Donor DNA is present in recipient tissues after grafting also in graft-versus-host disease-free individuals.
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DNA Fingerprinting↗
Biomedical subjects
Waldemar L Olszewski
Publications and source records attributed to Waldemar L Olszewski.
Human skin preserved long-term in anhydric pulverized sodium chloride retains cell molecular structure and resumes function after transplantation.
BACKGROUND: Human skin is needed to cover large areas of the body lost through burns, trauma, and extensive maxillofacial surgery. Contemporary methods of skin storage are limited by the period of preservation to a few days. Our previous findings showed that fixation and storage of human skin in anhydric sodium chloride at room temperature for weeks or months preserves its morphological and molecular structure. In this study, we examined whether skin grafts preserved in sodium chloride may be successfully transplanted. METHODS: Skin was harvested from lower limbs of patients during elective surgery, placed in containers with anhydric salt powder, and kept at 22 degrees C for 3 to 12 weeks. Desalination and rehydration took place before transplantation. Desalinated fragments were transplanted onto the dorsum of scid mice. RESULTS: All grafts were accepted by recipients. Three weeks after transplantation, keratinocytes synthesized keratins 10, 16, and 17 and expressed antigens specific for stem (p63) and transient (CD29) cells. Moreover, they proliferated vigorously, their basal layer cells incorporated bromdeoxyuridine and expressed proliferative cell nuclear antigen. Isolated from transplants and cultured in vitro, they remained viable and produced enzymes. Dermis retained its structure and expressed fibroblast-specific antigen. All graft cells remained human leukocyte antigen I. CONCLUSION: Human skin preserved in anhydric sodium chloride at room temperature for months can be successfully transplanted to scid mice. We propose the concept of "spore-like" keratinocyte stem cells to explain the long-term ex vivo survival of keratinocytes. The mechanism of survival of fibroblasts remains to be determined.
Animals↗
Neurogenic factors in the impaired healing of diabetic foot ulcers.
BACKGROUND: We hypothesize that the reduced innervation of skin can be observed both in clinically neuropathic and non-neuropathic diabetic foot ulcers and can contribute to low inflammatory cell infiltration. MATERIALS AND METHODS: Twenty patients with type 2 diabetes and active foot ulcers, without clinical evidence of peripheral sensory neuropathy (n = 12) and with sensory neuropathy (n = 8) were involved in this study. Biopsies from ulcer margin were examined immunohistochemically. RESULTS: Studies revealed presence of protein gene product 9.5 (PGP9.5)+ nerve endings only in reticular dermis in 3 of 12 non-neuropathic subjects, however, regenerating GAP-43+ endings were seen in dermis of almost all specimens. Lack of substance P+ nerve endings was characteristic for both groups. The reduced distribution of calcitonin gene-related peptide+ nerves in epidermis and dermis was seen mainly in neuropathic group. In neo-epidermis lack of nerve growth factor expression was observed in both groups, whereas neurotrophin 3 immunostaining was characteristic for neuropathic specimens (P < 0.03). Expression of trkA and trkC receptors did not differ significantly between groups. Low inflammatory cell infiltration and moderate presence of fibroblasts was characteristic for all studied specimens. CONCLUSIONS: The observed reduction of foot skin innervation and neurogenic factors expression can be correlated with low inflammatory cell accumulation and subsequently leads to the observed chronicity of diabetic foot ulcer healing process in both neuropathic and non-neuropathic patients.
Aged↗
Mass kinetics of apolipoprotein A-I in interstitial fluid after administration of intravenous apolipoprotein A-I/lecithin discs in humans.
Apolipoprotein kinetics are customarily determined by modeling time curves of specific radioactivity or isotopic enrichment in plasma after intravenous infusion of radiolabeled lipoproteins or stable isotope-enriched amino acids. However, this provides no information on the fractional rate of transfer of the apolipoprotein from plasma to interstitial fluid (k(p-if)) or its mean residence time in interstitial fluid (MRT(if)). To determine these parameters for a pharmacologic dose of exogenous apolipoprotein A-I (apoA-I) given intravenously as apoA-I/lecithin discs, we measured apoA-I in plasma and prenodal leg lymph in five healthy men before, during, and after a 4 h infusion at 10 mg/kg/h. ApoA-I concentrations in plasma and lymph were modeled by linear compartmental models (SAAM II version 1.1), using lymph albumin to adjust for the effects of variations in lymph flow rate. k(p-if) averaged 0.75%/h (range, 0.33-1.32), and MRT(if) averaged 29.1 h (14.1-40.0). Neither parameter was correlated with the distribution volume (57-105 ml/kg) or the fractional elimination rate (1.44-2.91%/h) of apoA-I, determined by modeling plasma apoA-I concentration alone. Although used here to study the mass kinetics of apoA-I, if combined with infusion of a tracer, analysis of lymph could also expand the modeling of endogenous apolipoprotein kinetics.
