Skin own bacteria may aggravate inflammatory and occlusive changes in atherosclerotic arteries of lower limbs.

AIM: Seroepidemiological studies have given rise to the hypothesis that microorganisms like Chlamydia pneumoniae (CP), Helicobacter pylori (HP), cytomegalovirus (CMV), HCV types 1 and 2, and bacteria involved in dental or other unspecified infection sites may initiate or maintain the atherosclerotic process in lower limb arteries. However, not much attention has been attached to the patient's own limb skin and deep tissues bacterial flora, activated in ischemic tissues. This flora may enhance the inflammatory and thrombotic process in the atherosclerotic arteries. Lower limb tissues are exposed to microorganisms from the environment (foot) and microbes on floating epidermal cells from the perineal and anal regions. The aim of this paper was to identify microbial cells and their DNA in perivascular tissues and arterial walls of lower limbs. METHODS: Bacterial cultures and PCR method for detection of 16sRNA and immunohistopathological staining for identification of immune cells infiltrating vascular bundles. RESULTS: 1) specimens of atherosclerotic calf and femoral arteries contained bacterial isolates and/or their DNA, whereas, in control normal cadaveric organ donors' limb arteries or patients' carotid arteries and aorta bacteria they were detected only sporadically; 2) lower limb lymphatics contained bacterial cells in 76% of specimens, whereas controls only in 10%; 3) isolates from limb arteries and lymphatics belonged in majority to the coagulase-negative staphylococci and S.aureus, however, other highly pathogenic strains were also detected; 4) immunohistopathological evaluation arterial walls showed dense focal infiltrates of granulocytes and macrophages. CONCLUSION: Own bacterial isolates can be responsible for dense neutrophil and macrophage inflitrates of atherosclerotic walls and periarterial tissue in lower limbs and aggravate the ischemic changes.

Human splenic dendritic cells react differently to bacterial and allogeneic antigens.

We investigated the ability of bacterial (cells, LPS, and DNA) or allogeneic antigens to stimulate splenic dendritic cells (DCs) and expression of Toll-like receptors (TLRs), CD14 (co-functional molecule to TLR), CD83 (activation molecule on migrating DCs), CD123 (IL-3R specific for myeloid DC), Hsp60 and Hsp90 (heat shock proteins) involved in recognizing pathogen-associated molecular patterns (PAMP) and other pathogen recognition receptors (PRR) ligands. Allogeneic stimulation of DC TLRs was weak compared with that of bacterial products. This suggests that the highly conserved TLRs destined to react to bacterial products do not recognize donor products differing at the major histocompatibility complex (MHC) locus, at least using in vitro culture conditions.

Endogeneous sources of infection in transplant recipients.

The mammal organisms carry on their surfaces and in their tissues cohorts of microorganisms of various nature. There is a balance of interests and profits between the host and microbial inhabitants. The bacteria and fungi behave like comensals, colonizers, dormants, however, under certain, mostly unknown, conditions may evoke reaction of the host. This process is damaging both for the host and microbes. Large surgical trauma and allograft itself, as well as, immunosuppression create favorable conditions for imbalance between inhabiting microorganism and the recipient. The host flora and that transplanted with the organ graft become activated. Active combating of the proliferating bacteria with antibiotics becomes necessary. Our knowledge of the bacterial flora of the so called "sterile" tissues remains rudimentary. There is still a great deal of prejudice on the sterility of deep tissues e.g. muscles, fat tissue, etc. This review cumulates pertinent literature data on the microorganisms-host interactions. Our own findings on colonization of arteries and adjacent tissues are discussed in the context of atherosclerosis and grafting.

Activation of splenic dendritic cells by bacterial and allogeneic antigens.

Allograft ischemia and cellular degradation accompanying rejection favor graft colonization by translocated microorganisms. Bacterial colonization adds to the graft destruction. The dendritic cells (DC) of allograft recipients engage in allogeneic and antibacterial reactions; they process and present to lymphocytes 2 types of antigens. This may lead to overstimulation of DCs that may nonspecifically intensify the rejection process. We investigated the effects of allogeneic and bacterial antigens on splenic DCs phenotypes. In vitro stimulation of a spleen DC-enriched population by E. coli, LPS, and CpG DNA brought about an increase in expression of OX6(+) (MHC class II) from 47.4% in the control cells to 65% in the E. coli-stimulated group (P < .05) and 85% in the LPS and CpGDNA groups (P < .05). Interestingly, a significant drop in the frequency of OX62(+) DC was observed after incubation with LPS. Allogeneic heart transplants brought about an increase of OX6(+) in DCs to 100% and a decrease of ED1(+) monocyte frequency. Simultaneously, an increase in expression of W3/13(+) T cells in DC-enriched splenic cells was observed. There was no significant change in the frequency of OX62(+) expression. Both types of antigens evoked splenic DC response; however, there were differences in the frequency of phenotype expression. Allogeneic but not bacterial antigens increased W3/13 antigen expression; the frequency of OX62(+) in cells decreased after LPS but not after bacterial stimulation.

