Randomized clinical trial of omega-3 fatty acid-supplemented enteral nutrition versus standard enteral nutrition in patients undergoing oesophagogastric cancer surgery.

BACKGROUND: Oesophagogastric cancer surgery is immunosuppressive. This may be modulated by omega-3 fatty acids (O-3FAs). The aim of this study was to assess the effect of perioperative O-3FAs on clinical outcome and immune function after oesophagogastric cancer surgery. METHODS: Patients undergoing subtotal oesophagectomy and total gastrectomy were recruited and allocated randomly to an O-3FA enteral immunoenhancing diet (IED) or standard enteral nutrition (SEN) for 7 days before and after surgery, or to postoperative supplementation alone (control group). Clinical outcome, fatty acid concentrations, and HLA-DR expression on monocytes and activated T lymphocytes were determined before and after operation. RESULTS: Of 221 patients recruited, 26 were excluded. Groups (IED, 66; SEN, 63; control, 66) were matched for age, malnutrition and co-morbidity. There were no differences in morbidity (P = 0·646), mortality (P = 1·000) or hospital stay (P = 0·701) between the groups. O-3FA concentrations were higher in the IED group after supplementation (P < 0·001). The ratio of omega-6 fatty acid to O-3FA was 1·9:1, 4·1:1 and 4·8:1 on the day before surgery in the IED, SEN and control groups (P < 0·001). There were no differences between the groups in HLA-DR expression in either monocytes (P = 0·538) or activated T lymphocytes (P = 0·204). CONCLUSION: Despite a significant increase in plasma concentrations of O-3FA, immunonutrition with O-3FA did not affect overall HLA-DR expression on leucocytes or clinical outcome following oesophagogastric cancer surgery. REGISTRATION NUMBER: ISRCTN43730758 (http://www.controlled-trials.com).

Anti-vimentin antibody detection in recipients of heart-beating and non-heart beating donor kidneys.

Alternative donor sources include non-heart-beating donors (NHBDs). There donors have been exposed to significant ischemia, so that it is common to utilize machine perfusion to either improve the organs or at least assess their viability. Both prolonged warm ischemia and machine perfusion can potentially damage the vascular endothelium, thereby exposing vimentin to antigenic recognition. The aim of this study was to determine whether anti-vimentin antibodies could be detected in the blood of renal transplant recipients at specific time points after transplant and whether they could be related to the donor source. Fifty-one recipients of NHBD kidneys were compared to 52 recipients of heart-beating donor (HBD) kidneys. All recipients had similar anti-vimentin levels pretransplant. However, at 1 month those kidneys from Maastricht category II NHB donors showed significantly higher levels. At 6 months both Maastricht category II and category III NHB donor recipients displayed significantly higher levels than recipients of HBD kidneys.

Cardiovascular risk assessment scoring system for the determination of cardiovascular mortality in renal transplant patients.

It is well known that the greatest risk for mortality post-renal transplant is cardiovascular death. We compared a modified cardiac risk assessment system among renal transplant patients who subsequently died versus the group that survived. There was a good correlation between the low, medium, and high scores with survival. The deceased group had significantly greater cardiovascular scores than case controls.

Vimentin antibodies: a non-HLA antibody as a potential risk factor in renal transplantation.

Chronic allograft rejection is the major problem encountered in solid organ transplantation and is the end point of several complex processes. A number of recent studies show both alloimmune and autoimmune responses may have roles to play. The importance of HLA antibodies in transplantation is well documented, but despite the introduction of very sensitive HLA screening assays, antibody-mediated allograft rejection still occurs without detectable HLA antibodies. The target for antibody-mediated allograft rejection in these circumstances remains elusive, perhaps due to the variety of potential targets presented on endothelial cells. Recent studies identifying C4d and immunoglobulin deposits in patients undergoing late allograft loss provide evidence that chronic rejection involves humoral as well as cellular components. Several endothelial cell antigens that might be important in chronic rejection have been suggested, including MHC class I chain-related genes; Lewis; and the intermediate filament protein, vimentin. Vimentin is an ideal candidate antigen for antibody-mediated rejection as it is found in endothelial cells and is exposed to the immune system following surgery or by chronic allograft rejection due to endothelial cell breakdown, where the development of antibodies may cause further damage. We have developed a flow cytometric assay for the detection of antibodies to vimentin and have investigated whether HLA or vimentin antibodies are present in renal transplant recipients undergoing chronic rejection.

