Reduced binding of human antibodies to cells from GGTA1/CMAH KO pigs.

Xenotransplantation using genetically modified pig organs could solve the donor organ shortage problem. Two inactivated genes that make humans unique from pigs are GGTA1 and CMAH, the products of which produce the carbohydrate epitopes, aGal and Neu5Gc that attract preformed human antibody. When the GGTA1 and CMAH genes were deleted in pigs, human antibody binding was reduced in preliminary analysis. We analyzed the binding of human IgM and IgG from 121 healthy human serum samples for binding to GGTA1 KO and GGTA1/CMAH KO peripheral blood mononuclear cells (PBMCs). We analyzed a sub population for reactivity toward genetically modified pig PBMCs as compared to chimpanzee and human PBMCs. Deletion of the GGTA1 and CMAH genes in pigs improved the crossmatch results beyond those observed with chimpanzees. Sorting the 121 human samples tested against the GGTA1/CMAH KO pig PBMCs did not reveal a distinguishing feature such as blood group, age or gender. Modification of genes to make pig carbohydrates more similar to humans has improved the crossmatch with human serum significantly.

In vitro monitoring of in vivo development of human anti-thymoglobulin antibodies by ELISA.

Thymoglobulin (rATG), polyclonal immunoglobulin, is prepared from rabbits immunized with human thymocytes. It is effective in prevention and treatment of renal allograft rejection. Human antibodies against antilymphocyte preparations can reduce efficacy by accelerating drug clearance or by inducing serum sickness. We developed an enzyme-linked immunosorbent assay (ELISA) to study posttreatment development of anti-rATG. In an Institutional Review Board-approved trial, we tested 101 allograft recipients for anti-rATG antibodies. Patients received rATG intravenously at 1.25 to 2.0 mg/kg/d for 2 to 14 days. Serum samples were obtained pretreatment and at weeks 1, 2, 4, 6, and months 3 and 6 post-rATG. ELISA plates were coated with rATG (10 microg/mL). Samples were diluted 1:100 and tested in quadruplicate. Positive samples were titrated. Horseradish peroxidase-conjugated (HRPO) affinity-purified goat anti-human immunoglobulin G (H&L) antibody reacted with bound human antibody. A chromagenic substrate for HRPO was added and optical density (OD, 490 nm) was read. An OD of twice the negative control was considered positive. Mean ODs of negative and positive controls were 0.113 +/- 0.030 and 1.042 +/- 0.196, respectively. Ten patients had detectable anti-rATG before rATG administration (1:100). Thirty-five of 101 patients (35%) developed anti-rATG antibody. Patients showed an initial positive anti-rATG antibody from days 8 to 59 after infusion and titers from 1:100 to 1:4000. In spite of rATG's postulated anti-B-cell activity, this study confirms that rATG induces sensitization at a frequency and titer seen with other xenogeneic antilymphocyte antibodies. Formation of such antixenoantibodies can have a negative impact on treatment response and hence warrant monitoring.

New crossmatch technique eliminates interference by humanized and chimeric monoclonal antibodies.

Humanized and chimeric antilymphocyte antibodies (Ab) are used to prevent and treat rejection and for treatment of human disease. Rituximab (RIT, anti-CD20), daclizumab (DAC; anti-CD25), alemtuzumab (ALE; anti-CD52), or infliximab (IFX) may interfere with Ab detection methods such as complement-dependent cytotoxicity (CDC) and flow cytometric crossmatch (FCXM). These agents are recognized as anti-human Ab or fix complement and are not differentiated from anti-allo-Ab. A new enzyme-linked immunosorbent assay crossmatch (XM) utilizing class I and II HLA antigens from donor cells called Transplant Monitoring System (TMS; GTI, Waukesha, Wisc) potentially precludes interference by eliminating non-major histocompatability complex antigens. To test this, normal sera (nonsensitized volunteers) were supplemented with 0.1 or 10 microg/mL of RIT, DAC, IFX or ALE, and were tested using three methods: the TMS T-cell CDCXM with antihuman globulin (AHG); and B-cell CDCXM without AHG; and FCXM with mean channel shifts of 45 and 150 indicating positive T-cell and B-cell crossmatch, respectively. No reactivity occurred with normal sera using any crossmatch technique. At 0.1 and 10 microg/mL, RIT interfered with CDC B-cell, but not T-cell crossmatch. RIT at 10, but not 0.1 microg/mL interfered with B-cell FCXM. No interference occurred with RIT in T-cell FCXM or TMS. ALE interfered with B-cell and T-cell CDC and FCXM but neither class I nor II TMS. DAC did not interfere with CDC or FCXM at 0.1 microg/mL, but gave false positive B-cell FCXM and CDCXM with some samples. No interference by DAC occurred using TMS. TMS may be useful to differentiate de novo donor-specific Ab after treatment with humanized or chimeric Ab.