DNA released from rejecting organs is an indicator of the degree of graft cellular damage.

Warm and cold ischemia as well as rejection of the transplanted organ or tissue cause destructive changes in the graft parenchyma. Fragments of disintegrated cellular organelles are phagocytized and digested by recipient scavenger cells in lymph nodes, spleen, and liver. Some fragments engulfed by dendritic cells are processed including donor DNA present in the ingested cellular debris. The question arises as to whether the DNA from the disintegrated cells may be used as a measure of graft damage. In this study we provide evidence that both syngeneic and allogeneic organ transplantation followed by "seeding" of donor DNA from graft cells is internalized in recipient macrophages and dendritic cells in lymphoid organs. The kinetics of accumulation of donor DNA in recipient tissues reflected the degree of ischemic and immune graft damage. Immunosuppression with cyclosporine or tacrolimus did not significantly attenuate the DNA release. Measurements of the concentration of donor DNA gives insight into the kinetics of allograft rejection. Real-time polymerase chain reaction for donor DNA in recipient serum and blood leukocytes that have engulfed donor cell debris may be useful for clinical diagnostic application.

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.

DNA released from ischemic and rejecting organs as an indicator of graft cellular damage.

Donor cellular debris contains fragments of nuclei with genetic material. The question arises whether the amount of donor graft released DNA accumulating in the recipient lymphoid tissues after transplantation could be a measure of donor organ damage caused by ischemia and preservation as well as rejection. We found that donor heart passenger cells do not contribute to the DNA disseminated in the recipient. All donor DNA was, then, derived from the damaged graft cells. Immediately and 1 day after transplantation, it was present in blood (plasma and cells) to accumulate later in the spleen. Higher values of donor DNA in the syngeneic than allogeneic combination, most evident on day 7, were presumably due to better perfusion of graft not undergoing rejection. Immunosuppression attenuated donor DNA release and accumulation in recipient tissues, nevertheless, relatively high concentrations could still be detected. Further studies are in progress on the usefulness of measuring DNA concentration for evaluation of the graft damage.

Profiling of normal human leg lymph proteins using the 2-D electrophoresis and SELDI-TOF mass spectrophotometry approach.

The parenchymatous cells are supplied by nutrients transported in fluid from blood across the capillary wall. This fluid, called tissue fluid (TF), contains proteins originating from plasma as well as those synthesized and secreted by tissue cells. The protein composition of TF remains largely unknown. The TF which has entered lymphatics is called lymph (L). Harvesting L and measuring its proteins concentrations and identifying them provide an insight into biochemical processes in the TF. Here we describe our initial evaluation of the normal human prenodal L protein profile of m.w. 2.5 to 12.5 kDa using the ProteinChip SELDI MS system and compare it with that of plasma (P) protein. This is the first study in the literature providing evidence for the presence of the so far non-identified proteins in L as well as proteins identified in L but absent from P and conversely present in P but not in L. Evident differences between paired L and P samples have been found, along with similarities. Thirteen proteins were detected in P and seven in L in the region of 2.5 to 12.5 kDa. Five identical proteins, although of different relative intensity, were found in L and P. The proteins specific for L but not P had 7070 and 8619 ion values. P proteins absent from L were of 3890, 3969, 4078, 6863, 7676, 7778, 7847 and 7937 ion values. In addition to detecting some so far unknown proteins in L, these preliminary findings throw a new light on our understanding of the mechanism of transcapillary transport of low m.w. proteins. They challenge the commonly accepted notion of unlimited free diffusion of peptides across the capillary membrane.

Similar response of dendritic cells to bacterial and allogeneic antigens.

The dendritic cells (DC) of an allograft recipients become engaged not only in an allogeneic but also antibacterial reaction. They react to the alloantigens and microorganisms which colonize the rejecting grafts. This leads to overstimulation of DCs what may non-specifically intensity the rejection process. We investigated the effects of allogeneic and bacterial antigens on splenic DCs phenotypes. In vitro stimulation of spleen DC-enriched population by E. coli, LPS and CpG DNA brought about an increase in expression of OX6 (MHC class II) from 47.4% in the control population to 65% in the E. coli stimulated group (p < 0.05) and to 85% in the LPS and CpGDNA groups (p < 0.05). Interestingly, a significant drop in the frequency of OX62+ antigen was observed after incubation with LPS. Allogeneic heart transplants brought about an increase of OX6+ (MHC class II) DCs to 100% and a decrease of EDI+ cells. Simultaneously, an increase in expression of W3/13 on DC-enriched splenic cells was observed. There was no significant change in the frequency of OX62+ expression in conclusion, both bacterial and alloantigens strongly activate splenic DCs what may add to the intensity of the rejection process.

