Environmental survival of vancomycin-sensitive ampicillin-resistant Enterococcus faecium (AREfm).

Ampicillin-resistant Enterococcus faecium (AREfm) has gained increased footholds in many hospital intensive care units (ICUs) and belongs to specific hospital-adapted E. faecium sub-populations. Three AREfm strains survived in an in vitro survival setting for approximately 5.5 years. These findings have important consequences for the epidemiology of AREfm in hospital settings and stress the importance of maintaining a good level of hospital hygiene.

Immunohistological observations in rat kidney allografts after local steroid administration.

In this report we investigated local regulatory mechanisms in graft rejection and their response to local immunosuppressive therapy. For this purpose local immunosuppression was induced in rat kidney allografts by intrarenal infusion of prednisolone. Intrarenal drug delivery resulted in high drug levels within the graft and low systemic drug levels. Systemic drug levels were by themselves not sufficiently immunosuppressive to induce graft survival, and local prednisolone levels within the graft proved to be responsible for prolongation of graft survival. During intrarenal drug delivery, systemic responsiveness to the renal allograft proved normal, since intrarenally treated grafts were infiltrated by MHC class II-positive host cells and, except for a somewhat lower percentage of macrophages, cellular infiltration in intrarenal treated grafts was comparable to untreated grafts. However, T cells and macrophages present in intrarenally treated grafts were not able to destroy the grafted tissue. Local immunosuppressive therapy resulted in inhibition of IL-2-R expression, absence of IFN-gamma, and prevention of MHC class II induction on grafted tissue. These observations strongly indicate the presence of local regulatory mechanisms in graft rejection. The experimental model described can be used for further analysis of these intragraft events. Moreover, the results demonstrate that local immunosuppressive therapy can contribute to effective inhibition of cellular immune response in graft rejection.

Inhibition of fibroblast collagen synthesis and proliferation by levamisole and 5-fluorouracil.

Experimental studies indicate that anastomotic healing in the intestine is compromised by the immediate postoperative administration of 5-fluorouracil and levamisole. Since fibroblast functions are crucial to healing, we investigated the effects of (combinations of) both drugs on proliferation and collagen synthesis of rat skin fibroblasts in vitro. Proliferation was measured in actively dividing cells by cellular [3H]thymidine uptake and collagen synthesis in non-dividing cells by [3H]proline incorporation into collagenase-digestible protein. 5-Fluorouracil strongly and significantly (P < 0.05) reduced DNA synthesis and collagen synthesis at concentrations of 1 microM or more. The latter effect was not specific for collagen since total protein production was affected similarly. Both effects depended on the duration of exposure to the drugs. Levamisole also inhibited fibroblast proliferation dose-dependently, but less effectively than 5-fluorouracil: 50% inhibition was observed at approximately 0.1 mM. Collagen synthesis was unaffected by levamisole. If levamisole was added together with a low (0.1 microM) concentration of 5-fluorouracil, which in itself did not decrease thymidine incorporation, levamisole's antiproliferative effects became apparent at concentrations as low as 1 microM. A similar effect, but at a much higher concentration (1 mM) was noted on fibroblast collagen synthesis. These results indicate that levamisole potentiates 5-fluorouracil effects in fibroblast cultures and that direct effects of these drugs, alone or in combination, on fibroblast proliferation and collagen synthesis may be responsible for their negative influence on wound repair.

Hepatocyte transplantation for enzyme deficiency disease in congenic rats.