Enhanced recovery of cryopreserved islets using SIS.

Although cryopreservation of pancreatic islets would add flexibility to transplantation, the recoveries are only 60% to 90% and function is decreased. Islets are multicellular structures approximately 50 to 250 microm in diameter organized into a network of cells and vascular channels. Due to this complexity, islets are more susceptible to damage during cryopreservation than an individual cell. This study investigated porcine small intestinal submucosa (SIS) as a matrix to support islets recovery and function post-thaw. Groups of frozen/thawed human islets (150 IE/condition; n = 4 preparations) were cultured for 5 weeks in plates containing noncoated Biopore membrane inserts alone or inserts covered with SIS. Islets were placed directly on the insert post-thaw (SIS(1)), or cultured overnight in standard plates, washed, and then transferred to the SIS (SIS(2)). Function was assessed by determining glucose-stimulated release of insulin, which was measured by radioimmunoassay. Analysis of basal insulin secretion showed time and treatment to be significantly different (P =.0043 and P =.0123, respectively) but without an interaction (P >.05). The two SIS treatments were not significantly different (P >.05); however, both SIS(1) and SIS(2) were significantly different from controls (P =.0108 and P =.0420, respectively). Similar results were obtained for stimulation indices; time and treatment were significantly different (P =.0161 and P =.0264, respectively) but not an interaction (P >.05). The two SIS treatments were not significantly different (P =.05); however, both SIS(1) and SIS(2) differed from controls (P =.0248 and P =.0407, respectively). The results indicate that SIS enables frozen-thawed islets to exhibit superior post-thaw function compared with a non-SIS-supported condition.

Improved in vitro function of islets using small intestinal submucosa.

Transplantation of human pancreatic islets has been demonstrated to be a viable alternative to exogenous insulin therapy for diabetes mellitus. However, optimum results require transplantation of islets from two to three pancreas donors after a minimum number of days in culture. This implies that a substantial part of the transplanted islet mass may be nonfunctional. This study investigates the ability of an optimized technique to retain islet function using porcine-derived small intestinal submucosa (SIS) during in vitro culture. Groups of purified human islets were cultured for 3 weeks in modified standard islet culture conditions of CMRL = 1066 tissue culture medium supplemented with 25 mmol/L HEPES, penicillin/streptomycin, and a commercial insulin-transferin-selenium (ITS) supplement. Islets (50 to 200 IE/condition; n = 5 preparations) were cultured in plates containing noncoated Biopore membrane inserts alone, or on inserts that had been covered with SIS. Function was assessed by static incubation with low (4 mmol/L), or high (20 mmol/L) glucose at the end of each week. Glucose-stimulated release of human insulin was measured by radioimmunoassay (Linco, St. Charles, Missouri). Remaining islets were stained and evaluated visually. Neither culture condition resulted in significantly different basal secretion until week 3 (P =.05). However, by the end of week 2 and for the duration of the experiment thereafter, SIS-treated islets exhibited a higher SI (P <.05). At the end of the experiment, islets cultured on the SIS exhibited excellent morphology, with greater than 90% staining positive with Dithizone. Islets cultured on the inserts alone lost their initial morphology, becoming "loose" in appearance. The results of this study indicate that SIS enables enhanced function of islets in vitro as compared to non-SIS supported culture conditions.

Prospective study of microchimerism in transplant recipients.

