Biomedical subjects
Publications and source records attributed to W L Olszewski.
Recirculation of lymphocytes labeled with 51Cr and 125IUDR was studied in the non-lymphoid tissues of normal rat. About 10% of the 51Cr and 40% of the 125IUDR-labeled cells were found in the extralymphoid tissues. The 125IUDR-labeled cells showed predilection toward the gut, lungs and skin. Migration of these cells to skin is an original observation, but the mechanism of the phenomenon is unknown so far.
Oil induced mice peritoneal cells were stored at -196 degrees C for 3 months, using dimethyl sulfoxide (DMSO) as a cryoprotective medium. Recovery counts upon thawing were about 90%. Macrophages retained their phagocytic activity of SRBC in 89% and there were no significant differences in spontaneous migration, expression of membrane immune receptors and non-specific cytotoxicity of frozen-thawed cells compared with the controls.
Preservation of liver for transplantation into allogeneic recipient affects the integrity and function of Kupffer cells. Rapid method for obtaining pure macrophages by dispersing the liver with collagenase and pronase would open possibility for different investigations. The experiments concerning morphology and phagocytic function were performed on Kupffer cells after 21 hours in vitro culture in Eagle medium with 20% inactivated calf serum. Studies on endocytosis using human red cells and colloidal carbon indicated that Kupffer cells revealed high phagocytic activity after culture.
Low concentration of immunoglobulins G, A and M and complement component proteins were found in leg lymph of normal men. The lymph/serum ratios of different proteins ranged from 0.07 to 0.24 and depended on the molecular weight of the measured protein. Major variations in concentration were found dependent on the rate of capillary filtration and lymph formation. Hemolytic activity of total complement and of the 9 components were low, with the L/S ratio from 0.06 to 0.25. The C3H50 in lymph was much lower than of the remainder of components. Remarkably low immune adherence capacity of lymph C was noted.
The haemolytic activity of nine individual components of complement and the concentrations of C1q, C1s, C4, C3, C3PA and C9 proteins were measured in the leg lymph and serum of four normal men. The mean lymph/serum ratio for total haemolytic complement was 0.257, for C1H50 it was 0.138, for C4H50 0.105, for C2H50 0.279, for C3H50 0.063, for C5H50 0.266, for C6H50 0.145, for C7H50 0.25, for C8H50 0.244 and for C9H50 0.253. The mean lymph/serum ratio for complement proteins was: for C1q 0.048, for C1s 0.06, for C4 0.199, for C3 0.225, for C9 0.244 and for C3PA 0.263. The low haemolytic activity of total complement and of all components in lymph seems to be dependent on the low complement protein concentration. The remarkably low lymph C1q protein concentration may play a physiological role in controlling total complement activity in lymph and interstitial fluid. The total complement haemolytic activity in lymph exceeded the haemolytic activity of C1 and C3, which may indicate the possibility of independent activation of C5-7 and C8-9. The considerably low C3H50 level, with high C3 protein concentration, at present lacks a proper explanation.
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Injection of Lipiodol UF into the distal epiphyses of tibia and femur in the dog was followed within several minutes by visualization of lymphatics draining the bone. Some of the radioopaque material was retained in the bone marrow cavity and tiny lymph vessels originating in that area could be seen. Contrast medium filled also the popliteal lymph node. The problem of migration of bone marrow lymphocytes through the lymphatics to the regional lymph nodes is discussed.
Methods used for collection and measurement of physiological parameters of peripheral lymph and interstitial fluid in man have been described, basing on the experience of lymphological groups from Warsaw and Oslo and on data from the relevant literature. The review consists of description of methods for chronic cannulation of leg lymphatics, collection of lymph from skin lymphatic vessels, measurement of peripheral lymph flow, lymph hydrostatic and oncotic pressures, collection of interstitial fluid with the capsular, liquid-paraffin cavity, wick and skin window methods, interstitial fluid hydrostatic and oncotic pressure measurement.