Do recipients of kidneys from donors treated with streptokinase develop anti-streptokinase antibodies?

Streptokinase is used for preflush for non-heart-beating donors (NHBDs) in our center. The aim of this study was to evaluate whether the use of thrombolytic streptokinase results in the production of anti-streptokinase antibodies in the recipients after renal transplantation. Recipient sera taken prior to and at 1 and 6 months posttransplant were tested for the presence of antibodies to streptokinase using an enzyme-linked immunosorbent assay assay. No differences were detected between a group of 18 recipients who had kidneys from thrombolytic-treated NHBDs and a further group of 18 who received NHBD kidneys without such treatment.

Examination of multidrug resistance in cell lines and primary breast tumours by flow cytometry.

The aim of this study was to measure multidrug resistance (MDR) by flow cytometry and quantify the expression of P-glycoprotein (using antibody) glutathione transferase (using alpha-GSTpi antibody) in alpha-JSB-1 and alpha-GSTpi of a series of cell lines and primary breast cancers, and to assess the relationship between these MDR proteins and a selection of oncogene and prognostic markers in breast cancer. Flow cytometry was performed using permeabilised cells stained with fluorescent antibodies using well-established methods. Antibody staining was confirmed for JSB1, but not GSTpi by use of known positive and negative controls. No correlation was seen when comparing the number of molecules of alpha-JSB-1 with alpha-GSTpi (P = 0.1, r2 = 0.4, n = 14) using a selection of cell lines. Examination of 45 breast tumours for expression of JSB-1 and GSTpi revealed a significant association between these two antibodies (P < 0.00001, r2 = 0.5, n = 45). On examining the breast tumours, alpha-JSB-1 showed a positive association with c-erbB-2 (P = 0.003), c-myc (P = 0.0004) and c-jun (P = 0.02) but not ER or EGF-R expression. alpha-GSTpi showed a positive association with c-erbB-2 (P = 0.03) and c-myc (P = 0.0004) but not ER, EGF-R or c-jun. Flow cytometric MDR levels were not related to tumour grade or axillary node status. In solid tumours, a relationship between the two antibodies used has been clearly demonstrated, however, specificity of alpha-GSTpi is questioned. Both antibodies show an association with c-erbB-2, which is associated with poor prognosis and with c-myc which is involved in cell cycling and differentiation. Monitoring MDR markers (Pgp) using this methodology may be useful for evaluation of prognosis in breast cancer.

The macrophage electrophoretic mobility test (MEM) some technical considerations.

The following parameters of the cytopherometric test system have been studied. 1. Temperature is less important for the production of macrophage slowing factor (MSF) by lymphocyte-antigen interaction than it is for the activity of MSF on macrophages. 2. Lymphocyte number titration curves allow discrimination between 'weak' and 'strong' antigen responses. 3. Lymphocyte-antigen reaction reaches a plateau after 30 min incubation, whilst maximum MSF-macrophage reaction requires 60 min. 4. Interaction of MSF with macrophages is sensitive to cycloheximide. 5. Irradiation of peritoneal exudate cells (200 rad) is essential in order to obtain a maximal result in the MEM test. In the 'split MEM test, 200 rad to the indicator peritoneal exudate cells produces a greater result, i.e. it in some way amplifies the macrophage response to MSF. 6. Excess of peritoneal exudate cells, especially when non-irradiated, suppresses the test result. This is not due to consumption of MSF produced. Probably macrophages rather than lymphocytes are responsible for the suppression.

A comparison of the kinetics of the macrophage electrophoretic mobility (MEM) and the tanned sheep erythrocyte electrophoretic mobility (TEEM) tests.

In this study the macrophage electrophoretic mobility (MEM) test was modified by using tanned sheep erythrocytes in place of guinea pig peritoneal macrophages as the indicator cells of lymphocyte sensitization to antigens. This modification is named the tanned sheep erythrocyte electrophoretic mobility (TEEM) test, and a comparison of the kinetics of the two systems allowed the following conclusions to be made: 1) Treatment of freshly drawn sheep red blood cells with a concentration of 1/40,000 tannic acid produced optimum results in the TEEM test. 2) Lymphocyte-antigen and lymphocyte-number response curves show similarity in the two test systems. 3) A plateau response with slowing factor is achieved at a lower dilution in the TEEM test than in the MEM test. 4) Whilst similarity in the first stage reaction was found in the two systems, in the second stage of the test (at 37 degrees C) tanned sheep red cells gave a plateau response after 45 min instead of the 60 min found in the MEM test. 5) The two slowing factors showed similar gel filtration patterns with molecular weights between 13,000 and 15,000 daltons, and had equivalent activity in both test systems. 6) The disadvantages of the guinea pig macrophage as an indicator cell are discussed. 7) The TEEM test seems simpler to perform than the MEM test and may be widely applicable in clinical immunology for the estimation of lymphocyte sensitization.

