A pilot study of mitochondrial DNA point mutation A3243G in a sample of Croatian patients having type 2 diabetes mellitus associated with maternal inheritance.

In this work, patients having type 2 diabetes mellitus and diabetic mothers were tested for the presence of mitochondrial DNA point mutation A3243G. This mutation is associated with the MELAS syndrome (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes), diabetes and deafness. Twenty-two diabetic persons were screened. DNA was isolated from peripheral blood lymphocytes and from swabs of oral mucosa. The mitochondrial DNA point mutation A3243G was detected using PCR-RFLP test. The mutation was detected in oral mucosal DNA of two patients (but not from lymphocyte DNA). One patient was a man with hearing and visual impairments and proteinuria; the other was a woman having proteinuria but no hearing impairment. The mutation was not detectable in oral mucosal DNA from the control persons: 20 diabetic patients having diabetic fathers and 22 healthy, nondiabetic volunteers. The incidence of mitochondrial DNA point mutation A3243G in this study of Croatian diabetic patients is in line with data in the literature.

The effect of the zeolite clinoptilolite on serum chemistry and hematopoiesis in mice.

Zeolites are natural or synthetic crystalline alumosilicates with ion exchanging properties. Supplied in fodder, they promote biomass production and animal health. Our aim was to assess the effects of the natural zeolite, clinoptilolite, on hematopoiesis, serum electrolytes and essential biochemical indicators of kidney and liver function in mice. Two preparations differing in particle size were tested: a powderized form obtained by countercurrent mechanical treatment of the clinoptilolite (MTCp) and normally ground clinoptilolite (NGCp). Young adult mice were supplied with food containing 12.5, 25 or 50% clinoptilolite powder. Control animals received the same food ration without the clinoptilolite. After 10, 20, 30 and 40 days, six animals from each group were exsanguinated to obtain blood for hematological and serum for biochemical measurements as well as to collect femoral bone marrow for determination of hematopoietic activity. Clinoptilolite ingestion was well tolerated, as judged by comparable body masses of treated and control animals. A 20% increase of the potassium level was detected in mice receiving the zeolite-rich diet, without other changes in serum chemistry. Erythrocyte, hemoglobin and platelet levels in peripheral blood were not materially affected. NGCp caused leukocytosis, with concomitant decline of the GM-CFU content in the bone marrow, which was attributed to intestinal irritation by rough zeolite particles. The mechanically treated clinoptilolite preparation caused similar, albeit less pronounced, changes. In a limited experiment, mice having transplanted mammary carcinoma in the terminal stage showed increased potassium and decreased sodium and chloride levels, severe anemia and leukocytosis, decreased bone marrow cellularity and diminished content of hematopoietic progenitor cells in the marrow. The clinoptilolite preparations ameliorated the sodium and chloride decline, whereas the effects on hematopoiesis were erratic.

Thiorphan, an inhibitor of neutral endopeptidase/enkephalinase (CD10/CALLA) enhances cell proliferation in bone marrow cultures of patients with acute leukemia in remission.

Thiorphan, (DL-mercapto-2-benzylpropanoyl)-glycine is a potent and specific inhibitor of membrane metallo-endopeptidase (EC 3.4.24.11, CD10). We explored its effects in short-term clonal cultures of the bone marrow from 10 patients with acute leukemia in remission. The cell suspensions were incubated with thiorphan (10(-13) to 10(-5) M) and seeded for the granulocyte/macrophage-colony forming unit (GM-CFU) assay. In normal bone marrow samples the median seeding efficiency was 119 colonies and clusters per 10(5) cells and thiorphan caused slight stimulation of the clonal growth in concentrations above 10(-9) M. In the leukemic samples, the median seeding efficiency varied from 10 to 366 colonies and clusters per 10(5) seeded cells. Meaningful alterations of the clonal growth were noted in 32 out of 83 thiorphan-treated cultures (39%). In those 32 cultures the stimulatory effects outnumbered the inhibitory effects (24 versus 8). Thus, thiorphan stimulated the progenitor cell proliferation in bone marrow samples from the normal donor and from the patients with acute leukemia in remission. Thiorphan binding to CD10 might interfere with the processing of neuropeptide hemoregulatory factors and thus influence the progenitor cell proliferation.