Adult↗
Lymphoscintigraphic monitoring of the lower limb lymphatic system response to bone fracture and healing.
Closed bone fractures, and torn muscles and tendons are "internal wounds". What kind of reaction do they evoke in the local and systemic immune system? Cellular debris of damaged tissue and extravasated blood cells are removed by scavenger cells. They are transported via lymphatics to the lymph nodes. There elimination of self antigens takes place. Clinically, no enlargement of lymph nodes is observed after closed fractures and soft tissue damage. The question arises whether there is really no enlargement of regional lymph nodes, in other words, no reaction to damaged cell antigens. This question was studied by using lymphoscintigraphy to visualize lymphatics and lymph nodes draining the site of closed bone fracture. The lymphoscintigraphic pictures of two groups of patients, those with a rapid noncomplicated healing of leg fractures, and those with protracted healing and undergoing surgical reconstructions, were evaluated. The surface area of lymphatic pathways and inguinal lymph nodes on the injured and contralateral normal limb were measured. Enlarged superficial lymphatics and inguinal lymph nodes were found in limbs with healed bone fractures, and decreased inguinal lymph nodes and visualization of deep lymphatics and popliteal nodes in the majority of patients with nonhealing fractures. There was a lack of correlation between age of patients, duration of healing, and surgical interventions and the lymphoscintigraphic changes. These findings suggest that the fracture gap tissue is a dominant source of signals to the lymph nodes, releasing cellular and humoral regulatory factors. Taken together, there is a strong immune reaction of lymph node to the fracture, although it cannot be recognized clinically.
Adult↗
The innate reaction of the human skin lymphatic system to foreign and self-antigens.
The skin lymphatic system is the constitutive anatomical organization of the innate immune system. It reacts immediately to penetrating foreign antigens and presents them to the organized lymphoid tissue, where subsequently the adaptive immune response develops. Both phylogenetically and ontogenetically, defense mechanisms ascribed to the lymphatic system developed earlier than the nutritive function of blood elements. Resident and migrating immune cells, regulatory proteins, neuroregulatory factors, complement components, coagulation factors, and antimicrobial peptides react immediately to foreign antigens. Moreover, they most likely participate in elimination of shed autoantigens. Thus far, the knowledge of specific events and mechanisms operative in the innate response is still in a premordial stage. Further studies will elucidate not only how we immediately recognize what is nonself and react to it but also learn more about evolution of local immune memory in the immune and parenchymatous cells.
Antigens↗
Transplantation of organs is transplantations of donor DNA: fate of DNA disseminated in recipient.
Microchimerism after allogeneic organ transplantation has been widely documented using DNA identification techniques. However, the question as to whether the detected donor DNA is present in the surviving donor passenger cells, recipient macrophages phagocytizing rejected donor cells, or dendritic cells (DC) internalizing donor apoptotic bodies or cell fragments has not been answered. We provide evidence that allogeneic organ transplantation is followed not only by cellular microchimerism caused by release of graft passenger cells but also dissemination of donor DNA from the ischemic rejecting graft cells and its internalization in recipient DC. The high levels of donor DNA at the time of heart rejection were inversely proportional to the concentration of donor passenger cells detected with use of flow cytometry. Depending on the type of graft, the kinetics of DNA distribution in recipient tissues were different. Immunosuppressive drugs attenuated the rejection reaction and release of DNA from grafts. Allogeneic but not syngeneic donor DNA fragments were found in recipient splenic DC-enriched population. Interestingly, that donor DNA fragments could be detected in recipient tissue at high levels on day 30. This challenges the notion that fragments of DNA are immediately cleaved by cell plasmatic enzymes. The biologic significance of our findings is not clear. We speculate that donor DNA fragments in recipient DC may play a, so far unknown, role in the immunization/tolerance process to allogeneic antigens.
Animals↗
Expression of natural antimicrobial peptide beta-defensin-2 and Langerhans cell accumulation in epidermis from human non-healing leg ulcers.