BACKGROUND: We evaluated peripheral blood microchimerism in 48 consecutive organ transplant recipients (35 kidneys, ten livers, one kidney-liver, one kidney-pancreatic islet, one kidney pancreas) up to 12 months post-transplantation. Patients were categorized according to the presence or absence of rejection episodes, and the patterns of microchimerism in the two groups were then compared. METHODS: DNA was extracted from donor, pre-transplant, and post-transplant peripheral blood samples. Several polymerase chain reaction (PCR)-based assays were developed for the detection of microchimerism. Assay sensitivities ranged from 0.0001 to 3%. RESULTS: Microchimerism was detected only in sex-mismatched cases (male donors and female recipients) using nested PCR for a Y-chromosome marker. There were ten such cases (six kidneys, two livers, and two combined organ transplants). In patients without rejection (n = 7), there was a peak of donor-DNA at 1-3 wk post-transplantation followed by a second peak between 3 wk and 4 months. In patients with biopsy-proven rejection (n = 3), the peaks were absent and the levels of microchimerism were extremely low (< 0.001%). Microchimerism levels declined in all 10 patients and were barely detectable 1 yr post-transplantation. Microchimerism was not detected in the remaining 38 patients despite using a battery of sensitive PCR-based assays. CONCLUSIONS: In our study, microchimerism was detected using the Y-chromosome PCR assay only and the level of donor-DNA in a given patient varied over time. This study highlights the difficulties in establishing a correlation between microchimerism and transplant tolerance.

Production of interferon-gamma by lung lymphocytes in HIV-infected individuals.

A CD8(+) lymphocytic alveolitis occurs in up to 60% of asymptomatic human immunodeficiency virus (HIV)-infected individuals. Early in HIV infection, lymphocytes consist predominantly of cytotoxic T lymphocytes directed against HIV-infected targets. As HIV disease progresses, they are replaced by CD8(+)CD57(+) suppressor cells. Virus-specific cytotoxic T lymphocytes secrete interferon-gamma (IFN-gamma), an important cytokine in upregulating immune responses, primarily through macrophage activation. We examined the ability of lung and blood lymphocytes from HIV-positive patients at various stages of HIV infection to secrete IFN-gamma spontaneously and in response to phytohemagglutinin A. IFN-gamma production and secretion were determined with ELISA, Western blot, immunoprecipitation, and Northern blot techniques. Lung lymphocytes from HIV-infected individuals secreted large amounts of IFN-gamma. However, this ability was lost in patients with late-stage disease. Correlation between blood and lung lymphocyte IFN-gamma secretion was poor, suggesting regional differences in lymphocyte function. These data suggest that lung levels of IFN-gamma are high until late in HIV disease. These findings support the concept of administering exogenous IFN-gamma to patients with late-stage HIV disease and opportunistic infections.

Lymphocyte subset analysis for the diagnosis of rejection and infection in recipients of liver transplants.

We retrospectively investigated the predictive value of lymphocyte subset changes in the peripheral blood of 37 consecutive liver transplant recipients for rejection and infection, both bacterial and cytomegalovirus, during a period of 2 years. Total lymphocyte count correlated with biopsy-proven rejection. There was moderate elevation of CD16+ count during the course of bacterial infection.

Expression of the human papillomavirus type 11 E5A protein from the E1E4,E5 transcript.

The abundant human papillomavirus type 11 (HPV 11) E1E4,E5 transcript potentially encodes the E1E4,E5a and E5b proteins. It is not known if either of the E5 proteins are expressed from this transcript. For HPV 16, E5 is a single open reading frame (ORF), and the E5 protein is expressed from an unspliced E2,E5 transcript but not from the spliced E1E4,E5 transcript. This study was undertaken to determine if the HPV 11 E5a protein is expressed from the E1E4,E5 transcript. To detect E5a expression in eukaryotic cells, the green fluorescent protein (GFP) gene was fused to the 3' end of the E5a gene in the pEGFP-N1 vector. Several recombinant plasmid constructs were made to determine if E5a translation is influenced by upstream sequences present in the E1E4,E5 transcript. COS-7 cells were transfected with each construct, and flow cytometry was performed after 24 h of growth. The amount of E5a-GFP expressed from each construct was determined by the mean fluorescence of 2,000 transfected cells. Although the E5a-GFP fusion was expressed by all but one construct, the quantity of expressed E5a-GFP varied considerably. The most abundant expression was detected in cells transfected with the E1E4,E5a construct that lacked the 5' noncoding sequence between nucleotides (nts) 714 and 831 that is present in the authentic transcript. Other constructs expressed E5a-GFP in variable amounts, suggesting that sequences between nt 714 and the start of the E5a ORF affect expression of the E5a protein. An E2,E5a construct was made to compare the HPV 11 E5a expression to that of HPV 16. In contrast to HPV 16, no E5a-GFP was expressed from the HPV 11 E2,E5a construct. E1wedgeE4 protein was detected by immunofluorescence in COS-7 cells transfected with a construct that expressed E1E4 as a T7-epitope-tagged protein, and E5a as a GFP fusion. We conclude that the abundant HPV 11 E1E4,E5 transcript is a functional message that can support both E1E4 and E5a expression in eukaryotic cells.