[What a physician should know about zeolites]. / Sto lijecnik treba znati o zeolitima.

Zeolites are natural and synthetic hydrated crystalline aluminosilicates endowed with absorptive and ion exchange properties. They have found numerous and multifarous applications--in industry as catalysts and absorbents, in water sanitation for the removal of ammonia and heavy metals, in agriculture as fertilizers, and in animal husbandry as the absorbents of excreted material and as food additives. Medical applications have included the use in filtration systems for anesthesia or dialysis and as the contrast materials in NMR imaging. Recently, zeolite powders for external use have found application as deodorants, antimycotic agents and wound dressings. Peroral use of encapsulated zeolite powders enriched with vitamins, oligoelements or other ingredients has been claimed to exert beneficial medical effects. Ingestion of zeolites may be considered analogous to the clay eating (geophagia), considered in traditional medicine as a remedy for various illnesses. Being amphoteric, zeolites are partly soluble in acid or alkaline media, but within the physiological pH range the solubility is generally low. Minimal amounts of free aluminium or silicium from the ingested zeolites are resorbed from the gut. The bulk of ingested zeolite probably remains undissolved in the gut. In view of the ion exchange properties, zeolites may be expected to change the ionic content, pH and buffering capacity of the gastrointestinal secretions and to affect the transport through the intestinal epithelium. In addition, zeolites could affect the bacterial flora and the resorption of bacterial products, vitamins and oligoelements. The contact of zeolite particles with gastrointestinal mucosa may elicit the secretion of cytokines with local and systemic actions. Reactive silicium ions might react with biomolecules of the intestinal epithelium, and if resorbed, do so in other cells. Mutagenic and carcinogenic effects of zeolite particles have been described, resembling such effects of asbestos fibers. Thus, local and systemic effects of zeolites may be complex and interrelated, and an objective assessment requires appropriate experimental models.

Effects of a membrane-metallopeptidase blocking agent thiorphan in long-term cultures of human bone marrow.

Thiorphan [(DL-3-mercapto-2-benzylpropanoyl)-glycine], a drug blocking the activity of membrane metalloendopeptidase EC 3.4.24.11 (CD10, CALLA), was added to long-term cultures of human bone marrow. Progression of the cultures was assessed by cell counts, cytology and clonogenic (GM-CFU) ability of the non-adherent cells in the supernatant and by morphology of the adherent stromal layer. A stimulatory effect on hematopoiesis was noted.

Effect of opioid peptide methionine-enkephalin in long-term cultures of human bone marrow.

Methionine enkephalin, an opioid peptide belonging to the family of neuropeptides, has been shown to function as a neurotransmitter, hormone and growth factor. The present work explored its effects in long-term culture of bone marrow cells, harvested from a patient with acute lymphoid leukemia (ALL-L3) in the second complete remission. Nine cultivation flasks were established and maintained for five weeks, with medium renewal once a week. At each re-feeding, methionine-enkephalin was added to the cultures in final concentrations 10(-8), 10(-10) or 10(-12) M, and granulocyte-macrophage progenitor cells (GM-CFU) were determined among the harvested, nonadherent cell populations. The total number of nonadherent cells was 8% to 42% higher in the treated cultures than in the control, nontreated cultures, and the GM-CFU counts were three to four times higher. Those changes, although evident, did not reach statistical significance because of the small group sizes. In 1 of 9 cultures the adherent cell layer was atypical, the cell population consisted of small cells resembling the lymphoblasts, and the cell count was 2-8 times higher than in the controls. That aberrant culture has presumably arisen from residual leukemic cells remaining in the bone marrow after chemotherapy. The findings support the idea that opioid peptides, including methionine-enkephalin, participate in regulation of hematopoiesis. Two mechanisms may have accounted for the observed effects of enkephalin on cultured bone marrow cells: an indirect action, via interleukins secreted from the stromal cells upon stimulation of the opioid receptors, or a direct action on hematopoietic precursors.