Chronic wounds like venous calf and diabetic foot ulcers are frequently contaminated and colonized by bacteria and it remains unclear whether there is sufficient expression of defensins and recruitment of epidermal Langerhans cells in the margin of ulcer compared to normal skin. The aim of this study was to examine immunohistochemically the expression of beta-defensin-2 (hBD2), GM-CSF, VEGF growth factors and accumulation of CD1a+ Langerhans cells (LC) in epidermis from chronic skin ulcers and to compare it to normal skin from the corresponding areas. Studies were carried out in 10 patients with diabetic foot, 10 patients with varicous ulcers of the calf and 10 patients undergoing orthopedic surgery (normal skin for control). Biopsy specimens were immunostained using specific primary antibodies, LSAB+ kit based on biotin-avidin-peroxidase complex technique and DAB chromogen. Results were expressed as a mean staining intensity. Statistical analysis of staining showed significantly higher staining of hBD2 in both normal and ulcerated epidermis from foot sole skin compared to calf skin (normal and ulcerated, p < 0.05). Chronic ulcers showed the same expression of hBD2 as normal skin. There was significantly lower accumulation of CD1a+ LC in normal epidermis from foot sole skin compared to normal calf skin (p<0.05). Accumulation of CD1a+ LC and GM-CSF upregulation at the border area of diabetic foot ulcer and reduction of LC concentration at the margin of venous calf ulcer compared to normal skin were observed. It seems that normal calf and sole epidermis is, unlike in the mechanisms of innate immunity, influenced by the different keratinocyte turnover and bacterial flora colonizing these regions. Insufficient upregulation of hBD2 in both diabetic foot and venous calf ulcers may suggest the pathological role of this protein in the chronicity of ulcers.
Diabetic Foot↗
Innate immunity processes in organ allografting--their contribution to acute and chronic rejection.
The innate immune processes proceeding in organ allografts and their contribution to acute and chronic rejection have been described. Ischemia/reperfusion phenomenon with damage of the endothelial and parenchymal cells and reaction of granulocytes and macrophages as well as humoral factors including complement, coagulation factors, free radicals, nitrous oxide have an impact on late function of the transplanted organs. Participation of granulocytes and macrophages in acute allogeneic rejection is another example of the role of the innate system in transplant damage. Recurrent basic disease, diabetes, hyperlipidemia, adverse effects of immunosuppressive drugs in allograft recipients are factors evoking reaction of the innate system. Investigations of the innate immune system have shown an essential role, beside of the adaptive system stimulated by allogeneic mismatch, in organ graft rejection.
Acute Disease↗
[The role of sentinel node biopsy in breast cancer surgery].
Mastectomy with axillary lymph node dissection remains the routine surgical treatment of breast cancer in Poland. Lymph edema of the upper extremity is one of the major long-term complications of axillary dissection. Axillary lymph node status is the most valuable prognostic indicator and decision factor on adjuvant chemotherapy or radiotherapy for breast cancer patients. Level I and II axillary lymph node dissection provides prognostic information, maintains local control in the axilla and determines the need for adjuvant systemic treatment, but it is also associated with 30% rate of lymph edema. Multiple studies confirm that sentinel lymphadenectomy accurately stages cancer advancement and is associated with less morbidity than axillary dissection. Over 40% of breast cancer patients in Japan are submitted to breast conserving therapy with sentinel node biopsy. In our opinion sentinel node biopsy may be accepted as an alternative staging procedure for the axilla in breast cancer. Sentinel node biopsy is especially valuable tool for breast cancer patients undergoing breast sparing surgery (IIA), due to excellent cosmetic outcome, minimal morbidity and high degree of histological accuracy associated with the procedure. This article reviews current literature in breast conserving therapy and sentinel node biopsy. Author would like to thank to Professor Kenji Ogawa, Chairman of Surgical Department of Tokyo Women's Medical University Daini Hospital, Professor Fujio Kasumi Chief of Breast Surgical Department of Cancer Institute Hospital and the Japan Society for the Promotion of Science for the scientific support during research visit in Tokyo.
Breast Neoplasms↗
The new approaches to preservation of graft cell integrity in preservation for transplantation.
Restoration of cell plasma membrane integrity after injury is essential for the survival of animal cells. In case of graft preservation or during chemotherapy in cancer, cell membrane integrity and the process of its repair are disrupted. Cytoprotective substances are important in such cases, as well as in other diseases, for example in myocardial infarction, acute insults and in chronic neurodegenerative diseases. Hyperosmolarity is a condition in which cell membrane stability may be damaged in vivo but preserved in the in vitro conditions. Hypertonicity causes water leaving from cells by osmosis, decreasing cell volume and increasing of intracellular ionic strength. High intracellular ionic strength perturbs cellular function by decreasing the rates of biochemical reaction. We review the new experimentally studied cytoprotective substances and their application in cell membrane protection. Moreover, we present our data on the effects of hyperosmolarity and its protective effect on cell internal structure.
