Modeling RAS phenotype in colorectal cancer uncovers novel molecular traits of RAS dependency and improves prediction of response to targeted agents in patients.

PURPOSE: KRAS wild-type status is an imperfect predictor of sensitivity to anti-EGF receptor (EGFR) monoclonal antibodies in colorectal cancer, motivating efforts to identify novel molecular aberrations driving RAS. This study aimed to build a quantitative readout of RAS pathway activity to (i) uncover molecular surrogates of RAS activity specific to colorectal cancer, (ii) improve the prediction of cetuximab response in patients, and (iii) suggest new treatment strategies. EXPERIMENTAL DESIGN: A model of RAS pathway activity was trained in a large colorectal cancer dataset and validated in three independent colorectal cancer patient datasets. Novel molecular traits were inferred from The Cancer Genome Atlas colorectal cancer data. The ability of the RAS model to predict resistance to cetuximab was tested in mouse xenografts and three independent patient cohorts. Drug sensitivity correlations between our model and large cell line compendiums were performed. RESULTS: The performance of the RAS model was remarkably robust across three validation datasets. (i) Our model confirmed the heterogeneity of the RAS phenotype in KRAS wild-type patients, and suggests novel molecular traits driving its phenotype (e.g., MED12 loss, FBXW7 mutation, MAP2K4 mutation). (ii) It improved the prediction of response and progression-free survival (HR, 2.0; P < 0.01) to cetuximab compared with KRAS mutation (xenograft and patient cohorts). (iii) Our model consistently predicted sensitivity to MAP-ERK kinase (MEK) inhibitors (P < 0.01) in two cell panel screens. CONCLUSIONS: Modeling the RAS phenotype in colorectal cancer allows for the robust interrogation of RAS pathway activity across cell lines, xenografts, and patient cohorts. It demonstrates clinical utility in predicting response to anti-EGFR agents and MEK inhibitors.

Suppression of interleukin-12 production through endogenously secreted interleukin-10 in activated dendritic cells: involvement of activation of extracellular signal-regulated protein kinase.

Our recent study suggested the reverse relationship between the production of interleukin-10 (IL-10) and IL-12 in dendritic cells (DCs) activated by lipopolysaccharide (LPS) or LPS plus interferon (IFN)-gamma. In the present study, a series of experiments were performed to investigate the mechanisms responsible for this reverse relationship. Our results showed that neutralization of the secreted IL-10 by antibody could enhance the production of IL-12. Neutralization of IL-12 by antibody did not affect the IL-10 production. Addition of exogenous IL-10 suppressed the production of IL-12 by activated DCs, and addition of exogenous IL-12 did not affect IL-10 production. TaqMan real-time reverse transcriptase-polymerase chain reaction supported the fact that the observed effects occurred at mRNA transcription level. We also found that LPS or LPS plus IFN-gamma significantly enhanced the phosphorylation of extracellular signal-regulated protein kinase (ERK) and p38 mitogen-activated protein kinase. In addition, inhibition of ERK by PD98059 significantly suppressed IL-10 and increased the IL-12 production. Exogenous IL-10 reversed the upregulated production of IL-12 induced by PD98059. The above findings suggest a unidirectional negative autocrine regulation of IL-12 by IL-10 in activated DCs and that activation of ERK involves the differential production of IL-10 and IL-12 by activated DCs. Thus, the regulation of differential production of IL-10 and IL-12 may play an important role for DCs in priming T helper 1 (Th1) or Th2 in the immune responses.

Monocyte-derived CD1a+ dendritic cells generated in two different culture systems: immunophenotypic and functional comparison.

