Lymphocyte profile in peripheral blood of patients with multiple myeloma.

Multiple myeloma (MM) is a disease which remains incurable. One of the main reasons is a weakened immune system that allows MM cells to survive. Therefore, the current research is focused on the study of immune system imbalance in MM to find the most effective immunotherapy strategies. Aiming to identify the key points of immune failure in MM patients, we analysed peripheral lymphocytes subsets from MM patients (n = 57) at various stages of the disease course and healthy individuals (HI, n = 15) focusing on T, NK, iNKT, B cells and NK-cell cytokines. Our analysis revealed that MM patients exhibited immune alterations in all studied immune subsets. Compared to HI, MM patients had a significantly lower proportion of CD4 + T cells (19.55% vs. 40.85%; p < 0.001) and CD4 + iNKT cells (18.8% vs. 40%; p < 0.001), within B cells an increased proportion of CD21LCD38L subset (4.5% vs. 0.4%; p < 0.01) and decreased level of memory cells (unswitched 6.1% vs. 14.7%; p < 0.001 and switched 7.8% vs. 11.2%; NS), NK cells displaying signs of activation and exhaustion characterised by a more than 2-fold increase in SLAMF7 MFI (p < 0.001), decreased expression of NKG2D (MFI) and NKp46 (%) on CD16 + 56 + and CD16 + 56- subset respectively (p < 0.05), Effective immunotherapy needs to consider these immune defects and monitoring of the immune status of MM patients is essential to define better interventions in the future.

Human cells with osteogenic potential in bone tissue research.

Bone regeneration after injury or after surgical bone removal due to disease is a serious medical challenge. A variety of materials are being tested to replace a missing bone or tooth. Regeneration requires cells capable of proliferation and differentiation in bone tissue. Although there are many possible human cell types available for use as a model for each phase of this process, no cell type is ideal for each phase. Osteosarcoma cells are preferred for initial adhesion assays due to their easy cultivation and fast proliferation, but they are not suitable for subsequent differentiation testing due to their cancer origin and genetic differences from normal bone tissue. Mesenchymal stem cells are more suitable for biocompatibility testing, because they mimic natural conditions in healthy bone, but they proliferate more slowly, soon undergo senescence, and some subpopulations may exhibit weak osteodifferentiation. Primary human osteoblasts provide relevant results in evaluating the effect of biomaterials on cellular activity; however, their resources are limited for the same reasons, like for mesenchymal stem cells. This review article provides an overview of cell models for biocompatibility testing of materials used in bone tissue research.

The origin and development of fat droplet and its role in health and illness.

Lipid droplets are found in most cells, where they are needed for lipid metabolism and energy homeostasis. Their biogenesis is still not entirely described process, in which an organelle with monomolecular layer on the surface originates from two-layer membrane of the smooth endoplasmic reticulum. Large lipid droplets can be formed either by growth or by fusion of smaller lipid droplets. Their basic functions are controlled lipid deposition and lipid metabolism and the protection of the intracellular environment from lipotoxicity. Currently, dysfunction of lipid droplet is associated with a number of diseases such as obesity, cardiometabolic syndrome, tumor and neurodegenerative diseases, lipodystrophy and cachexia. Keywords: lipid droplet, adipocyte, endoplasmic reticulum, perilipin, obesity, type 2 diabetes mellitus, lipodystroph.

Some immunological properties of female saliva and its effect on sperm motility.

BACKGROUND: In this study, we investigated pH, levels of IgA, IgG, IgM, lysozyme and spermagglutinating antibodies in human saliva of healthy women, with or without hormonal contraception (HC) and their effect on sperm motility. METHODS AND RESULTS: Saliva was collected immediately after waking up from 59 healthy randomly selected women. We measured pH in sera and in saliva as well as immunoglobulin G, A and M levels in saliva by radial immunodiffusion. Sperm - saliva capillary penetration test in native saliva served for examination of sperm motility, indirect-MAR test for detection of sperm antibodies in sera, and in saliva, both observed by inverted microscope. The average pH values in native saliva according to the menstrual cycles were: from 2-5 days 6.896, from 6-17 days 7.027, and from 18 and more days 7.17. Rapid decrease of sperm motility was registered in acidic saliva in comparison with alkaline. The average concentration of IgA, IgG, IgM and lysozyme slightly differed in women with or without hormonal contraception. Moderate decreasing of sperm motility was found in saliva with higher levels of IgA. No sperm agglutinating antibodies in saliva and in serum were detected in all subjects. CONCLUSIONS: The pH of saliva is influenced by several causes (hormonal, immunological, microbiological factors, by meal and drinks, smoking, etc.). We demonstrated that sperm motility is lower in acidic saliva. pH is markedly changed during various days of menstrual cycles. We proved individual high levels of saliva IgA. Human saliva used as coital lubricant decreases sperm motility, but does not replace a local form of contraception.