Long-term effects of hepatocyte transplantation (HTX) in the treatment of enzyme deficiency disease were studied. Congenic enzyme-deficient (R/APfd-j/j) and non-enzyme-deficient (R/APfd) rats were used as recipients and donors, respectively. The R/APfd-j/j rat strain is congenitally deficient of bilirubin uridyldiphosphate (UDP)-glucuronyl transferase. R/APfd-j/j rats underwent HTX by intrasplenic injection of 10(7) isolated R/APfd hepatocytes (group 1A). Another group of R/APfd-j/j rats was treated similarly, but underwent splenectomy after 11 weeks (group 1B). Controls consisted of R/APfd-j/j rats grafted with 10(7) R/APfd-j/j hepatocytes (group 2), and R/APfd-j/j rats that underwent a sham operation (group 3). Total plasma bilirubin (TB) levels were significantly reduced in groups 1A and 1B during the experiment (both P less than 0.01). In the control groups TB reduction was not observed. Bile analyses at 30 weeks after HTX showed that in group 1A 13.7 +/- 2.7% of total biliary bilirubin was conjugated. In group 1B a significantly lower fraction was conjugated: 6.6 +/- 1.1% (P less than 0.05). Conjugated bilirubin was not found in bile of groups 2 and 3. Histology showed survival of hepatocytes in all spleens of rats of groups 1A, 1B and 2. It is concluded that congenic hepatocytes from R/APfd donors are not rejected after transplantation into the R/APfd-j/j rat, and maintain long-term function. Splenectomy does not abolish, but does reduce, the therapeutic effect significantly, indicating that part of the transplanted hepatocytes maintains function in the enzyme-deficient host liver. The congenic R/APfd-j/j and R/APfd rat strains represent a new animal model for research in metabolic deficiency disease.

Prolongation of canine renal allograft survival. A study on the effect of donor pretreatment.

Treatment of kidney donors with procarbazine hydrochloride and methylprednisolone, respectively, 5 and 2 1/2 hr before harvesting the kidney, improved renal allograft survival in dogs significantly. Pretreatment of the donor did not have a deleterious effect on the early function of the kidney grafts. Donor blood transfused peroperatively into the recipient caused a significant reduction in survival of kidney grafts from pretreated donors, although it did not influence the survival of nontreated kidneys. Furthermore, it appeared that a peroperative injection of a suspension of nonirradiated donor lymphocytes as well as donor lymphocytes irradiated with 2,500 rad significantly decreased the survival time of pretreated kidneys. A peroperative transfusion of leukocyte-poor blood prepared with a leukocyte filtration column, which leaves erythrocytes, thrombocytes, and plasma and eliminates most of the leukocytes (99.9%), also abolished the effect of donor pretreatment. Thus, administration of donor blood constituents, whether lymphocytes or leukocyte-poor blood, can abrogate the beneficial effect of donor pretreatment on kidney graft survival. These data indicate that the effect of donor lymphocytes on the survival of pretreated kidneys is not because of a specific immunological activity of these lymphocytes but merely because of the presence of antigens on their cell surface.

Effect of blood transfusions on canine renal allograft survival.

In this study significantly prolonged canine renal allograft survival has been demonstrated after transfusion of 100 ml of third-party whole blood given peroperatively. Peroperative transfusions of third-party leukocyte-free blood or pure lymphocyte cell suspensions did not influence graft survival. Furthermore, no improvement in graft survival has been found after a peroperative transfusion of irradiated whole blood (2500 rad). These data suggest that delayed graft rejection after blood transfusions can only be expected after the administration of whole blood. The role of competent lymphocytes in whole blood is questionable, since a transfusion or irradiated whole blood in combination with nonirradiated lymphocytes did not lead to prolonged graft survival. Immunosuppression of the recipient directly after transfusion seems to be essential to induce the beneficial effect of blood transfusions. This has been demonstrated for a transfusion of whole blood 14 days before transplantation. A single transfusion of 100 ml of whole blood 14 days before transplantation could effectively prolong graft survival if immunosuppression with azathioprine and prednisone was started on the day of transfusion. No improvement in graft survival has been found with such a transfusion if preoperative immunosuppression has been omitted.

Lymphocyte stimulation by canine kidney cells.

Lymphocyte stimulation in mixed kidney cell-leukocyte cultures (MKLC) has been investigated in a canine model. Canine kidney cells were obtained by perfusion trypsinization. Cultured kidney cells, which appeared to be of epithelial origin by several criteria, have been used as stimulator cells. Maximal stimulation was obtained in the MKLC and mixed leukocyte culture (MLC) at stimulator to responder (S:R) cell ratios of 1:20 and 1 1/2:1, respectively. Lymphocyte proliferation has been observed in cultures with kidney cells in S:R cell ratios lower than 1:20. Stimulation has not been observed in MLCs at these low ratios. The addition of graded numbers of kidney cells of the responder to a one-way MLC inhibited the response gradually. The fact that kidney cells have both strong stimulator capacities and inhibitor capacities could explain the lower optimal S:R ratio. Lymphocyte stimulation has not been obtained in mixed kidney leukocyte cultures between major histocompatibility complex (MHC)-identical closely bred animals. The nature of the antigens present on canine kidney epithelial cells stimulatory to allogeneic lymphocytes is discussed.

