Immunoglobulin E antibodies from pancreatic cancer patients mediate antibody-dependent cell-mediated cytotoxicity against pancreatic cancer cells.

In addition to allergy and parasitic infections, immunoglobulin E (IgE) has been shown recently to possess anti-viral and anti-cancer effects. We investigated serum levels of IgE, its low-affinity receptor, soluble CD23 (sCD23) in patients with pancreatic cancer and the effect of IgE against pancreatic cancer cells. Twelve patients were evaluated for pancreatic cancer by imaging and confirmed by biopsy. Fifteen healthy volunteers served as controls. Serum Igs (IgG, IgM, IgA, IgE) and sCD23 levels were determined (enzyme-linked immunosorbent assay, nephelometry) and the presence of cancer-specific IgE was assessed (fluorescence microscopy, Western blot). IgE anti-cancer activity was determined by antibody-dependent cell-mediated cytotoxicity (ADCC). Serum levels of IgE and sCD23 were elevated significantly in patients with pancreatic cancer versus controls, whereas no differences were observed in other Ig isotypes (IgG, IgM, IgA). Flow cytometry and immunofluorescence microscopy demonstrated similar presence of IgG and IgE pancreatic cancer Igs. However, Western blot analysis indicated differences in IgG and IgE antigen-specific antibodies; IgE antibody recognized a 50 kD protein. ADCC studies demonstrated that serum and purified IgE-mediated cytotoxicity against pancreatic cancer cells, effects which were reversed with anti-IgE neutralizing antibody and IgE depletion (immunoaffinity); greater cytotoxicity was observed in patient serum when compared with healthy controls. These data suggest that IgE and sCD23 may serve as useful biomarkers for patients with pancreatic cancer and may be important in the immune response to this disease in that IgE-directed therapy may help to direct treatment.

B-cell receptor and Fas-mediated signals for life and death.

A series of B-cell lymphoma lines with an immature phenotype has been used as a model system to study molecular events associated with receptor ligation induced death. B-cell receptor (BCR) cross-linking with antibodies to membrane IgM (but not with anti IgD) induces c-Myc downregulation via nuclear factor kappaB inactivation and p27(Kip1) accumulation in these B lymphomas. Anti-mu-treated cells then undergo G1 arrest and die by apoptosis independent of Fas. Steroids and retinoids similarly downregulate c-Myc and induce apoptosis in these B cells and synergize with anti-mu. Rescue from apoptosis induced by anti-mu or steroids occurs with T-cell signals, like CD40L, or a broad-range caspase inhibitor, but only CD40L prevents the loss of c-Myc, p27 accumulation and growth arrest. Both IgM and IgD signaling lead to modulation of phosphatidylinositol 3-kinase (PI3K) signals, including the activation of p70(S6K), but this pathway recovers under anti-IgD treatment. Blockade of the PI3K pathway augments anti-mu-induced death and converts anti-delta to an apoptotic signal. Resistance to Fas-mediated death may be an important factor in B-cell transformation in vivo. Many of our panel of lymphomas are insensitive to Fas-mediated death signals, although all can form a death-inducing signaling complex (DISC). Additional studies suggest that some lymphomas can be blocked at the DISC complex by anti-apoptotic proteins, whereas others are inhibited downstream of caspase 8 activation. Anti-Ig treatment of a Fas-sensitive line, A20.2J, activated a number of genes whose products may block apoptosis proximally (like FLICE-inhibitory protein (FLIP1)) or at late points, such as bcl-2-family members. Our data suggest that B lymphomas develop multiple pathways of resistance to Fas-mediated signals during lymphomagenesis, in part via signaling through the BCR.

Distinct molecular mechanisms of Fas resistance in murine B lymphoma cells.