Previous studies demonstrated that CD1a+ dendritic cells (DCs) could not be prepared ex vivo without using fetal calf serum (FCS). Recently, we developed a method of using heparin to induce differentiation of human monocytes into CD1a+ DCs without using FCS. In order to determine the potential clinical applicability of heparin-induced CD1a+ DCs, we conducted this study to compare both types of CD1a+ DCs, immunophenotypically and functionally. Our results showed that the expression of CD1a on heparin-DCs was lower than that on FCS-DCs. Both types of DCs expressed similar levels of CD11c, HLA-DR, CD40, CD83, CD80 and CD86 before and after lipopolysaccharide stimulation. Immature heparin-DCs and FCS-DCs had similar phagocytic activities. Heparin-DCs consistently secreted higher interleukin-10 (IL-10) and lesser IL-12 than FCS-DCs after activation. Mature heparin-DCs were slightly more active than mature FCS-DCs in stimulating the proliferation of allogeneic CD4+ T cells. Both types of mature CD1a+ DCs primed the naïve CD4+ T cells to produce large amount of interferon-gamma (IFN-gamma). However, naïve CD4+ T cells stimulated with FCS-DCs produced more IFN-gamma, while the naïve CD4+ T cells stimulated with heparin-DCs produced more IL-5. The results indicate that both types of CD1a+ DCs do not have identical function in the priming of CD4+ T cells and have minor difference in immunophenotypes.

A prospective randomized trial of prophylactic platelet transfusion and bleeding incidence in hematopoietic stem cell transplant recipients: 10,000/L versus 20,000/microL trigger.

An optimal platelet-count threshold for prophylactic platelet transfusion in hematopoietic stem cell transplant (HSCT) recipients has yet to be determined. Between July 1997 and December 1999, we performed the first prospective randomized clinical trial addressing this issue in 159 HSCT recipients who received a prophylactic platelet transfusion when the morning platelet count fell below a 10,000/microL (10K) or 20,000/microL (20K) threshold. Subsequent prophylactic transfusions were administered according to a predetermined algorithm. The number of prophylactic and therapeutic transfusions and the incidence of minor and major bleeding were compared between the 2 groups. The groups were matched according to patient and transplantation characteristics. There were no significant differences in bleeding incidence or severity. Fourteen percent of patients in the 10K arm compared to 17% in the 20K arm had major bleeding events. Only 3 central nervous system bleeds occurred, 2 in the 10K group and 1 in the 20K group. No deaths were attributed to bleeding. An average of 11.4 days of bleeding occurred in both groups. An average of 10.4 platelet transfusions per patient were administered in the 10K group compared to 10.2 in the 20K group (P = .94). More transfusions were given above the assigned transfusion threshold in the 10K group than in the 20K group (4.3/patient versus 1.9/patient, respectively, P = .05). Safety measures incorporated into our study may have precluded demonstration of significant differences in platelet use between the groups. In conclusion, a platelet transfusion trigger of 10K was found to be safe; however, a decrease in platelet use was not achieved because of safety measures incorporated into our study design.

Obtaining blood samples for anti-factor Xa quantification through umbilical artery catheters.

OBJECTIVE: Neonates who are treated with low-molecular-weight heparin require repeated venipunctures to monitor anti-factor Xa (anti-FXa) levels. Generally, blood withdrawn from umbilical artery catheters (UACs) is not useful for such monitoring because heparin in the fluids running through the catheters contaminates the results. We tested methods for collecting blood for anti-FXa levels from UACs through which heparin-containing fluids were running, in an attempt to reduce the need for repeated venipunctures of anticoagulated neonates. STUDY DESIGN AND METHODS: A new blood drawing port, through which no heparin was run, was added to the UAC system. Qualifying neonates with UACs were randomized to have anti-FXa levels drawn after line-clearing volumes of 0.5, 3.0, or 4.0 ml. RESULTS: Twelve patients with UACs were enrolled and all completed the study with no adverse events. When 0.5, 3.0, or 4.0 ml of blood was cleared from the UAC before withdrawing the test sample, heparin contaminated the test (anti-FXa levels > or = 0.1 U/ml) in 66%, 16%, and 8% of samples, respectively. Differences between the 0.5- vs. 3.0-ml line-clearing volumes and between the 0.5- vs. 4.0-ml clearing volumes were significant (p = 0.0026 and 0.0006, respectively). CONCLUSION: Blood samples for anti-FXa can be drawn from UACs through which heparin-containing solutions are infusing if a port is added through which no heparin-containing fluids are run, a line-clearing volume of at least 4.0 ml is drawn, and a contamination rate of 8% of samples is acceptable.