DNA from rejecting donor organs can be detected in recipient lymphoid and non-lymphoid tissues.

Rejecting tissue and organ grafts shed cellular debris from damaged cells. Cellular debris contains fragments of nuclei with their genetic material. The question arises what is the fate of donor DNA in recipient fluids and tissues. Is it enzymatically disintegrated and becomes a waste product or it is utilized by recipient cells. We have shown, using sex-mismatched transplants and the Sry-gene fragment PCR detection method, that at the time of rejection recipient tissue contain donor DNA. The concentration of donor DNA did not parallel the concentration of live donor passenger cells. There were differences in donor DNA concentration depending on whether heart, skin or BMC were transplanted. Moreover, there was more donor DNA in recipient tissues than in control syngeneic transplants. Interestingly, a relatively high donor DNA concentration was detected in syngeneic recipients reflecting the extent of non-immune pre-transplantation ischemic damage of the graft.

Interaction of E. coli with dendritic cells--a model for studies of graft infections.

The rejection process of skin allografts is mediated by dendritic cells (DC) and lymphocytes. The recipient DCs are engaged not only into an allogenic but also antibacterial reaction to the penetrating bacteria. The capacity of these cells to sample sites of pathogen entry, respond to microbial signals and activate naive T cells suggests a critical role for DC in initiating antimicrobial immunity. In our study, we investigated the ability of the green fluorescent protein (Gfp) labelled E. coli to infect DC. We studied kinetics of in vitro and in vivo adherence and incorporation of E. coli by rat spleen and bone marrow (BM) DC. Bacterial adherence to the cell surface was observed after 2 h incubation of DC with bacteria. A 24-hour culture of DC from both sources was followed by bacterial adherence to all cells and engulfment by at least 50% of cells. There was an increased expression of the phenotypic markers on the DC cultured with E.coli. The Gfp-labelled E.coli should be useful for studies of the activation of dendritic cells. The method will allow to study the process of simultaneous activation of DC by allo- and bacterial antigens.

Donor DNA can be detected in recipient tissues during rejection of allograft.

The main source of donor DNA in recipients of allograft are "passenger" cells. It is claimed that they are responsible for the posttransplantation microchimerism and prolongation of allograft survival. We have observed that besides cellular microchimerism, donor DNA can be found in the recipient tissues at the time of rejection of the allograft. In this study, we provide evidence for the presence in the recipient of both DNA in "passenger cells" and free DNA in tissues at the terminal stage of rejection. Male BN (RT1 n) rat heart or skin was transplanted to female LEW (RT1 l) rats followed by a vascularized bone marrow in a hindlimb transplant. In another group, heart and skin were transplanted followed by immediate i.v. infusion of donor-type bone marrow cells. CsA was given in a dose of 17 mg/kg body weight for 30 days, then the rats were followed up until day 100 unless rejection occurred earlier. LEW blood, spleen, mesenteric node and bone marrow cells were stained with moAb OX27 specific for BN but not LEW. Genomic male DNA was isolated and amplified with SRY oligonucleotide. At day 30 and day 100 cellular microchimerism was detected in blood, spleen, nodes and bone marrow cells. Donor DNA was detected in recipient skin, liver and heart extracts, as well as lymphoid organs, at the time of rejection of allograft, but not when the rats were maintained on CsA. Taken together, donor DNAwas detected in recipient tissues at the time of heart or skin rejection. It appeared to be released from cells of rejecting grafts and not from "passenger" cells, representing only a minor cellular mass compared with the graft.

Vascularized bone marrow transplanted in orthotopic hind-limb stimulates hematopoietic recovery in total-body-irradiated rats.