Oligopeptide fragments of the enkephalin molecule interfere with hematopoietic cell colony formation.

Dipeptide Tyr-Gly and tripeptide Tyr-Gly-Gly representing the NH(2)-end of the Met-enkephalin molecule inhibited the GM-colony formation in clonal cultures of mouse bone marrow cells. Intermediate or C00H-terminal dipeptides Gly-Gly and Phe-Met respectively were inneffective. The suppressive effects were not abolished by opioid receptor blocking agent naloxone and only partly so by depletion of the accessory (adherent) cells. The results are congruent with idea that neuropeptides and products of their enzymatic degradation participate in the regulation of hematopoiesis.

[Neuropeptides, endogenous opioid peptides and cell proliferation]. / Neuropeptidi, endogeni opioidni peptidi i stanicna proliferacija.

Neuropeptides are oligo- or polypeptidies having a number of common features including biosynthesis, metabolism and biological effectiveness at extremely low concentrations. They function as cotransmitters in the central, peripheral and autonomic nervous system. As autocrine, paracrine, neurocrine or endocrine factors, neuropeptides also directly modulate functions of many types of cells in different tissues, including the lymphoid tissue. Neuropeptides influence cell proliferation and differentiation. Those functions are accomplished by neuropeptide binding to specific receptors. Recent studies emphasize the participation of neuropeptides in the control of organogenesis in embryonal, fetal and early postnatal periods as well as in tumor growth control. The family of neuropeptides includes endogenous opioid peptides, found in nervous and many other tissues. Lymphoid and hematopoietic tissues produce opioid-like oligopeptides, and the membrane marker CD10/CALLA (enkephalinase) expressed on lymphoid, myeloid and stromal bone marrow cells functions as an enzyme processing the enkephalins and other neuropeptides. It might be assumed therefore that opioid peptides participate among other cytokines in the regulation of hematopoiesis.

[Skin cell culture: utilization in plastic surgery and laboratory studies]. / Kultura koznih stanica: primjena u plasticnoj kirurgiji i laboratorijskom istrazivanju.

Skin is the largest organ of the human body (8-10 kg, 1.5-2.0 m2, 10(11) cells of epidermal, mesenchymal and neural origin). Although endowed with remarkable regeneration ability, the recovery after major injuries viz. burns requires appropriate surgical treatment, temporary coverage of defects and supportive measures. Large defects are covered with viable transplants of autologous or allogeneic skin, frozen or lyophilized human and animal skin, bioartificial tissues made of synthetic or biodegradable materials, sheets of keratinocytes cultured in vitro. The use of autotransplants is limited by the size of preserved skin areas as well as by general condition of the patient. Allotransplants collected from cadavers or volunteers are rejected after 1 or 2 weeks and thus afford only temporary coverage. Grafts of human or animal skin devitalized by lyophilization or freezing in glycerol accommodate connective tissue and blood vessels ingrowing from the graft bed but eventually dissolve. Artificial skin consists of collagen, chondroitin or similar fiber network (substituting the dermis) covered by semipermeable silicon foil (substituting the epidermis). After healing in, the silicon foil is peeled off and replaced by skin autotransplants or autologous keratinocytes grown and expanded in vitro. The technique for massive production of human keratinocytes, invented some twenty years ago, has been applied for clinical purposes by several specialized centers. During the culture period of approximately three weeks the keratinocyte population may enlarge five to ten thousandfold. Keratinocytes obtained from a 1.5 cm2 piece of skin (half of a postal stamp) may thus yield progeny sufficient for the coverage of 1.5 m2, which is almost the total body surface. The period required for culturing autologous keratinocytes is bridged by temporary transplants and vigorous supportive treatment of the patient. Cultured keratinocytes display all essential features of keratinocytes in situ. They divide and differentiate, express membrane structures required for intercellular communication and reception of signals regulating cell division and differentiation, secrete cytokines. In addition to clinical application, the culture of human keratinocytes is a convenient and useful model for studies of cellular biology. This review is illustrated by first examples of keratinocyte cultures grown in our laboratory.