The autoimmune regulator (AIRE) is a DNA-binding protein.

The autoimmune regulator (AIRE) protein is a putative transcription regulator with two plant homeodomain-type zinc fingers, a putative DNA-binding domain (SAND), and four nuclear receptor binding LXXLL motifs. We have shown here that in vitro, recombinant AIRE can form homodimers and homotetramers that were also detected in thymic protein extracts. Recombinant AIRE also oligomerizes spontaneously upon phosphorylation by cAMP dependent protein kinase A or protein kinase C. Similarly, thymic AIRE protein is phosphorylated at the tyrosine and serine/threonine residues. AIRE dimers and tetramers, but not the monomers, can bind to G-doublets with the ATTGGTTA motif and the TTATTA-box. Competition assays revealed that sequences with one TTATTA motif and two tandem repeats of ATTGGTTA had the highest binding affinity. These findings demonstrate that AIRE is an important DNA binding molecule involved in immune regulation.

Characterization of immunologic tolerance induced by transfusion of UV-B--irradiated allogeneic mononuclear leukocytes.

Transfusions of UV-B--irradiated peripheral blood mononuclear cells (UV-B--PBMCs) from BALB/c (H-2(d)) mice into CBA (H-2(k)) mice can induce humoral immune tolerance to H-2(d) antigens, and the induced tolerance is partially mediated by negative regulatory PBMCs. To further identify which subset of spleen mononuclear leukocytes (MNLs) in the tolerant CBA mice is responsible for the negative regulatory activity, adoptive transfer experiments were conducted using spleen MNLs from the tolerant CBA mice. Results showed that only CD4(+) T cells could transfer the negative regulatory activity in a dose-dependent manner. This negative regulatory activity was significantly reduced when CD25(+) helper T cells were removed. Further study suggested that inhibition of IL-12 production by UV-B--irradiated PBMCs played a role in the induction of immune tolerance. In vitro study of the cytokine production profile by CBA CD4(+) T cells, after stimulation with gamma-irradiated BALB/c spleen cells, revealed an enhanced production of the type 2 T-cell cytokines after tolerance induction. Induction of tolerance also prevented the development of cytotoxic T cells in CBA mice against BALB/c MNLs. Adoptive transfer study suggested that the cellular immune tolerance was also mediated by CD4(+) negative regulatory T cells. The induced immune tolerance was nullified after 400 cGy sublethal gamma irradiation. These results suggest that the ex vivo study of cytokine production by T cells may be used to monitor tolerance induction and the selection of gamma radiation dose is critical for potential clinical application of the tolerance induced by UV-B--PBMCs. (Blood. 2001;98:1239-1245)

Structural studies of a neuropeptide precursor protein with an RGD proteolytic site.

The snail Lymnaea stagnalis produces a neuropeptide precursor protein that contains seven Arg-Gly-Asp (RGD) sites. These sites are recognized and cleaved by one or more prohormone convertases in the first processing step to yield mature neuropeptides in the secretory pathway. Conformations of two synthetic RGD-containing peptides derived from the L. stagnalis precursor protein were determined by NMR spectroscopy. The peptides were tested in a platelet aggregation assay for RGD activity and were processed in vitro by PC2 and furin. The native peptide with a proline following the RGD site has minimal structure around the RGD region, does not inhibit platelet aggregation, and is properly processed by the enzymes PC2 and furin. A variant of the native fragment with a serine following the RGD sequence has a significant amount of a reverse turn around the RGD region, is a potent inhibitor of platelet aggregation, and is processed with the same specificity as the native fragment. The large conformational differences between the two peptides provide a molecular mechanism for effects of proline residues following the RGD site and suggest that precursor processing is influenced more by flexibility than by the conformation of the processing site.

Platelet transfusions in the neonatal intensive care unit:factors predicting which patients will require multiple transfusions.