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The output of lymphocytes, monocytes, granulocytes and erythrocytes in peripheral leg lymph from healthy volunteers has been followed during night and everyday activity, during and after venous stasis, ergometer cycling and warm foot bath and during immobilisation in horizontal position over two 24-hour periods. The output of lymphocytes exceeded the output of monocytes and erythrocytes. Granulocytes were not found except immediately after cannulation. The cell output was low during night rest and the horizontal position for 24 hours. After assumption of the upright position the output of lymphocytes increased about 80 times and the output of erythrocytes 40 times probably because of washout of cells accumulated in tissue during rest. During ergometer cycling and venous stasis the output of cells increased and even more in the one hour rest period following this experiment. During venous stasis the output dropped but increased after the stasis had been released. The pattern of variation in lymphocyte, erythrocyte and monocyte output was the same. This indicate that the fluctuation in output depends on variation in blood flow in the tissue. The output of cells during and after heating seems to be dependent also on increased capillary permeability for cells as the output of erythrocytes exceeded the output of lymphocytes. The high erythrocyte/lymphocyte ratio in blood versus a low ratio in lymph shows that these two cell types pass through the capillary wall by different mechanisms.
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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.
Transplants of isolated syngeneic and allogeneic hepatocytes are rapidly disintegrated, irrespective of the site of engraftment; be it spleen, liver, portal vein, peritoneum, or subcutaneous tissue. Host scavenger cells are responsible for this reaction. We designed a method overcoming early disintegration of the grafted hepatocytes. It consisted of administration of anti-asialoGM1 antiserum eliminating natural killer cells; sublethal whole body irradiation; and reconstitution with syngeneic bone marrow cells, ligation of host bile duct, intrasplenic hepatocyte transplantation, and three consecutive partial hepatectomies. Six months after transplantation a glycogen-rich, trabeculae-forming, dividing hepatocytes, situated along strands of newly-formed fibrous tissue and numerous dilated blind bile cannaliculae were observed. There was evidently more bile canaliculae in hosts with ligated bile duct than nonligated controls. This is the first study showing fibrous tissue formed at the site of hepatocyte implantation, and stellate cells are presumably involved in this process.
Human skin fragments can be preserved in anhydric sodium chloride at room temperature for periods of weeks or months and successfully transplanted, retaining normal morphological structure. Skin fragments of 10 x 10 x 6 mm were harvested during elective vascular and orthopedic surgery of lower limbs, dried of blood, and placed in anhydric sodium chloride powder in tightly sealed containers. Prior to transplantation to scid mice, the specimens were desalinated and rehydrated. Specimens preserved for 1-6 months and harvested 3-4 weeks after transplantation revealed intensive incorporation of bromdeoxyuridine (BdUR) into basal keratinocytes (stem cells). They expressed p63 and CD29 (stem cells and transient cells antigens), proliferating cell nuclear antigen (PCNA), and cytokeratin 16 specific for proliferating keratinocytes. We conclude that human epidermal stem cells can survive in a dehydrated state in sodium chloride for months and after transplantation give rise to keratinocyte progenies. Skin fibroblasts and some resident immune cells can also survive.
Experimental studies on allogeneic transplantation have shown that recipient dendritic cells (DC) play a role in peripheral tolerance as well as in rejection of allografts. It is not known whether DCs exert their tolerogenic function in the graft or in recipient lymphoid tissue. To answer this question we created a chimeric heart model deprived of its own DCs and repopulated with recipient DCs. The rationale for this model was to observe whether recipient mature and immature DCs located in the graft attenuate recruitment and stimulation of recipient lymphocytes, subsequently prolonging graft survival. Vascularized bone marrow transplants from the prospective recipient to the lethally irradiated heart donor, which function for a period of 14 days, were used to replace donor DCs with prospective recipient either mature or immature DCs. Replacement of the donor heart with either of these cells did not prolong graft survival. The intragraft microchimerism did not mitigate the allogeneic rejection reaction.
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.
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.