Organotypic skin cultures: a human model for basic studies.

AIM: To produce organotypic skin cultures using human skin samples as a source of keratinocytes and fibroblasts. METHODS: Keratinocytes and fibroblasts from human skin samples were separated by warm trypsine and collagenase, respectively. Keratinocytes were plated in tissue culture dishes in keratinocyte serum free medium supplemented with epidermal growth factor and bovine pituitary extract, and were grown until confluence. Fibroblasts were cultured in Dulbecco's medium (DMEM) supplemented with fetal bovine serum and hydrocortisone. A mixture of fibroblasts, rat dermal collagen type I, E tissue culture medium, and reconstitution buffer were used as a dermal equivalent. Keratinocytes were plated on the top of the dermal equivalent and cultured for 10 days in organotypic culture dishes on stainless steel grids in the supplemented DMEM medium. The cultures were fixed in formaline, embedded in paraffin, stained with hematoxylin and eosin, and immunohistochemically stained with a nti-cytokeratin and anti-HLA-DR antibody. RESULTS: Cultured keratinocytes in organotypic skin cultures expressed the majority of the cytokeratins seen in the normal stratified epithelium. Consistent with previous studies, organotypic skin cultures did not show antigen-presenting Langerhans cells. CONCLUSION: Human skin from patients who underwent thoracic surgery can be used to produce organotypic skin cultures. This artificial skin can serve as a basis for future basic science studies and as a skin transplantation model.

Thiorphan stimulates clonal growth of GM-CFU in short-term cultures of bone marrow from a healthy donor and from patients with non-Hodgkin lymphoma.

Thiorphan, a specific inhibitor of membrane neutral endopeptidase (NEP, EC 3.4.24.11) also known as the common acute lymphoblastic leukemia antigen (CALLA, CD10) was added into short-term clonal cultures of the buffy coat concentrates of human bone marrow obtained from a healthy donor (six experiments) and from ten patients with non-Hodgkin lymphoma (NHL) (eight in complete remission, one in partial remission and one in relapse). Thiorphan concentrations ranged from 10(-5) to 10(-13) M. Nanomolar and higher concentrations of the drug mildly stimulated the granulocyte-macrophage colony-forming unit (GM-CFU) counts in the cultures of normal bone marrow, reaching the significance at 10(-7) M. Meaningful alterations of the GM-CFU counts were noted in 31 of 79 thiorphan-treated cultures of NHL bone marrow (39%). In those cultures the stimulatory effects (33%) outnumbered the inhibitory ones (6%). The stimulatory effects occurred mainly in the bone marrow samples of the patients with highly malignant NHL. The observations are compatible with the idea that the membrane endopeptidase (CALLA, CD10) participates in processes controlling the proliferation and differentiation of hematopoetic cells by cleaving the neuropeptides and related hemoregulatory peptides.

[Membrane metalloendopeptidase (CD10/CALLA): distribution, physiologic and pathophysiologic functions and its inhibitors]. / Membranska metaloendopeptidaza (CD10/CALLA): rasprostranjenost, fizioloske i patofizioloske funkcije i inhibitori.