BACKGROUND: Previous studies suggest that recombinant thrombopoietin (rTPO) will increase platelet production in thrombocytopenic neonates. However, the target populations of neonates most likely to benefit should be defined. Studies suggest that rTPO will not elevate the platelet count until 5 days after the start of treatment. Therefore, the neonates who might benefit from rTPO are those who will require multiple platelet transfusions for more than 5 days. This study was designed to find means of prospectively identifying these patients. STUDY DESIGN AND METHODS: A historic cohort study of all patients in the neonatal intensive care unit (NICU) at the University of Florida who received platelet transfusions from January 1, 1997, through December 31, 1998, was conducted. RESULTS: Of the 1389 patients admitted to the NICU during the study period, 131 (9.4%) received platelet transfusions. Seventeen were treated with extracorporeal membrane oxygenation and were excluded from further analysis. Of the remaining 114 patients, 55 (48%) received one transfusion and 59 (52%) received more than one transfusion (21 had >4). None of the demographic factors examined predicted multiple platelet transfusions. However, two clinical conditions did; liver disease and renal insufficiency. Neonates who received one platelet transfusion had a relative risk of death 10.4 times that in neonates who received none (p = 0.0001). Neonates who received >4 platelet transfusions had a risk of death 29.9 times that in those who received no transfusions (p = 0.0001). CONCLUSION: NICU patients with liver disease or renal insufficiency who receive one platelet transfusion are likely to receive additional transfusions. Therefore, these patients constitute a possible study population for a Phase I/II rTPO trial.

Mechanisms for genetically predetermined differential quantitative expression of HLA-A and -B antigens.

Previous studies showed that different HLA-A and -B antigens are differentially expressed in cells. Their relative quantities are genetically predetermined and inherited according to Mendelian laws. To investigate mechanisms responsible for this differential expression, a correlation study between the relative quantities of different HLA-A and -B proteins and their mRNA levels in eight different HLA-phenotyped lymphoblastoid cell lines (LCLs) were performed. The results show proportional correlation in all the studied cell lines except those that are positive for HLA-A24. Study of the turnover of HLA antigens reveals that different HLA-A and -B antigens are proportionally degraded. Measurement of the relative quantities of HLA-A and -B mRNAs in six LCLs before and after treatment with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), an inhibitor of RNA polymerase II, demonstrates that HLA-A and -B mRNAs are proportionally degraded except slight differences in two LCLs. Measurement of the relative quantities of different HLA-A and -B pre-mRNAs in nuclei shows that they are not proportional to the relative quantities of their respective mature mRNAs in cytoplasm in four of six LCLs. These results indicate that combinations of different regulatory steps which include gene transcription, pre-mRNA splicing and mRNA degradation are involved in the genetically predetermined quantitative differential expression of HLA-A and -B antigens. Transcription of HLA genes and splicing of HLA pre-mRNAs appear to be the dominant regulatory steps.

Management of a patient with a mechanical aortic valve and antibodies to both thrombin and factor V after repeat exposure to fibrin sealant.

We describe a patient who developed a markedly prolonged PT, PTT, and thrombin time 13 days after repeat exposure to fibrin sealant during coronary artery bypass grafting and aortic valve replacement. Evaluation revealed an inhibitor to bovine thrombin that cross-reacted with human thrombin. In addition an inhibitor to human coagulation factor V was identified. Despite coagulation abnormalities there was no evidence of bleeding. Nevertheless, effective anticoagulation was required to minimize the thrombotic complications associated with the patient's prosthetic valve. We elected to take a conservative approach and not utilize pharmacologic anticoagulation until there was diminution in the effect of the acquired inhibitors. We report on our patient's course and review the available literature addressing the management of patients demonstrating inhibitors to blood coagulation factors after repeat exposure to fibrin sealants.

Measurement of tissue factor activity in whole blood.