Intracerebellar application of P19-derived neuroprogenitor and naive stem cells to Lurcher mutant and wild type B6CBA mice.

BACKGROUND: Neurotransplantation has great potential for future treatments of various neurodegenerative disorders. Preclinically, the Lurcher mutant mouse represents an appropriate model of genetically-determined olivocerebellar degeneration. The aim of the present study was to assess survival of naïve and neurally differentiated P19 carcinoma stem cells following transplantation into the cerebellum of Lurcher mice and wild type littermates. MATERIAL/METHODS: Adult normal wild type (n=51) and Lurcher mutant mice (n=87) of the B6CBA strain were used. The mean age of the animals at the time of transplantation was 261.5 days. Suspension of naive and neurally differentiated P19 carcinoma stem cells was injected into the cerebellum of the mice. In the Lurcher mutants, 2 depths of graft injection were used. Three weeks after implantation the brains of experimental animals were examined histologically. RESULTS: Survival of neuroprogenitor grafts at a depth of 1.6 mm was significantly higher in wild type vs. Lurcher mutant mice. In wild type mice, the typical graft localization was in the middle of the cerebellum, whereas in Lurcher mice the graft was never found inside the degenerated cerebellum and was primarily localized in the mesencephalon. CONCLUSIONS: We conclude that the appearance and low survival rate of cerebellar P19 carcinoma stem cell grafts in the Lurcher mutant mice weigh against the therapeutic value of this cell line in preclinical studies of neurodegeneration.

Laser Capture Microdissection Coupled Capillary Immunoassay to Study the Expression of PCK-2 on Spatially-Resolved Islets of Rat Langerhans.

The platform for precise proteomic profiling of targeted cell populations from heterogeneous tissue sections is developed. We demonstrate a seamless and systematic integration of LCM with an automated cap-IA for the handling of a very small-sized dissected tissues section from the kidney, liver and pancreatic Langerhans islet of rats. Our analysis reveals that the lowest LCM section area ≥ 0.125 mm2 with 10 µm thickness can be optimized for the detection of proteins through LCM-cap-IA integration. We detect signals ranging from a highly-abundant protein, ß-actin, to a low-abundance protein, LC-3AB, using 0.125 mm2 LCM section from rat kidney, but, so far, a relatively large section is required for good quality of results. This integration is applicable for a highly-sensitive and accurate assessment of microdissected tissue sections to decipher hidden proteomic information of pure targeted cells. To validate this integration, PCK2 protein expression is studied within Langerhans islets of normal and diabetic rats. Our results show significant overexpression of PCK2 in Langerhans islets of rats with long-term diabetes.

Role of Polymorphisms of NKG2D Receptor and Its Ligands in Acute Myeloid Leukemia and Human Stem Cell Transplantation.