A study on the mechanism of donor pretreatment: effect of procarbazine hydrochloride and methylprednisolone in immunocompetent cells.

Significantly prolonged canine renal allograft survival can be obtained by donor pretreatment with procarbazine hydrochloride and methylprednisolone. This is thought to be caused either by a reduced antigenicity of the graft or by a local immunosuppressive effect by drugs transplanted with the graft. In this study a decrease in the number of peripheral donor T and B lymphocytes was observed at the time of procuring. Leukocytes harvested from dogs pretreated with a combination of procarbazine hydrochloride and methylprednisolone showed a decrease in their ability either to stimulate or respond to mixed leukocyte cultures (MLCs). Complete restoration of MLC responses was obtained however by purification and washing of these leukocytes. Sera of pretreated animals were not able to reduce MLC responses. It was concluded that drug metabolites in or on the cells were apparently responsible. A local inhibition of the immunocompetence of host lymphocytes by small amounts of transplanted drug metabolites in or on the graft cells might be responsible for the beneficial effect of donor pretreatment with procarbazine hydrochloride and methylprednisolone. Furthermore, this postulation explains the abrogation of prolonged survival of pretreated grafts after systemic administration of nontreated donor blood or donor leukocyte-free blood, as we reported earlier.

Cellular proliferation at the site of organ allografts and the influence of immunosuppressive therapy.

In this study we investigated cellular proliferation of T cells and macrophages at the site of rat cardiac allografts and determined the influence of immunosuppressive therapy on the proliferative characteristics of these cell types. A bromodeoxyuridine-labeling technique was used that allowed both the accurate detection of proliferative activity and the phenotypic characterization of cellular infiltrates within grafted tissues. In untreated recipients (BN----Lewis), T cytotoxic/suppressor cells as well as T helper cells showed proliferative activity at the site of the graft. The percentage of OX8-positive cells within the graft that showed proliferation ranged from 15% to 37%. The percentage of W3/25-positive cells within the graft that showed proliferation ranged from 25% to 30%. In contrast, macrophages hardly showed proliferative activity within the graft; only 1-4% of the macrophages stained positive for bromodeoxyuridine. From these observations it is concluded that the graft serves as a nonlymphoid tissue site, wherein lymphocytes can freely proliferate and expand. To study the influence of immunosuppressive therapy on cellular proliferation, the steroid budesonide, 120 micrograms/kg/day, was administered for 13 days (MST, 20 days). During effective immunosuppressive therapy, still a remarkable amount of infiltrating cells was present within the grafts. Moreover, immunosuppressive treatment did not primarily appear to affect the proliferative capacity of the individual cell types. OX8-positive cells as well as W3/25-positive cells clearly showed proliferative activity within the treated grafts. However, despite the presence of these proliferative cells, signs of graft destruction were absent during immunosuppressive therapy. This finding may shed new light on the effect of steroids at the site of the graft and their role in the prevention of tissue destruction.

Effect of cyclosporine on MHC class II antigen expression on arterial and venous endothelium in vitro.

MHC class II antigens play a crucial role in immunological responses. The expression of MHC class II antigens on monocytes and endothelial cells is reported to be variable and able to be induced by gamma-interferon. In this study we report on MHC class II antigen expression in vitro by arterial and venous canine endothelial cells, as detected with FACS analysis and indirect immunofluorescence with a monoclonal antibody against canine MHC class II antigens. It appears that cultured endothelial cells do not express MHC class II antigens. Their expression could be induced during a three-day incubation period in lymphokine-containing supernatant produced in mixed leukocyte culture (MLC). Cyclosporine (CsA) added to allogeneically stimulated or unstimulated canine lymphocytes in MLC inhibited the induction of expression by the MLC supernatant. The addition of CsA to MLC supernatant did not have an inhibitory effect. It is concluded that CsA inhibits the production of an MHC class-II-antigen-inducing lymphokine produced by lymphocytes in mixed cultures; allogeneic stimulation is not necessary for production of the lymphokine. It is postulated that a possible mode of action of CsA in prolongation of allograft survival is based on prevention of the induction of MHC class II antigen expression by endothelial cells.