High circulating levels of the procoagulant molecule tissue factor (TF) are associated with thrombosis in a variety of diseases including unstable angina, cancer, and sepsis. Currently, there are no clinical assays to measure the level of TF activity in whole blood. We present an assay called Tissue Factor Clotting Time ("TiFaCT") that detects fibrin formation in human blood. The mean baseline clotting time in a healthy population was 472 +/- 94 s (mean +/- SD, n = 150). Bacterial lipopolysaccharide (LPS or endotoxin) shortened the clotting time in a time-dependent manner. Inhibitory anti-TF antibodies prolonged the clotting time of LPS-stimulated blood, indicating that the shortened clotting time was due to induction of TF expression. Patients with unstable angina had shortened mean baseline clotting time (284 +/- 86, n = 13) compared with healthy volunteers (474 +/- 98, n = 30), suggesting that these patients had elevated levels of circulating TF. The TiFaCT assay should prove clinically useful in quantifying the levels of circulating TF in patients at risk of thrombosis.

Prevention of graft-versus-host disease by induction of immune tolerance with ultraviolet B-irradiated leukocytes in H-2 disparate bone marrow donor.

Transfusions (Tx) of Ultraviolet B (UVB)-irradiated peripheral blood mononuclear leukocytes (MNL) have been shown to induce humoral immune tolerance to major histocompatability complex (MHC) antigens (Blood 88:4375, 1996). To determine whether cellular immune tolerance to MHC antigens can be induced by the same approach, transplantation of bone marrow and spleen cells from tolerant donors across the H-2 barrier was conducted to study its effect on prevention of graft-versus-host disease (GVHD). After immune tolerance induction by four weekly Tx of UVB-irradiated BALB/c (H-2(d)) peripheral blood MNL into CBA/HT6 (H-2(k)) mice, bone marrow cells (BMC) and spleen MNL from tolerant or naive CBA mice were transplanted into lethally irradiated BALB/c mice. The transplanted mice were followed by measuring body weight, peripheral leukocyte counts, GVHD, survival, and cytokine response. All BALB/c recipient mice were fully engrafted with H-2(k) CBA donor cells after transplantation. The severity of GVHD was significantly attenuated in BALB/c mice transplanted with BMC and spleen MNL from tolerant CBA donor mice. The recovery of peripheral leukocyte and lymphocyte counts were faster and more complete in mice transplanted with cells from the tolerant donors. The serum cytokine profile after transplantation with tolerant donor cells showed increased interleukin-4 and reduced gamma interferon that are consistent with a polarized Th2 response. The results pooled from three separate experiments showed that BALB/c mice transplanted with 5 x 10(6) BMC and 4 x 10(5) spleen MNL from tolerant CBA donors had better overall survival than the control group (72% v 17%, P =.018). The findings show that transplantation with bone marrow and spleen cells from tolerant H-2 disparate donor mice is associated with significant attenuation of GVHD and better outcomes. The results also support that transfusions of UVB-irradiated leukocytes may induce cellular immune tolerance.

The Collaborative Hospital Transfusion Study: variations in use of autologous blood account for hospital differences in red cell use during primary hip and knee surgery.

BACKGROUND: Red cell use in patients undergoing Diagnosis Related Group (DRG) 209 procedures (major joint and limb reconstruction procedures of the lower extremities) has been shown to have large, unexplained interhospital variations. STUDY DESIGN AND METHODS: Abstracted records of 2590 consecutive DRG 209 patients at five university hospitals from January 1992 to December 1993 were stratified by procedure and preoperative blood deposit status. Patient characteristics and transfusion and in-hospital outcomes were compared across hospitals. RESULTS: Blood use among patients who did not preoperatively deposit blood was similar across hospitals. Significant differences were found across hospitals for total hip replacement patients in the percentage of patients preoperatively depositing blood (59-80%), percentage of patients receiving transfusion(s) (51 to > 99%), the mean number of units collected per patient (1.6-2.9), and the mean number of unused autologous units per 100 patients (1-185). No significant differences were found in the percentage of those who deposited blood and then required allogeneic units. There was little variability in length of hospital stay or in last hematocrits. Findings were similar for total knee replacement patients. CONCLUSIONS: Interhospital variations in red cell use for primary total hip and knee reconstruction are primarily due to hospital-specific differences in autologous blood collection and transfusion.

Role of class-II major histocompatibility complex (MHC)-antigen-positive donor leukocytes in transfusion-induced alloimmunization to donor class-I MHC antigens.