A rapid, objective method for the detection of lymphocytotoxic antibodies using flow cytometry.

Whilst several centres have reported lymphocytotoxic antibody detection using single and dual fluorescent stains with analysis of the fluorescence emitted from the cell population present in a well of a multiwell plate, problems are encountered with cell concentration and light emission overlap. A method we have developed using flow cytometry produces similar values of percentage cell death using single or double staining techniques (correlation coefficient = 0.9896). This method is not influenced by slight variation in cell number or light emission overlap. The effect of introducing red cell impurities into the normal lymphocyte preparation is described.

Use of real-time PCR to measure Epstein-Barr virus genomes in whole blood.

The measurement of the Epstein-Barr viral load in peripheral blood has been recognised as an important way of monitoring the response to treatment in patients with Epstein-Barr virus (EBV)-related malignancies. In particular, EBV load in transplant recipients can be used as a predictive parameter for Post-transplant Lymphoproliferative Disorder (PTLD). The aim was to develop a rapid and reliable PCR protocol for the quantification of the cell-associated EBV genome. Real-time PCR using TaqMan methodology was established. This technique was applied to determine the EBV load in various study groups including healthy controls, transplant recipients, patients on haemodialysis, and patients with infectious mononucleosis. The baseline level of EBV genomes in the immunosuppressed renal transplant recipients was significantly different from that in the healthy controls.

Rapid detection of low levels of donor specific IgG by flow cytometry with single and dual colour fluorescence in renal transplantation.

Lymphocytotoxic immunoglobulin is routinely assayed before human renal transplantation. If IgG directed against donor T cells is detected in the serum of the potential recipient, transplantation is not performed as it is associated with a poor graft outcome. Poor sensitivity of the conventional assay has been postulated as being the cause of some graft failures. Two new flow cytometric assays are described which are more sensitive than the conventional test. The first assay requires manual separation of T and B lymphocytes and therefore takes a similar time to perform as the conventional assay. The second assay utilises a two-colour system and lymphocyte's separation is by fluorescence. This assay takes half the time to perform, thereby decreasing graft ischaemic time before transplantation.

A flow cytometric technique for simultaneous analysis of human mononuclear cell surface antigens and DNA.

A method for dual staining of mononuclear cells for lymphocyte phenotypic markers and DNA is described. The cells were stained with fluorescein-conjugated monoclonal antibodies and then rendered permeable to propidium iodide using saponin. Propidium iodide stains DNA and, using flow cytometry, cell cycle analysis of individual lymphocyte subpopulations can be determined. Saponin acts within 1 min, preserves expression of surface antigens and is effective at all concentrations from 0.001% to 1%. This technique is simple, rapid and gives reproducible results.

The flow cytometric crossmatch in liver transplantation.

The role of the cytotoxic crossmatch in liver transplantation is generally considered controversial. The development of the flow cytometric crossmatch has allowed the detection of lower levels of donor-directed IgG than is possible with the conventional crossmatch. This assay has been shown to be useful in renal transplantation. However, with the controversial role of the standard cytotoxic crossmatch, the flow cytometric crossmatch has not been used in liver transplantation. Twenty-seven human orthotopic liver allograft recipients were tested for donor-directed IgG using the flow cytometric crossmatch. Thirteen recipients were identified with either T or B lymphocyte-directed IgG. This group had a significantly increased incidence of clinical rejection (75%) as compared with the negative group (29%, P = 0.02, Fisher's exact test). The differences were greatest with B lymphocyte-directed IgG and the rejections were generally steroid sensitive. In this series, the flow cytometric crossmatch proved to be a better prognostic indicator of rejection than the conventional cytotoxic crossmatch. In addition, the association of a positive flow cytometric crossmatch with rejection indicates that the liver follows the same pattern seen in renal and cardiac grafts.

