B Interewicz

Bacteriology of Callus of Closed Fractures of Tibia and Femur

BACKGROUND More than 1% of closed fractures of lower limbs and 6% of orthopedic implants are complicated by inflammation caused by infection despite of all precautionary methods taken. The question arises whether this clinical complication is not caused by bacteria dwelling in limb tissues. METHODS Skin, subcutaneous fat, muscle, and fracture gap callus were obtained from 71 adult patients operated on due to closed fractures of tibia or femur; 28 because of comminuted fractures and mal-alignment of bone axis with nonoperative means, and 43 due to delayed healing and unstable union. RESULTS Aerobic bacteria were isolated from gap callus of 14% healing and 35% nonhealing fractures. No isolates were found in subcutis and only in 3% in muscles. No anaerobic bacteria were detected. Polymerase chain reaction amplifications of 16S rRNA were found positive in 42% of callus specimens proving presence of bacterial DNA even when no isolates were found. The 95% similarity of the genetic pattern of some strains from foot skin and callus, estimated with random amplification of polymorphic DNA technique, suggested their foot skin origin. CONCLUSIONS The colonizing bacterial cells and their DNA were detected in fracture callus but not in other deep tissues. Contamination was precluded by lack of isolates in disinfected cutis, subcutis, muscles, and materials used for sampling cultured after surgery. We suggest that certain strains of bacteria dwell in normal tissues of lower limbs and may cause inflammation upon stimulation by trauma. Their source may be tissue fluid, superficial and deep lymphatics, and lymph serving the physiologic transport to the regional lymph nodes of microorganisms penetrating foot skin during microinjuries.

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

Abstract 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 × 107 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.

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.

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

Abstract 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 1) rats followed by a vascularized bone marrow in a hind‐limb 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 DNA was 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.