Animals↗
Contemporary methods for detection of microbial infections in transplanted tissues.
Infective processes developing after transplantation are diagnosed mainly in the specimens harvested from blood and exudates. Not much attention has been focused on identification of microorganisms present in the graft or penetrating the graft from the recipient. Although a progress has been made with respect to detection of viruses there is still little knowledge on the pathological role of bacteria, transplanted together with the graft or acquired from the recipient, in evoking or enhancing the rejection reaction (heterologous immune response). Recently developing molecular techniques allow us to detect bacterial genetic materials in clinical specimens. This article reviews commonly used methods for detection, identification and typing bacterial pathogens. It focuses on specific and universal methods based on gene sequencing, PCR and techniques based on polymorphic DNA. We also present the examples of genes and microorganisms analysed in laboratories. Together with conventional microbiological methods the molecular tests can serve as a key for better understanding the bacterial presence in tissues, not necessarily causing inflammation, quite often being commensal, but also evoking host reaction under certain so far not defined circumstances.
Bacterial Infections↗
The response of spleen dendritic cell-enriched population to bacterial and allogeneic antigens.
The dendritic cells (DC) play crucial role in initiation and modulation of immune response especially innate immune response. We investigated the influence bacterial (E. coli and S. epidermidis) and allogeneic antigens (heart, skin and bone marrow transplants) on splenic DC- enriched population. We found that 1) the in culture stimulation of rat splenic DC-enriched population by E. coli, S.epidermidis, LPS and CpG DNA caused increase in class II-positive cells. Simultaneously, a decrease in percentage of EDI, B cells and OX62 migrating DC upon treatment with S.epidermidis was observed. LPS caused decreased frequency of OX62 and NK cells. 2) Similarly to the in vitro the in vivo stimulation by E. coli, S.epidermidis, LPS and CpG DNA increased the percentage of class II-positive cells. There was a decrease in the ED1, OX62 and B cell populations following stimulation by S. epidermidis. 3) Mixed DC-enriched population and donor PBM culture showed high level of response in both populations. 4) Syngeneic and allogeneic transplants of heart, skin and BMC caused increase in class II-positive cells. Moreover, there was an increase in frequency of the ED1 and W3/13 populations after both syn- and allogeneic transplantation. The OX62 cells did not react, whereas the B cell frequency rose only after allogeneic transplantation. A significant decrease in NK cell population was noticed. 5) The in vitro and in vivo bacterial stimulation brought about expression of TLR receptors and Hsp. Mixed recipient DC with donor PBM culture caused expression of Hsp 90 but not TLRs. Allogeneic stimulation by transplanted tissues did not evoke expression of the investigated receptors and proteins. 6) Recipient DC-enriched population produced IFN gamma upon stimulation with bacteria and skin but not heart and BMC. Further studies on simultaneous stimulation of splenic DCs by bacterial and allo-antigens will throw light on additive effects of bacterial activation in allograft rejection.
Animals↗
Keratinocyte and dermal vascular endothelial cell capacities remain unimpaired in the margin of chronic venous ulcer.
The role of endogenously produced cytokines and growth factors in the impaired healing of chronic leg ulcers remains uncertain. The aim of this study was to determine the functional capacity of skin cells in ulcer bed tissue compared to those in the edge of ulcers and skin distal to ulcers. Biopsies from leg ulcers of ten randomly selected patients were examined immunohistochemically for cytokines and growth factors produced by keratinocytes (KC) and vascular endothelial cells (EC). The phenotype of leukocytes infiltrating venous ulcers and the expression of vascular adhesion molecules responsible for extravasation were also studied. The expression of cytokines and growth factors by KC was similar in areas adjacent and remote from an ulcer. In the dermis adjacent to an ulcer, the expression of IL-1alpha, IL-1beta, IL-1Ra, EGF and PDGFa by EC was higher than the levels of expression in EC from the distant dermis. The expression of IL-6, TNFalpha and GM-CSF was comparable to that in cells from intact dermis. For all these factors staining was cytoplasmic, suggesting production in these areas. Ulcer bed tissue contained few fibroblasts and blood capillaries showing a high staining intensity for CD62E and CD106 EC adhesion molecules but no FGF2 expression (P<0.05). The intensity of staining for scavenging CD15+ elastase+ granulocytes and CD35+ (C3bR) activated macrophages in the ulcer bed was comparable to that in the margin but higher than that in the distant dermis (P<0.05), whereas staining for CD68+, HLA DR+, TGFbeta+ and CD54+ dermal macrophages was similar in all areas. There was reduced staining for CD4+ and CD8+ cells in the ulcer bed (P<0.05). There were no CD1a+ Langerhans cells in the epidermis encroaching upon the granulation tissue and there was reduced CD1a staining in the adjacent epidermis (P<0.05). In conclusion, there is chronic accumulation of scavenging cells with lack of remodeling of the granulation tissue and, at the same time, preserved cytokine and growth factor secretory potential of KC and dermal EC in non-healing venous leg ulcers.