The relevance of a more sensitive crossmatch assay to renal transplantation.

While the importance of the standard preoperative crossmatch in predicting renal graft success is accepted, a more rapid and sensitive assay may be of additional clinical benefit. We have developed a flow cytometric assay to detect the presence of antibodies (IgG) in the recipient sera directed against donor lymphocytes, prior to transplantation. This assay is more rapid and sensitive than the conventional cytotoxic test. In a clinical study the sera of 75 renal graft recipients were tested, all of which were negative in their conventional crossmatch; 12 of these were identified as having T cell-directed IgG, and 4 had B cell antibody. Graft failure was not significantly different in the positive and negative antibody groups, as defined by flow cytometry (P = 0.147, chi square test). The incidence of postoperative complications was studied in the 60 grafts functioning at three months. Recipients with donor B or T cell directed antibodies had a longer primary nonfunction (P = 0.0098, Mann-Whitney U test), and showed a higher number of rejection episodes (P = 0.014, Mann-Whitney U test); accordingly they were more likely to require strong immunosuppressive agents such as OKT3 or ATG (P less than 0.05, chi square test). Patients with donor-directed antibodies were also hospitalised for a longer period (P = 0.015, Mann-Whitney U test) and had a higher creatinine level 3 months after transplantation (P = 0.021 Mann-Whitney U test). This study shows that the described preoperative flow cytometric crossmatch is capable of defining a population of renal transplants who form an at-risk group. Thus this assay has considerable potential in pretransplant matching of recipients with a particular graft donor.

The value of posttransplant monitoring of interleukin (IL)-2, IL-3, IL-4, IL-6, IL-8, and soluble CD23 in the plasma of renal allograft recipients.

Over the past few years, the central role of cytokines in the amplification of the immune response has been reported and several studies have examined the relationship between the plasma level of individual lymphokines during renal allograft rejection. The aim of the present investigation was to study simultaneously IL-2, IL-3, IL-4, IL-6, IL-8, and soluble CD23. Analysis of results has allowed both the prognostic value and any possible interrelationships between the measured cytokines to be determined. We studied 16 renal transplant recipients for the first 14 days after transplantation. Seven patients showed clinical evidence of acute allograft rejection and 5 showed excellent stable graft function with no signs of rejection. Primary nonfunction was seen in 4 patients. The plasma levels of each cytokine were measured by commercially available ELISA and immunoradiometric assay kits. As reported in previous studies, plasma IL-2 levels, whenever found at detectable levels, were predictive of impending graft rejection. Serial monitoring of IL-4 and IL-6 was more reliable for the differential diagnosis of rejection, particularly toward the end of the first week after transplantation. IL-3, IL-8, and soluble CD23 were not diagnostic or predictive of rejection, due to the occurrence of significantly high levels in transplant patients who showed no evidence of clinical rejection. While the value of cytokine monitoring has been shown in this study, it should be remembered that infection, although not seen in these studies, may have a profound affect on the results obtained.

Renal allograft rejection. Possible involvement of antibody-dependent cell-mediated cytotoxicity.

We have demonstrated that serum from appropriately sensitized patients can contain IgG antibodies that bind to cultured renal epithelial cells. The presence of such antibodies on the surface of renal cells enables otherwise nonlytic PBMC to lyse these renal cells by an antibody-dependent cell-mediated cytotoxicity (ADCC) mechanism. Experiments involving cell-sorting and specific complement-mediated lysis showed that the ADCC effector cells were of the CD3 -ve, C16 +ve phenotype characteristic of NK cells. In this report it is argued that an ADCC mechanism may be of importance in mediating chronic renal cell damage in the absence of acute allograft rejection.

Renal allograft rejection. Functional impairment of kidney epithelial cell monolayers mediated by lymphokine-activated killer cells and by antibody.

A continuous line of human kidney epithelial cells was cultured to confluency on porous membranes, and the formation of intercellular tight junctions was monitored by measuring increases in the trans-monolayer electrical resistance. Typical monolayers developed functioning tight junctions within 4 days of culture and showed an increase in resistance of 1840 +/- 440 ohms (mean +/- SD; n = 5) at this time. Conventional 51Cr release assays showed that suspended kidney cells were lysed by specific antibody and complement but were relatively resistant to lysis mediated by lymphokine-activated killer cells. However, when antibody and complement or LAK cells were added to functioning kidney cell monolayers the electrical resistance of the monolayers was rapidly reduced in both cases. In the absence of trans-monolayer resistance the ion gradients essential for renal tubular function will not be supported. These results indicate that the ability of a cytotoxic effector cell to liberate 51Cr from suspended kidney cells may not be a sensitive assay for the ability of these effector cells to impair the function of structured kidney cell monolayers. It is possible that significant kidney dysfunction may occur during renal allograft rejection by failure of trans-epithelium resistance in the absence of widespread epithelial cell lysis.