Membrane metalloendopeptidase EC 3.4.24.11 (Enkephalinase, neutral endopeptidase, NEP) is a cellular ectoenzyme, immunophenotypically identified as the leukocyte cluster of differentiation CD10 or CALLA (common acute lymphoblastic leukemia antigen). Immunological, biochemical and molecular biology techniques have identified tis cell membrane feature in various organs: brain, cardiovascular system, lung, placenta, kidney etc. The CD10 immunophenotype is a common feature of lymphoblasts in acute lymphoid leukemia not expressing the T- or B-markers. The enzymatic activity of CD10/NEP possibly influences normal lymphocyte ontogeny by proteolytic cleavage of the regulatory peptides. The substrates of CD10/NEP in the kidneys are (see the list of abbreviations) ANP, adrenomedullin and PAMP; in the brain, the substrates are enkephalins and oxytocin; in the lung, bombesin, BLP, GRP, neuromedin C, substance P and neurokinin A; in the cardiovascular system, angiotenisin II, bradykinin and CGRP; in the gut, VIP; on the neutrophil membrane, fMLP etc. Some substrates are not strictly tissue-specific, e.g. substance P. Preclinical and clinical trials explore possibilities of therapeutic application of the inhibitors of neutral endopeptidase, such as thiorphan in the management of pain, diarrhoea, depression, arterial hypertension and asthma. Other possibilities of application include the treatment of hyalinomembranous disease and prevention of neurotoxicosis in tetanus and botulism.

[[Long-term bone marrow culture]. / Dugotrajna kultura kostane srzi.

Long-term bone marrow culture is an experimental in vitro model of hematopoiesis imitating conditions in vivo. It contains hematopoietic elements at various stages of differentiation as well as a supportive stromal microenvironment. Primitive hematopoietic stem cells of mesenchymal origin, the long-term culture initiating cells proliferate and differentiate into different cell types, giving rise to the adherent stromal layer and to various hematopoietic elements attached to it or floating freely in the supernatant medium. The stromal layer keeps the hematopoietic cells aggregated, helps their mitosis, differentiation and maturation by cell-to-cell contact, produces hematopoietic growth factors (cytokines), and forms the extracellular matrix required for cell attachment. Hematopoiesis occurs without exogenous growth factors. The appearance and development of the stroma, the proliferation and differentiation of hematopoietic progenitor cells, and the production of cytokines differ in long-term cultures of the normal and of pathologically altered bone marrow. Long-term bone marrow culture is applied in fundamental studies of normal and pathologically altered hematopoiesis, in pharmacological research, in the purging of residual leukemia cells from bone marrow autotransplants, and in the gene transfer. It is also suitable for testing carcinogenic and toxic chemicals causing hematopoietic damage through occupational or habitual exposure.

Enkephalins in hematopoiesis.

Recent data support the view that neuropeptide mediators, in particular opioid peptides, participate in the control of hematopoiesis. The main arguments are: neuropeptides modulate the functions of lymphoid cells, macrophages and mature granulocytes; they control cell proliferation and differentiation in many tissues, particularly during embryogenesis; lymphoid cells, macrophages, polymorphonuclear granulocytes and bone marrow stromal elements express neuropeptide receptors; bone marrow cells produce opioid-like neuropeptides; the CD10/CALLA marker of lymphoid, myeloid and marrow stromal cells is an enzyme, endopeptidase, which cleaves- and thus activates/inactivates-opioid and other neuropeptides. We have shown that opioid peptides enkephalins, opioid antagonist naloxone, and the inhibitor of enkephalin-degrading endopeptidase, thiorphan, modulate the proliferation and differentiation of hematopoietic cells in clonal and long-term cultures of mouse bone marrow. The effects partly depended on the presence of the accessory hematopoietic elements, and followed a circadian pattern. The dose-responses were irregular, showed strain-dependent and individual variations, and apparently reflected the state of the activity of target cells, cellular interactions and simultaneous signals by other mediators. The enkephalins were shown to bind to specific (opioid) receptors on the target cells, and their signals to be transmitted to the cell interior by a cascade of secondary messengers including diacyl-glycerol (DAG), protein-kinase C (PKC) and Ca++ ions. Neuropeptide regulation of hematopoiesis might belong to a complex immuno-neuroendocrine network including melatonin.