Natural killer cells possess key regulatory function in various malignant diseases, including acute myeloid leukemia. NK cell activity is driven by signals received through ligands binding activating or inhibitory receptors. Their activity towards elimination of transformed or virally infected cells can be mediated through MICA, MICB and ULBP ligands binding the activating receptor NKG2D. Given the efficiency of NK cells, potential target cells developed multiple protecting mechanisms to overcome NK cells killing on various levels of biogenesis of NKG2D ligands. Targeted cells can degrade ligand transcripts via microRNAs or modify them at protein level to prevent their presence at cell surface via shedding, with added benefit of shed ligands to desensitize NKG2D receptor and avert the threat of destruction via NK cells. NK cells and their activity are also indispensable during hematopoietic stem cell transplantation, crucial treatment option for patients with malignant disease, including acute myeloid leukemia. Function of both NKG2D and its ligands is strongly affected by polymorphisms and particular allelic variants, as different alleles can play variable roles in ligand-receptor interaction, influencing NK cell function and HSCT outcome differently. For example, role of amino acid exchange at position 129 in MICA or at position 98 in MICB, as well as the role of other polymorphisms leading to different shedding of ligands, was described. Finally, match or mismatch between patient and donor in NKG2D ligands affect HSCT outcome. Having the information beyond standard HLA typing prior HSCT could be instrumental to find the best donor for the patient and to optimize effects of treatment by more precise patient-donor match. Here, we review recent research on the NKG2D/NKG2D ligand biology, their regulation, description of their polymorphisms across the populations of patients with AML and the influence of particular polymorphisms on HSCT outcome.

Chronic DDE Exposure Modifies Mitochondrial Respiration during Differentiation of Human Adipose-Derived Mesenchymal Stem Cells into Mature Adipocytes.

The contribution of environmental pollutants to the obesity pandemic is still not yet fully recognized. Elucidating possible cellular and molecular mechanisms of their effects is of high importance. Our study aimed to evaluate the effect of chronic, 21-day-long, 2,2-bis (4-chlorophenyl)-1,1-dichlorethylenedichlorodiphenyldichloroethylene (p,p'-DDE) exposure of human adipose-derived mesenchymal stem cells committed to adipogenesis on mitochondrial oxygen consumption on days 4, 10, and 21. In addition, the mitochondrial membrane potential (MMP), the quality of the mitochondrial network, and lipid accumulation in maturing cells were evaluated. Compared to control differentiating adipocytes, exposure to p,p'-DDE at 1 µM concentration significantly increased basal (routine) mitochondrial respiration, ATP-linked oxygen consumption and MMP of intact cells on day 21 of adipogenesis. In contrast, higher pollutant concentration seemed to slow down the gradual increase in ATP-linked oxygen consumption typical for normal adipogenesis. Organochlorine p,p'-DDE did not alter citrate synthase activity. In conclusion, in vitro 1 µM p,p'-DDE corresponding to human exposure is able to increase the mitochondrial respiration per individual mitochondrion at the end of adipocyte maturation. Our data reveal that long-lasting exposure to p,p'-DDE could interfere with the metabolic programming of mature adipocytes.

Long-Term Cryopreservation Does Not Affect Quality of Peripheral Blood Stem Cell Grafts: A Comparative Study of Native, Short-Term and Long-Term Cryopreserved Haematopoietic Stem Cells.

Cryopreserved haematopoietic progenitor cells are used to restore autologous haematopoiesis after high dose chemotherapy. Although the cells are routinely stored for a long period, concerns remain about the maximum storage time and the possible negative effect of storage on their potency. We evaluated the effect of cryopreservation on the quality of peripheral stem cell grafts stored for a short (3 months) and a long (10 years) period and we compared it to native products.The viability of CD34+ cells remained unaffected during storage, the apoptotic cells were represented up to 10% and did not differ between groups. The clonogenic activity measured by ATP production has decreased with the length of storage (ATP/cell 1.28 nM in native vs. 0.63 in long term stored products, P < 0.05). Only borderline changes without statistical significance were detected when examining mitochondrial and aldehyde dehydrogenase metabolic activity and intracellular pH, showing their good preservation during cell storage. Our experience demonstrates that cryostorage has no major negative effect on stem cell quality and potency, and therefore autologous stem cells can be stored safely for an extended period of at least 10 years. On the other hand, long term storage for 10 years and longer may lead to mild reduction of clonogenic capacity. When a sufficient dose of stem cells is infused, these changes will not have a clinical impact. However, in products stored beyond 10 years, especially when a low number of CD34+ cells is available, the quality of stem cell graft should be verified before infusion using the appropriate potency assays.

Mitochondria in White, Brown, and Beige Adipocytes.