Aged↗
[Stem cells - biology and therapeutic application].
Enormous hope is connected with stem cells with regard to cell therapy, and this has become one of the most dynamically developing areas of science at the moment. A stem cell has unlimited potential for self-renewal. It appears that it can be a source of in vitro differentiated progeny cells capable of repairing damaged tissue. These review provides information about the biological properties of embryonic stem cells, i.e. ESs (embryonic stem cells), EGs (embryonic germ cells), and ECs (embryonic carcinoma cells). Possible human embryonic stem cell applications are described, with consideration of the desired cell line and the signals involved in their differentiation. The information about adult stem cells present - hemopoietic stem cells and the cells residing in selected tissues and organs: endothelium, pancreas, liver, epithelium, and gastrointestinal tract. Methods of their identification using the cell surfaces are also presented: the possibilities of in vitro transdifferentation, the phenomenon of in vivo plasticity, as well as morphological and genetic properties. Some topics of cell therapy and its clinical application in diabetics amplification are included.
Adaptation, Biological↗
Migration of liver sinusoidal leukocytes to the liver colon adenocarcinoma metastases.
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Journal Article↗
Variations in lipid and apolipoprotein concentrations in human leg lymph: effects of posture and physical exercise.
We studied the variations in the concentrations of cholesterol, triglycerides, phospholipids, apolipoproteins (apos) (A-I, A-II, B, C-III, E), free glycerol and albumin in human prenodal leg lymph during the 24 h cycle. Lymph was collected continuously for up to 96 h from nine healthy males on a low-fat isocaloric diet. In three free-living subjects, all lipid and apolipoprotein concentrations underwent synchronous variations, rising during the night and decreasing during the day. In three subjects who remained in supine rest for 48 h, the amplitude of circadian variation was much smaller. In three who alternated periods of supine rest with upright exercise, the highest concentrations occurred during rest. Lipid, apolipoprotein and albumin concentrations were inversely related to lymph flow rate. Free glycerol, much of which in tissue fluid is derived from local adipocytes, did not follow this pattern. On multiple regression, concentrations in lymph were related independently to the corresponding concentration in plasma (positive) and to lymph flow rate (negative) or lymph albumin concentration (positive). These results show that lipoprotein concentrations in human tissue fluid are determined only partly by their concentrations in plasma. They are also strongly affected by hemodynamic factors via their effects on fluid transport.
Adult↗
Effects of intravenous apolipoprotein A-I/phosphatidylcholine discs on paraoxonase and platelet-activating factor acetylhydrolase in human plasma and tissue fluid.
We have previously shown that intravenous apolipoprotein (apo) A-I/phosphatidylcholine (apo A-I/PC) discs increase plasma high-density lipoprotein (HDL) concentration in humans. We have now studied the associated changes in two enzymes, paraoxonase (PON) and platelet-activating factor acetylhydrolase (PAF-AH) that are carried in whole or in part by HDLs, and are thought to influence atherogenesis by hydrolyzing oxidized phospholipids in lipoproteins. Apo A-I/PC discs (40 mg/kg over 4 h) were infused into eight healthy males. Although plasma apo A-I and HDL cholesterol increased on average by 178 and 158%, respectively, plasma total PON and total PAF-AH concentrations did not rise. By the end of the infusion, HDL-associated PAF-AH had increased by 0.56 +/- 0.14 microg/mL (mean +/- S.D., P < 0.01), and nonHDL-associated PAF-AH had decreased by 0.84 +/- 0.11 microg/mL (P < 0.05). These changes were accompanied by an increase in the HDL-associated PAF-AH/apo A-I ratio from 0.19 to 0.35 (P < 0.05), and by a decrease in the nonHDL-associated PAF-AH/apo B ratio from 2.1 to 1.4 (P < 0.05). No changes in PON or PAF-AH concentrations were detected in prenodal lymph (tissue fluid), collected continuously from the leg. Our results show that the total concentrations of PON and PAF-AH in plasma are uninfluenced by plasma HDL concentration. PAF-AH transfers readily between HDLs and LDLs in vivo, and its distribution between them is determined partly by their relative concentrations and partly by HDL composition.
1-Alkyl-2-acetylglycerophosphocholine Esterase↗