Complete suppression of skin allograft rejection in rats treated with continuous infusion of 2'deoxycoformycin.

Congenital deficiency of the enzyme adenosine deaminase (ADA) results in severe combined immunodeficiency. 2'deoxycoformycin (2'dcf) is a tightly binding inhibitor of ADA, and the drug makes it possible to mimic a state of ADA deficiency. In this study we tested the immunosuppressive effect of 2'dcf in a rat skin transplantation model. Rats treated with continuous infusion of 2'dcf at doses of 0.3 mg/kg, 0.5 mg/kg and 0.7 mg/kg body wt/day showed significant prolongation of graft survival. 2'dcf given by bolus injections did not prolong graft survival. In rats treated with continuous infusion of 2'dcf at a dose of 0.7 mg/kg body wt/day mean graft survival time (MST) after withdrawal of treatment was equal to MST in untreated animals, suggesting that during 2'dcf treatment allograft rejection was completely suppressed. In vitro, lymphocytes isolated from animals treated with continuous infusion of 2'dcf showed marked suppression of mitogen response. The 2'dcf preferentially effects lymphocytes, but neutrophils seem resistant to the effect of the drug. The lymphocytotoxic effect of the drug is extreme; during therapy splenic weight decreased by almost 50% and the differential lymphocyte count in blood decreased from 85% to 17%. Immunofluorescence studies showed that, within the spleen, the amount of T cells and B cells decreased markedly. Both T cell subsets were affected--OX8+ cells (suppressor/cytotoxic T cells) and W3/25+ (helper T cells). However OX8+ cells were more resistant to the drug than W3/25+ cells. Skin-grafted rats treated with 2'dcf showed a strong decrease in the W3/25: OX8 ratio. In contrast, untreated rats showed a slight increase in the ratio after skin transplantation. It is concluded that 2'dcf is a strong immunosuppressive drug in rats if given by continuous infusion.

Cell-mediated cytotoxicity patterns of cloned cytotoxic T lymphocytes. Cytotoxicity directed against canine lymphoblasts, monocytes, and endothelial cells.

Knowledge of the antigens recognized during allograft rejection is still incomplete. Cloned cytotoxic T lymphocytes were used to study the distribution of target determinants in the dog. CTL clones were obtained with a limiting dilution technique from effector cells generated in mixed lymphocyte culture. The clones have been tested for cell-mediated cytotoxicity against PHA-stimulated lymphoblasts, monocytes, and arterial and venous endothelial cells. A limited number of patterns of lysis of one, or more than one, of the four different target cells was observed. The nature of the possible target determinants recognized by these CTL-clones is discussed.

The cytokinetic behavior of donor hepatocytes after syngenic hepatocyte transplantation into the spleen.

The cytokinetic behavior of isolated hepatocytes transplanted into the spleen of syngenic normal Wistar rats was studied. Hepatocyte transplantation (HTX) was performed by the intrasplenic injection of 10(7) isolated hepatocytes. The proliferation index (PI) of intrasplenic donor hepatocytes was assessed by immunocytochemical visualization of DNA-synthesizing cells after pulse-labeling with bromodeoxyuridine (BrdU), a thymidine analogue. A method for determination of intrasplenic liver mass based on tissue glutamate dehydrogenase content was developed. The spontaneous PI of donor hepatocytes at 12 and at 20 weeks post-HTX amounted to around 3%. A significant increase of intrasplenic liver mass was demonstrated between the 12th and 20th week post-HTX (from 8.1 +/- 0.8% to 10.8 +/- 0.8% of spleen weight, P less than 0.05). After partial hepatectomy (PH) at 12 weeks post-HTX, the PI of liver cells in the spleen showed a transient increase up to about 10%, which rapidly declined to the "spontaneous" level of 3%. However, PH did not cause an additional increase in intrasplenic liver mass. This study shows that continuous mitotic activity of intrasplenic hepatocytes results in an actual increase of liver mass in spleen. Although a short-lived increase of proliferative activity of ectopically grafted hepatocytes was shown to occur after PH in the HTX-treated rat, this procedure did not result in an additional increase of intrasplenic liver tissue.

