Genetically modified mono-nuclear cell fraction from human umbilical cord blood (HUCB) expressing human vascular endothelial growth factor (VEGF) and mouse neural L 1 cell adhesion molecule (L 1 CAM) were used for gene-stem cell therapy of transgenic G 93 A mice adopted as an animal amyotrophic lateral sclerosis (ALS) model. We generated non-viral plasmid constructs, expressing human VEGF 165 (pcDNA-VEGF) and mouse neural L 1 cell adhesion molecule (pcDNA-mL 1 CAM). Mono-nuclear fraction of HUCB cells were transiently transfected by electro-poration with a mixture of expression plasmids (pcDNA-VEGF + pcDNA-mL 1 CAM). Sixteen transgenic female and male mice were randomly assigned to three groups: (1) transplantation of genetically modified HUCB cells expressing L 1 and VEGF ( n = 6), (2) transplantation of un-transfected HUCB cells ( n = 5), and (3) control group ( n = 5). In first two experimental groups 1 × 10 6 cells were injected retro-orbitally in pre-symptomatic 22–25-week-old G 93 A mice. Our results demonstrate that HUCB cells successfully grafted into nervous tissue of ALS mice and survived for over 3 months. Therefore, genetically modified HUCB cells migrate in the spinal cord parenchyma, proliferate, but instead of transforming into nerve cells, they differentiate into endothelial cells forming new blood vessels. We propose that: (A) expression of mouse neural L 1 CAM is responsible for increased homing and subsequent proliferation of transplanted cells at the site of neuro-degeneration, (B) expression of human VEGF directs HUCB cell differentiation into endothelial cells, and (C) neuro-protective effect may stem from the delivery of various neuro-trophic factors from newly formed blood vessels.

Isolated human hepatocytes cultured in liver biomatrix express cytokeratins 8 and 18. By the end of the first and during the second week in culture, hepatocytes, express these cytokeratins and react with antibodies to cytokeratin-19. Cytokeratin-7 was found in individual cholangiocytes contaminating the culture. The presence of cytokeratin-19 in hepatocytes during the second week of culturing can be regarded as its reexpression.

Dimephosphone injected on days 1-19 of gestation did not cause fetal death and specific abnormalities in rats and did not modulate sex differentiation of fetuses. Morphological study of fetal liver revealed no pathological changes.

We evaluated the relationship between pathological changes in the liver and the state of intestinal microflora in rats with experimental dysbiosis. Changes in the intestinal microflora were accompanied by alteration of the morphological structure in the liver. Enhanced proliferation of Ito cells served as an indirect evidence of damage to the liver. Ito cells did not undergo transformation into myofibroblasts that excluded the possibility of fibrosis.

We estimated the count of pancreatic alpha- and beta-cells and blood glucose level at various stages of alloxan-induced diabetes in rats. Alloxan decreased the count of insulin-producing beta-cells, but increased the number of glucagon-secreting alpha-cells in the pancreas (week 1 of diabetes). These changes were accompanied by hyperglycemia. The decrease in blood glucose level in diabetic rats was associated with an increase in beta-cell count against the background of high density of pancreatic alpha-cells.

The effect of Gram-negative bacterial lipopolysaccharide on rat hepatocytes and sinusoidal cells was studied. The damage and regeneration potential of the liver were evaluated by activation of perisinusoidal Ito cells and proliferative activity of liver cells. Compensatory and repair reactions in the liver induced by lipopolysaccharide manifested by proliferation of liver cells and reversible activation of Ito cells without their transdifferentiation into myofibroblasts.