It has been shown that peripheral-blood mononuclear leukocytes (MNL) are responsible for transfusion-induced alloimmunization to donor major histocompatability complex (MHC) antigens. However, it is not known which subset of MNL is responsible for this immune response. Because elimination of class-II MHC antigen-positive passenger leukocytes effectively prolongs the survival of allografts, it has been hypothesized that class-II positive MNL are responsible for immunizing transfusion recipients to donor MHC antigens. To test this hypothesis, two different approaches were used. First, we compared the alloantigenicity of BALB/c mice (H-2(d)) peripheral blood MNL before and after depletion of class-II positive cells. CBA mice (H-2(k)) were used as transfusion recipients. Antibody development to donor class-I H-2 antigens was determined by flow cytometry and enzyme-linked immunoassay. After four weekly transfusions of MNL depleted for class-II positive cells, only 25% of recipient mice developed antibodies to donor H-2(d) antigens. In contrast, all mice transfused with control MNL became immunized. Second, we studied the alloantigenicity of peripheral MNL from C57BL/6 mice (H-2(b)) with homozygous deficiency of class-II MHC molecules in H-2 disparate recipient mice. After transfusions with class-II MHC molecule-deficient MNL, 0% of BALB/c, 40% of C57BR, and 25% of CBA-recipient mice developed antibodies to donor H-2(b) antigen. All control recipient mice were immunized. The antibody activities of the controls were also higher than those in the treatment group who became immunized. Thus, our study shows that class-II MHC antigen-positive MNL play a significant role in transfusion-induced alloimmunization to donor class-I MHC antigens. The results also support the hypothesis that direct antigen presentation by donor class-II positive MNL to the immune system of transfusion recipients is critical for the initiation of humoral immune response to donor MHC antigens.

Neonatal alloimmune thrombocytopenia associated with maternal anti-HLA antibody: a case report.

PURPOSE: Although neonatal alloimmune thrombocytopenia (NAIT) due to maternal sensitization to human platelet antigens is well described, the role of maternal anti-human lymphocyte antigen (HLA) antibodies in NAIT is not yet firmly established. PATIENT: A 31-week-old girl born prematurely to a G2POA1 mother was noted to have thrombocytopenia which lasted 18 days without any evidence of infection. MATERIALS AND METHODS: Platelet-associated IgG, anti-platelet antibody, and platelet PL(A1) antigen typing were determined using a commercial solid-phase red cell adherent test. Antibodies to platelet glycoproteins human platelet antigen (HPA) 1 to 5 were determined using a commercial ELISA. Anti-HLA antibodies were assayed using a standard lymphocytotoxicity test. Activities and IgG subclass of anti-HLA antibodies in plasma of the mother and other postpartum mothers were measured using purified HLA antigens in an enzyme linked immunoassay. RESULTS: Both mother and infant were positive for HPA-1 (PL(A1)) antigens. The mother's HLA phenotype was A3, A31, B7, B27. The level of platelet-associated IgG was not increased on maternal platelets; however, increased platelet-associated IgG was detected on the infant's platelets. Antibodies to platelet glycoproteins HPA1 to 5 were not detectable in the maternal plasma. Maternal serum was positive for anti-HLA antibodies, which reacted to 23 of 27 panel cells. The presence of HLA antibodies was confirmed by enzyme-linked immunoassay. Of note, the maternal antibodies reacted positively to the infant's platelets and anti-IgG anti-HLA antibodies were detected in the serum sample from the infant collected at birth. When the activity and IgG subclass of the maternal anti-HLA antibodies were compared with those of other mothers known to have high anti-HLA antibody activity, no differences were noted. CONCLUSION: This report documents a patient with neonatal thrombocytopenia induced by maternal IgG anti-HLA antibody. Neither activity nor IgG subclass could explain the occurrence of NAIT. The factors that contribute to NAIT induced by maternal anti-HLA antibodies remain to be identified.

The specific hospital significantly affects red cell and component transfusion practice in coronary artery bypass graft surgery: a study of five hospitals.