The effect of steroids on the regulation of major histocompatibility complex-class II expression on nonlymphoid tissue.

The induction of MHC-class II antigens on human nonlymphoid tissues plays an essential role during the inhibition and augmentation of the immune response. Steroids have long been shown to possess strong immunosuppressive properties and successful steroid treatment has been associated with the absence of MHC-class II antigens on grated tissues. In this study we more specifically investigated the effect of steroids on the regulation of MHC-class II expression on nonlymphoid tissue. First, the influence of prednisolone on the induction process of the MHC-class II antigens on nonlymphoid tissue was determined. For this purpose vascular endothelial cells, kidney epithelial cells, fibroblasts, and a human colon tumor cell line were incubated with rIFN-gamma or primary MLC supernatant in the absence or presence of different concentrations of prednisolone. It was demonstrated that the induction process of MHC-class II antigens on these cell types was not affected by the drug at the different concentrations tested (1, 10, and 100 micrograms/ml). Next, the effect of prednisolone on the production of MHC-class II inducing factors was investigated. The drug was added at initiation of culture to primary MLC and the MHC-class II-inducing capacity of the supernatants was determined on day 7. Prednisolone at concentrations of 0.5-100 micrograms/ml clearly inhibited the overall production of factors responsible for MHC-class II induction on nonimmunological cells. The drug inhibited the production of IFN-gamma as well as non-IFN-gamma MHC-class II-inducing mediators. At a concentration of 1 microgram/ml the production of IFN-gamma and non-IFN-gamma MHC-class II-inducing mediators was reduced by, respectively, 85 per cent and 75 per cent. At a concentration of 10 micrograms/ml the production of both IFN-gamma and non-IFN-gamma mediators was almost completely inhibited. It is concluded that steroids downregulate the expression of MHC-class II antigens on nonlymphoid tissue by the inhibition of the production of MHC-class II-inducing mediators. However, once these mediators are present, the induction process itself is not affected by the drug.

Analysis of cytotoxic T lymphocyte response in rejecting allografted canine kidneys.

The involvement of cytotoxic T lymphocytes (CTL) in the rejection process of allografted canine kidneys was studied. The frequency of donor-specific (precursor) CTL was determined with a sensitive limiting dilution assay. Longitudinal sampling of peripheral blood and kidney aspiration biopsies were used to obtain information on the CTL response toward the graft. An accurate analysis of CTL kinetics in both kidney and peripheral blood of allografted dogs appeared to be technically possible. During the first days after transplantation precursor CTL (CTLp) frequencies decreased in both blood and kidney. A minimum CTLp frequency of 5-15% of the pretransplant value was reached in the peripheral blood at day 4 after transplantation. The cause of this decrease, which was observed in all 5 allografted dogs is discussed. CTLp frequencies increased after day 4 and showed an exponential rise in the kidney before serum creatinine increased due to loss of kidney function caused by rejection. The data obtained with the quantitative study of CTL show that rejection of a canine kidney allograft is accompanied by a rise in CTL numbers in the kidney. The methodology developed permits extensive functional analysis of cellular processes in allografted organs.

Local treatment of renal allografts, a promising way to reduce the dosage of immunosuppressive drugs. Comparison of various ways of administering prednisolone.

A method is described for continuous administration of immunosuppressive drugs directly into rat renal allografts. The drug is given via a catheter, introduced into the suprarenal or testicular artery of the transplanted kidney. The cannula is connected to an implantable osmotic minipump that delivers an immunosuppressive drug with continuous flow for 13 days. It is demonstrated that the technique as such has no detrimental or enhancing effects on renal allograft survival. Depending upon the pharmacokinetic properties of the drug administered, this technique allows a more or less selective treatment of renal allograft rejection. The method was used to test the effect of intrarenal administration of prednisolone on renal allograft survival. Intrarenal administration of this drug appeared to be superior to any other way of administration tested. A low dose of 4 mg/kg body weight per day given by continuous intrarenal infusion results in significant prolongation of graft survival, whereas continuous systemic administration of this dose is not effective. To induce prolongation of graft survival by i.p. administration the prednisolone dose had at least to be doubled. The results prove that during graft rejection local events within the graft are vulnerable to prednisolone. It is concluded that local treatment of allograft rejection is possible and that this approach represents a potentially important way to manipulate the immune response.