Suppressive effect of met-enkephalin on bone marrow cell proliferation in vitro shows circadian pattern and depends on the presence of adherent accessory cells.

The cellularity of femoral bone marrow and the content of the granulocyte-macrophage colony forming cells (GM-CFC) were followed in mice between 0600 h and 1800 h. The cellularity increased at the beginning of the light period, and the GM-CFC content at the end. Opioid pentapeptide methionine-enkephalin reduced the GM colony forming ability of the bone marrow cell suspensions in proportion to the GM-CFC content. Removal of the accessory cells reversed the enkephalin sensitivity pattern of the GM-CFC. The circadian variations have been ascribed to a neuroendocrine regulatory network involving the opioid peptides and affecting the bone marrow accessory cells. The work draws attention to the circadian activity pattern of hemoregulatory oligopeptides applicable as adjuvants to antineoplastic chemotherapy.

Serotonin and serotoninergic agents affect proliferation of normal and transformed lymphoid cells.

Blastogenic transformation of murine spleen cells elicited with concanavalin A was suppressed by serotonin 10(-12) to 10(-6) M, and marginally stimulated by its antagonists ketanserin and propranolol in low concentrations (10(-15) to 10(-11) M). Ketanserin (5-HT2 receptor ligand) and propranolol (5-HT1A and beta-adrenergic ligand) did not block the suppressive effect of serotonin if used along with it in equimolar concentrations (10(-9) M). Ergot-alkaloid dihydroergosine suppressed, whereas dihydroergotoxin stimulated the blastogenic transformation. Opposite effects of the agents were obtained in experiments with mouse myeloma X63/Ag 8.653 and hybridoma SHV 125 cell lines, which unlike normal lymphoid cells, are homologous cell populations and proliferate spontaneously. The data indicate that serotonin and its antagonists interfere directly with mitosis and/or autocrine stimulation of target cells.

Naloxone behaves as opioid agonist/antagonist in clonal cultures of mouse bone marrow cells.

The opioid peptide methionine (Met)-enkephalin and the opioid-receptor blocking agent naloxone were added to unseparated or to progenitor-enriched cell suspensions of mouse bone marrow before assay in clonal cultures. Bone marrow samples harvested at 18:00 hours produced more granulocyte-macrophage (GM) colonies than the 06:00 hour samples, and were more sensitive to the proliferation inhibition by both agents. Additive inhibitory effects of naloxone with the enkephalin were occasionally seen. Thus, in this experimental system, naloxone could behave as an opioid agonist. However, there were examples of naloxone diminishing (blocking) the suppressive effect of the enkephalin, as a true opioid antagonist. Significant naloxone/enkephalin interactions occurred in opioid-sensitive (18:00 h) samples of unseparated bone marrow. The interactions were virtually absent in progenitor cell-enriched populations, indicating a significant role of accessory cells in opioidergic regulation of hematopoietic progenitors.

Enkephalinase-blocking agent thiorphan affects cell growth and differentiation in long term culture of mouse bone marrow.

Enkephalinase-blocking agent thiorphan was added to long-term cultures of mouse bone marrow cells at the time of culture initiation (time 0) or 2 weeks thereafter, when the stromal layer appears. Cellularity, cell morphology (in cytospin smears) and the yield of granulocyte-macrophage progenitor cells (GM-CFC assay in agar) were recorded. Low concentrations of thiorphan accelerated recovery of the cultures after an initial drop of the cell count. Expansion and maturation of the granulocytic lineage was promoted, with parallel decline of the GM-CFC yield. Thiorphan probably interfered with the activity of enkephalinase (endopeptidase 24.11) in the cultures. That enzyme is the CD10 surface marker (CALLA) of lymphoid, myeloid and stromal elements.