Anatomic distribution of pulmonary vascular compliance.

Previously, the pressure changes after arterial and venous occlusion have been used to characterize the longitudinal distribution of pulmonary vascular resistance with respect to vascular compliance using compartmental models. However, the compartments have not been defined anatomically. Using video microscopy of the subpleural microcirculation, we have measured the flow changes in approximately 40-micron arterioles and venules after venous, arterial, and double occlusion maneuvers. The quasi-steady flows through these vessels after venous occlusion permitted an estimation of the compliance in three anatomic segments: arteries > 40 microns, veins > 40 microns, and vessels < 40 microns in diameter. We found that approximately 65% of the total pulmonary vascular compliance was in vessels < 40 microns, presumably mostly capillaries. The transient portions of the pressure and flow data after venous, arterial, and double occlusion were consistent with most of the arterial compliance being upstream from most of the arterial resistance and most of the venous compliance being downstream from most of the venous resistance.

Evaluation of seven PCR-based assays for the analysis of microchimerism.

OBJECTIVE: The presence of small numbers of cells of donor origin in the circulation of recipients of organ transplants (microchimerism) may correlate with immunologic tolerance. As part of our ongoing studies on microchimerism, we evaluated the utility of seven PCR-based assays for the detection of the less abundant DNA in paired mixtures (100 ng total DNA). DESIGN AND METHODS: DNA samples were screened to identify pairs informative for one or more PCR assays. DNA mixtures from the informative pairs were then analyzed using at least one assay. The assays were based on the X-Y homologous region; a Y chromosome microsatellite locus; three autosomal microsatellite loci; the D1S80 minisatellite locus; and sequence specific oligonucleotide probe (SSOP) analysis of the HLA DRB1 locus. RESULTS: About 0.1% of male DNA against a background of female DNA was detectable using primers for the X-Y homologous region, but the sensitivity was increased to 0.0001% using nested primers for the Y chromosome microsatellite marker. Analysis of the minor DNA component was difficult with the three autosomal microsatellite assays because of the presence of shadow bands. Similar problems with the D1S80 assay were resolved using more stringent PCR conditions, and the sensitivity was 0.1%. Using the DRB1 locus, we were able to detect 1% DNA in the mixed samples. CONCLUSIONS: These studies show that: (a) nested PCR for the Y chromosome is the most sensitive assay for the detection of microchimerism; (b) D1S80 is a useful marker for microchimerism; (c) additional optimization of analytical conditions is required if autosomal microsatellite markers and the SSOP assay are to be used for microchimerism analysis.

Effects of L-arginine supplementation on human lymphocyte proliferation in response to nonspecific and alloantigenic stimulation.

BACKGROUND: L-Arginine has been described as a potential immunostimulant in vitro and in vivo. Excessive arginine, however, may be counterproductive. Data support the concept of minimal arginine requirements for normal lymphocyte proliferation, but the results of supplementation with pharmacologic doses of arginine have been contradictory. We hypothesized that excessive arginine supplementation might result in a blunting of normal immune responses of human lymphocytes in vitro. MATERIALS AND METHODS: Peripheral blood mononuclear and T-cells were isolated from normal human donors. Cells were cultured in complete media with various concentrations of L-arginine, L-ornithine, and glycine. Lymphocytes were then stimulated with PHA or alloantigens, and proliferation was determined by measuring [3H]thymidine incorporation. RESULTS: Lymphocyte proliferation was inhibited by L-arginine at pharmacologic doses. The effects were completely reversible. This inhibition could not be prevented by lymphocyte stimulation with IL-2. Lymphocyte proliferation was more sensitive to inhibition by lower doses of arginine when alloantigens from irradiated fresh tumor cells or allogeneic lymphocytes were the stimuli. Finally, lymphocytes showed variable sensitivity to inhibition of proliferation in response to mitogen when treated with L-ornithine (little to no effect) or L-arginine (consistent inhibition at high doses). Pharmacologic doses of L-arginine result in reversible inhibition of normal lymphocyte proliferation in response to both mitogen and alloantigen. This inhibition could not be blocked by interleukin-2. CONCLUSIONS: We conclude that caution should be exercised when recommending aggressive L-arginine supplementation as a possible method to reverse clinical immunosuppression caused by cancer, malnutrition, or trauma.