Hematopoietic recovery after bone marrow transplantation (BMT) is reported to be slow with long-lasting immune deficiency. This may be attributable to lack of a proper microenvironment for hematopoietic cell proliferation and differentiation. We have designed a model in which complete hematopoietic reconstitution of lethally irradiated rats can be achieved by vascularized bone marrow transplantation (VBMT) in an orthotopic hind-limb graft. The aim of the study was to investigate the process of repopulation of bone marrow cavities and peripheral blood of irradiated rats after VBMT and, in particular, to follow the contribution of grafted BM cells and residual recipient BM cells in hematopoietic regeneration. Lewis hind-limbs were transplanted orthotopically to totally irradiated (8 Gy) syngeneic sex-mismatched recipients (VBMT). In the control group 8 x 10(7) BM cells in suspension were injected intravenously (BMCT). After 10 days BM and peripheral blood (PB) cells were collected from the recipient. For cell subset analysis cytomorphological evaluation of BM smears and flow cytometry of PB cells were performed. Additionally, PCR was performed using specific primers for rat Y chromosome (sex-determining region Y-Sry) to detect male (donor or recipient) cells in sex-mismatched BM graft recipients and the products were analyzed by electrophoresis. VBMT brought about much faster replenishment of nucleated cells in BM and PB than did BMCT. Cytometry analysis of PB cells revealed more lymphocytes in VBMT than in BMCT recipients. The amount of donor DNA of bands corresponding to Y-Sry was also higher in PB cells of VBMT than of BMCT recipients. The presence of host DNA was observed in PB cells of VBMT rats but was not detected in PB population of BMCT recipients. VBMT is highly effective in hematopoietic reconstitution of irradiated recipients. The fast cell maturation and repopulation may be due to the presence of stromal cells transplanted in a normal spatial relationship with donor hematopoietic cells in hind-limb graft. Self renewal of radioresistant host cells was seen after VBMT but not after BMCT.

DNA from rejecting allografts can be detected in recipient nonlymphoid tissues.

The main source of donor DNA in recipients of allograft are "passenger" cells. They are claimed to be responsible for the posttransplantation microchimerism and prolongation of allograft survival. We have noticed that beside of the cellular microchimerism, donor DNA can be found in the recipient tissues at the time of rejection of allograft. In this study we provide evidence for presence in the recipient of both, DNA in "passenger cells" and free DNA in tissues at terminal stage of rejection. Male BN (RTIn) rat heart or skin were transplanted to female LEW (RTII) rats followed by a vascularized bone marrow in hind-limb transplant. CsA was given in a dose of 17mg/kg b.w. for 30 days, then rats were followed until day 100 unless rejection occurred earlier. LEW blood, spleen, mesenteric node and bone marrow cells were stained with moAb OX27 specific for BN but not LEW. Genomic male DNA was isolated and amplified with SRY oligonucleotide. At day 30 and 100 cellular microchimerism was detected in blood, spleen, nodes and bone marrow cells. Donor DNA was detected in recipient skin, liver and heart extracts, beside of lymphoid organs, at the time of rejection of allograft but not when rats were maintained on CsA. Taken together, donor DNA can be detected in recipient tissues at the time of heart or skin rejection. It seems to be released from cells of rejecting grafts and not from "passenger" cells representing only a minor cellular mass compared with the graft.

Hepatocyte transplantation--in vitro cytotoxic reaction of autologous granulocytes and mononuclears to isolated hepatocytes.

UNLABELLED: Hepatocyte (HC) transplantation (tx) may be useful for bridging patients to whole organ transplantation and for providing metabolic support during liver failure and for replacing whole organ transplantation in certain liver metabolic diseases. In specific situations where the death rate of host hepatocytes is high, the transplanted cells can repopulate the native liver. Successful transplantation of hepatocytes is hampered by lack of proper cellular (stromal) and humoral (cytokines) environment at the site of implantation. We have found that another factor responsible for low in vivo survival rate of transplanted HC is their rapid destroying by host granulocytes and monocytes. AIM. In this study we investigated the in vitro process of destruction of HC by granulocytes and mononuclear cells, the phenotypes of effector mononuclears and the tempo of HC lysis. METHODS: In vitro cell-mediated cytotoxicity, HC-PMN and HC-PBM rosette formation rate and HC lysis, as well as phenotypes of HC-adhering cells were investigated. RESULTS: Granulocytes formed rosettes with HC almost immediately after the beginning of incubation and were found highly cytotoxic to HC. The cellular mechanism of lysis was not mediated by serum natural antibodies. Also the in vitro mixed HC-granulocyte 51Cr test showed high granulocyte cytolytic activity. Monoclonal antibodies to class I and II antigens, CD11/18 and 54 did not block the granulocyte cytotoxicity. Blood mononuclear cells also formed rosettes with HC and were cytotoxic to them, but the level of cytotoxicity was lower than of granulocytes. ED1+ monocytes revealed highest cytolytic activity toward HC. Hepatocyte contained only trace levels of endotoxin and no chemotactic activity of granulocytes and monocytes toward HC could be observed. CONCLUSIONS: A random physical contact of blood leukocytes seems necessary for adhesion to isolated HC. Taken together, granulocytes and monocytes recognize intercellular surface molecules on HC "exposed during isolation" from the hepatic trabeculae as "non-self" and lyse HC by direct contact.