Improved graft outcome and reduced complications due to flow cytometric crossmatching and DR matching in renal transplantation.

Several previous studies, including our own, have indicated that flow cytometric assays can identify an at-risk population of kidney transplant recipients. We used the assay for recipient selection for a period of twelve months. Recipients with donor T cell-directed IgG were excluded from transplantation and those with B cell-directed IgG were treated with increased immunosuppression. The transplants performed over this period (n = 126) were compared with an earlier series (n = 118) where, although the flow cytometric crossmatches were performed, the results did not influence patient management. In the series where the flow cytometric crossmatch was used in management, a lower failure rate was found at three months (P = 0.037 chi square), primary non-function was reduced (P less than 0.0001, Mann-Whitney), rejection episodes were reduced (P less than 0.0001, Mann-Whitney) and the hospital stay was shorter (P less than 0.0001, Student's t). The risk factors of ischemic times, panel reactivity, exposure to previous grafts and A/B locus matching were identical between the two groups. However DR matching was found to be higher in the series with the improved results (P less than 0.0001, Mann-Whitney). In view of the significant improvement in graft success and low complication rate, we intend to continue with the policy of recipient selection by flow cytometric crossmatching and DR matching.

Evaluation of the flow cytometric crossmatch. Preliminary results of a multicenter study.

The flow cytometric crossmatch is a technique that is increasingly being used by clinical transplant laboratories. In this multicenter study by the British Society for Histocompatibility and Immunogenetics Flow Cytometry Group, a series of crossmatches were carried out to determine whether different centers obtained same results when performing the same crossmatch. There was greater than 80% agreement among participating laboratories on the results of 35/54 tests. There was no clear agreement in the remaining 20 cases. Quantitative analysis, estimating the number of cell-bound fluorescein molecules, demonstrated that differences in the criteria used by each center to define a positive crossmatch were responsible for some discordant results. When applied, definition of positivity based on the molecules of fluorescein increased concordance from 57.5% to 81.4%.l. These results suggest that a criterion for the interpretation of results based on quantitative analysis of bound antibody may be more reliable than methods in current routine use.

Expression of chemokine receptors CXCR1 and CXCR2 during cardiopulmonary bypass.

OBJECTIVE: This study investigated the effects of cardiopulmonary bypass on neutrophil expression of chemokine receptors, CXCR1 and CXCR2, and the beta2 integrin CD11b. METHODS: Ten patients undergoing coronary artery grafting with cardiopulmonary bypass were studied. Blood samples were collected preoperatively, before bypass, at termination of bypass, and 12 to 18 hours postoperatively. In vitro studies were performed on control subjects to determine changes in the surface expression of CXCR1, CXCR2, and CD11b on stimulation with interleukin 8. Receptor expression was measured by flow cytometry. Plasma levels of interleukin 8 from the patients were determined by enzyme-linked immunoassay. RESULTS: After bypass, CXCR2 expression fell by 66% (P <.0001) and remained low postoperatively (P <.0001). CXCR1 expression persisted at preoperative levels. CD11b expression increased significantly after bypass (P <.0001), returning to prebypass levels postoperatively. In vitro studies showed a dose-related fall of both CXCR1 (P <.0001) and CXCR2 expression (P <.0001) and a significant rise in CD11b expression (P <.0001). Plasma interleukin 8 increased significantly after bypass (P <.0001), remaining elevated 12 to 18 hours postoperatively (P =.02). Correlations between interleukin 8 levels and CXCR2 expression (P <.0001) and CD11b expression (P <.03) were demonstrated. CONCLUSIONS: CXCR2 expression is significantly down-regulated after bypass; in contrast, CXCR1 expression remains unchanged. In addition, whereas interleukin 8 is an important determinant of both CXCR1 and CXCR2 expression in vitro, it only correlates with CXCR2 and CD11b expression in vivo. This has implications in the search for antagonists against CXC chemokines and their receptors.