Sensitivity of graft rejection in rats to local immunosuppressive therapy.

In this study we investigated whether allograft rejection is sensitive to local immunosuppressive therapy. In rats, cardiac transplantations (BN----Lewis) were performed with venous return on the portal vein of the recipient. For local treatment the topical steroid budesonide was infused with an osmotic minipump directly into the carotid artery of the transplant. Budesonide is rapidly cleared by the liver, and cardiac tissue binding of the drug is high. Hence, local budesonide administration, 120 micrograms/kg/day, resulted in high drug levels within the graft (29.6 ng/mg) and low systemic drug levels (0.34 ng/ml). Systemic drug levels were so low that systemic biological effects of the drug during local administration were not measurable. In contrast systemic drug delivery, via the jugular vein of recipient, resulted in similar drug levels within the graft (31.0 ng/mg), but with high systemic drug levels within the graft (31.0 ng/mg), but with high systemic drug levels (1.65 ng/ml) and important systemic side effects. Both local and systemic administration of budesonide, 120 micrograms/kg/day for 13 days, resulted in significant prolongation of graft survival; median graft survival time was respectively 19.5 days and 20.0 days, compared with 7 days in controls. These results demonstrate that allograft rejection can be treated locally without significant systemic immunosuppression.

In vitro stimulation of lymphocytes by vascular endothelial cells. A study with canine arterial and venous endothelial cells.

Data are presented on the ability of arterial and venous endothelial cells to stimulate allogeneic leukocytes. Mixed cultures of allogeneic endothelium and lymphocytes result in proliferation of lymphocytes and generation of cell-mediated cytotoxicity, which do not occur in cultures of syngeneic combinations of endothelium and lymphocytes. Studies of kinetics showed a peak in proliferation at days 6-7. The optimal responder-stimulator ratio appeared to be 15:1. Lymphocytes stimulated with venous endothelial cells were cytotoxic both for arterial and for venous endothelial cells and PHA blasts of the stimulator dog, whereas lymphocytes stimulated with arterial endothelial cells lysed only arterial endothelial cells and PHA blasts of the stimulator. Lysis of syngeneic or third-party allogeneic control targets was virtually absent. Optimal conditions for long-term culture of effector cells from mixed leukocyte endothelial cell cultures were analyzed. Addition of Il 2 every 3 days and the original stimulating antigen every 6 days permitted continuous proliferation of these cytotoxic lymphocytes with preservation of the cytotoxicity pattern.

Influence of a skin allograft on frequencies of CTL precursor in peripheral blood of the dog.

Alloantigen-specific cytotoxic T lymphocytes (CTL) and their precursors (CTL-P) have been determined in the peripheral blood of skin allografted dogs. CTL-P frequencies increased rapidly after transplantation and reached maximal values after complete rejection of the skin allograft. Differences in the time response kinetics of CTL-P frequencies between recipients were not correlated with the length of graft survival. The CTL-P frequencies declined after days 13-20 and appeared still to be elevated 30 days after rejection of the graft.

Limiting dilution analysis of canine alloantigen-reactive T lymphocytes.

A sensitive limiting dilution assay for the determination of canine cytotoxic T lymphocyte precursors (CTL-P) has been developed. Murine second MLC supernatant as a source of Interleukin 2 (IL 2) was used to expand stimulated CTL-P to numbers that were easily detectable with the classic 51Cr lysis assay. The frequencies of CTL-P that reacted to allogeneic stimulator cells in canine peripheral blood ranged from 1:1000 to 1:2000 lymphocytes. During in vitro stimulation in a mixed leukocyte culture a rapid increase in frequency was noted, and at day 7 a frequency as high as 1:5.4 was found. The presence of irradiated stimulator cells during limiting dilution culture restricted proliferation of the CTL-P; only those which recognized the stimulator cells proliferated. The determination of cytotoxicity in large numbers of individual microcultures with CTL-P seeded at clonal conditions toward 2 allogeneic target cells permitted quantitation of the frequency of CTL-P with specificity for different alloantigens. These techniques greatly facilitate monitoring of cellular immune reactions after renal allografting because both determination of the influence of cytostatic drug treatment on CTL-P frequency, and analysis of the specificity and function of allograft infiltrating cells are now possible.