Mitochondria play a key role in energy metabolism in many tissues, including cardiac and skeletal muscle, brain, liver, and adipose tissue. Three types of adipose depots can be identified in mammals, commonly classified according to their colour appearance: the white (WAT), the brown (BAT), and the beige/brite/brown-like (bAT) adipose tissues. WAT is mainly involved in the storage and mobilization of energy and BAT is predominantly responsible for nonshivering thermogenesis. Recent data suggest that adipocyte mitochondria might play an important role in the development of obesity through defects in mitochondrial lipogenesis and lipolysis, regulation of adipocyte differentiation, apoptosis, production of oxygen radicals, efficiency of oxidative phosphorylation, and regulation of conversion of white adipocytes into brown-like adipocytes. This review summarizes the main characteristics of each adipose tissue subtype and describes morphological and functional modifications focusing on mitochondria and their activity in healthy and unhealthy adipocytes.

Immunohistochemical detection of cancer stem cell related markers CD44 and CD133 in metastatic colorectal cancer patients.

AIM: The goal of this study was to semiquantitatively detect presence of cancer stem cells markers CD44 and CD133 in immunohistochemically stained paired samples of colorectal cancer (CRC) and colorectal liver metastases (CLM). Level of staining intensity was compared to clinical and pathological characteristics of tumors with the aim to identify impact of CD44 or CD133 expression on tumor behavior. PATIENTS AND METHODS: Formalin fixed paraffin embedded samples from 94 patients with colorectal tumor and liver metastases were collected at Sikl's Department of Pathology. Samples were stained by antibodies against CD44 and CD133. Presence and intensity of staining was assessed semiquantitatively by three trained researchers. RESULTS: Patients with higher level of CD133 staining in CRC had longer disease free interval (Cox-Mantel P = 0.0244), whereas we found no relation between CD44 expression and overall survival or disease free interval. CD133 expression in CRC and CLM differed based on CRC grading; in case of CD44 we found differences in staining intensity in individual stages of tumor lymph node invasion. CONCLUSION: Effect of cancer stem cell markers on prognosis of colorectal cancer can vary depending on pathological classification of tumor, and we have shown that CD133, generally considered to be a negative marker, can bear also clinically positive prognostic information in group of patients with colorectal liver metastases.

Effects of the czech propolis on sperm mitochondrial function.

Propolis is a natural product that honeybees collect from various plants. It is known for its beneficial pharmacological effects. The aim of our study was to evaluate the impact of propolis on human sperm motility, mitochondrial respiratory activity, and membrane potential. Semen samples from 10 normozoospermic donors were processed according to the World Health Organization criteria. Propolis effects on the sperm motility and mitochondrial activity parameters were tested in the fresh ejaculate and purified spermatozoa. Propolis preserved progressive motility of spermatozoa in the native semen samples. Oxygen consumption determined in purified permeabilized spermatozoa by high-resolution respirometry in the presence of adenosine diphosphate and substrates of complex I and complex II (state OXPHOSI+II) was significantly increased in the propolis-treated samples. Propolis also increased uncoupled respiration in the presence of rotenone (state ETSII) and complex IV activity, but it did not influence state LEAK induced by oligomycin. Mitochondrial membrane potential was not affected by propolis. This study demonstrates that propolis maintains sperm motility in the native ejaculates and increases activities of mitochondrial respiratory complexes II and IV without affecting mitochondrial membrane potential. The data suggest that propolis improves the total mitochondrial respiratory efficiency in the human spermatozoa in vitro thereby having potential to improve sperm motility.

Screening tests of reproductive immunology in systemic lupus erythematosus.

Female patients in reproductive age with systemic lupus erythematosus and fertility complications together are observed by rheumatologists, gynecologists, and reproductive immunologists. The paper notes the presence of autoantibodies to zona pellucida, to phospholipids (phosphatidyl serine, phosphatidyl ethanolamine, phosphatidyl inositol, phosphatidyl glycerol, phosphatidic acid, annexin V, beta-2 glycoprotein I, and cardiolipin) and of isoantibodies to sperm cells. Isoantibodies to sperm cells are not significantly predominant, but autoimmunity is well expressed in IgG positivity against phosphatidyl inositol, phosphatidyl ethanolamine, phosphatidyl serine, cardiolipin, and beta-2 glycoprotein I, as well as antizona pellucida antibodies in IgG isotype. According to the levels of autoantibodies we have to choose preventive treatment to protect mother and her foetus.