BACKGROUND: Interhospital differences in blood transfusion practice during coronary artery bypass graft (CABG) surgery have been noted, but the underlying issues have not been identified. STUDY DESIGN AND METHODS: Records of 3217 consecutive CABG cases in five university teaching hospitals in 1992 and 1993 were stratified by hospital, type of revascularization conduit, patients' sex, and other factors. Statistical methods were used to compare patient characteristics, transfusion outcomes, and hospital outcomes. RESULTS: Forward two-step logistic regression using patient likelihood of red cell transfusion factors in the first step and the specific hospital in the second step revealed a significant effect of hospital on the delta odds ratios for red cell transfusion. This finding was confirmed by analyses of a highly stratified subset of cases, males in diagnosis-related group 107 (primary cases of coronary bypass without coronary catheterization) who underwent revascularization with venous and internal mammary artery grafts, revealing variations among hospitals from 109 to 457 units of red cells transfused per hundred cases. Corresponding variations in transfusions of all blood components were from 324 to 1019 units by hospital. Variation in red cell transfusion practice among surgeons in the same hospital was not responsible for these interhospital differences. CONCLUSION: The effect of the specific hospital on transfusion practice is attributed to institutional differences that, through reasons of training or hierarchy, become ingrained in hospitals.

In vitro-generation of islets in long-term cultures of pluripotent stem cells from adult mouse pancreas.

Pancreatic islets of Langerhans exhibit an architecture and cellular organization ideal for rapid, yet finely controlled, responses to changes in blood glucose levels. In type I, insulin-dependent diabetes (IDD), this organization is lost as a result of the progressive autoimmune response which selectively destroys the insulin-producing pancreatic beta cells. Since beta cells are perceived as end-stage differentiated cells having limited capacity for regeneration in situ, individuals with IDD resulting from beta cell loss or dysfunction require life-long insulin therapy. Efforts to produce islet neogenesis or initiate islet growth in vitro from either fetal or adult tissue have had minimal success. We now report that pancreatic-derived, pluripotent islet-producing stem cells (IPSCs), isolated from prediabetic mice, can be grown in long-term cultures and differentiated into immature functional islet-like structures containing cells which express low levels of insulin, glucagon and/or somatostatin. When such in vitro grown islets were implanted into clinically diabetic NOD mice, the implanted mice were successfully weaned from insulin long-term (>50 days) without ill effects. The implanted mice maintained blood glucose levels just above euglycemic (180-220 mg/dl) and showed no signs of disease. Thus, this technical breakthrough provides new therapeutic approaches to diabetes as an alternative to insulin therapy.

Measurement of relative quantities of different HLA-A and -B mRNAs in cells by reverse transcription-polymerase chain reaction and denaturing gradient gel electrophoresis.

In order to determine the relative quantities of different HLA-A and -B mRNAs in cells, we have developed a simple and reliable method by using reverse transcription-polymerase chain reaction (RT-PCR), denaturing gradient gel electrophoresis (DGGE) and phosphor imaging analysis. Cytoplasmic RNA from lymphoblastoid cell lines with well-characterized HLA phenotypes are reversely transcribed with a primer specific for all HLA-A and -B antigens. The first-strand cDNA is used as template for quantitative PCR. The primer pair used for quantitative PCR are specific for all class I HLA and one of the primers is labeled with [gamma-32P]ATP. The amplified sequences include parts of exon 2 and exon 3 which contain most polymorphic residues in class I HLA molecules. The RT-PCR products containing the amplified HLA-A and -B sequences are separated by DGGE. The radioactivities of different DNA bands separated in denaturing gradient polyacrylamide gels are measured by phosphor imaging and used to determine the relative amounts of HLA-A and -B mRNAs. This approach is validated by using samples containing known quantities of different HLA-A and -B mRNA transcripts and confirmed by S1 nuclease protection assay. The combined RT-PCR/DGGE approach therefore provides a simple and reliable method for quantitation of relative amounts of different HLA-A and -B mRNAs in cells. This method should also be useful for studying the expression of other highly conserved and duplicated genes.