A panel of murine B lymphoma cell lines, which express different levels of Fas, was extensively studied for sensitivity to Fas-mediated death signals via an anti-Fas mAb and Fas ligand-bearing cell lines. Expression of the Fas receptor on the B lymphoma cell lines did not correlate with their capacity to undergo Fas-mediated apoptosis. Moreover, Fas-associated death domain protein recruitment to the death-inducing signaling complex (DISC) complex occurred in all cell lines expressing Fas, regardless of whether they were sensitive to Fas-mediated death. Interestingly, the protein synthesis inhibitor, cycloheximide, and protein kinase C inhibitors, such as bisindolylmaleimide, rendered one of the resistant cell lines, CH33, sensitive to signals from the Fas receptor, although the levels of Fas were unchanged. This suggests that constitutive PKC activation plays a role in Fas resistance, perhaps by up-regulating NF-kappaB or Bcl-2 family members. Interestingly, CH33 demonstrated caspase 8 activity upon engagement of the Fas receptor in the absence of pharmacological manipulation, suggesting that the block in apoptosis is downstream of the DISC complex. In contrast, the fact that Fas-associated death domain protein was recruited to the DISC complex in other resistant lines, such as WEHI-231, with no caspase 8 activation indicates that these cells may be blocked within the DISC complex. Indeed, Western blot analysis showed that WEHI-231 expressed an isoform of FLICE-like inhibitory protein (cFLIPL), an antiapoptotic protein within the DISC. These studies provide evidence that murine B lymphoma cells utilize different molecular mechanisms along the Fas-signaling cascade to block apoptosis.

Steroid- and retinoid-mediated growth arrest and apoptosis in WEHI-231 cells: role of NF-kappaB, c-Myc and CKI p27(Kip1).

IgM cross-linking induces NF-kappaB inactivation, c-Myc down-regulation, and cyclin kinase inhibitor p27(Kip1) accumulation in WEHI-231 murine B lymphoma cells. p27(Kip1) up-regulation leads to a decreased cyclin-dependent kinase 2 activity, retinoblastoma protein hypophosphorylation, G1 arrest and apoptosis. Similar to membrane (m) IgM cross-linking in B lymphoma cells, steroids and retinoids down-regulate c-Myc (via NF-kappaB inactivation) and induce apoptosis in T cell hybridomas and thymocytes. In this study, we determined if steroids and retinoids have similar effects in WEHI-231 cells. Our results show that steroids and retinoids induce NF-kappaB inactivation, c-Myc down-regulation, p27(Kip1) up-regulation, G1 arrest, and apoptosis. Importantly, these hormones enhance anti-IgM-induced apoptosis in WEHI-231 cells. Similar to mIgM signaling, all these effects are prevented by treatment with CD40 ligand. Caspase inhibition, on the other hand, rescues cells from steroid/retinoid-induced apoptosis, but has no effect on growth arrest, p27(Kip1), and c-Myc. Together, these findings suggest that steroids/retinoids and mIgM cross-linking share a common signal transduction pathway leading to G1 arrest and cell death.

Life and death decisions in B1 lymphoma cells.

Crosslinking of surface immunoglobulin (Ig) receptors with anti-IgM (anti-mu) but not anti-IgD (anti-delta) antibodies causes growth arrest and apoptosis in several extensively characterized B1-like lymphoma cell lines. While anti-mu stimulates a transient increase in c-myc mRNA and protein expression, followed by a rapid decline below the baseline level, anti-delta only causes a moderate increase in the expression of this oncogene, which returns to baseline levels within 24-48 hours. However, signals downstream from anti-delta can be converted into an apoptotic pathway by modulating PI3K activity, suggesting that PI3K is a critical rheostat controlling survival signals in B1 cell lines. Anti-mu-induced down-regulation of c-Myc is followed in time with an increase in the cyclin dependent kinase inhibitor, p27Kip1, in all anti-mu sensitive lymphoma lines. This increase correlates with growth arrest and apoptosis. The anti-mu-mediated decrease in c-Myc, increase in p27Kip1, growth arrest and apoptosis, can all be prevented via CD40/CD40L signaling. Inhibition of caspase activation, on the other hand, prevents anti-mu-induced apoptosis, but has no effect on c-Myc, p27Kip1, and G1 arrest. Interestingly, we also found that steroids and retinoids can mimic anti-mu-mediated signaling and lead to a loss of c-Myc, an increase in p27Kip1, G1 arrest, and apoptosis. Together, these data suggest that modulation of c-Myc and p27Kip1 protein levels is crucial for the life versus death decisions in murine immature B1-like lymphoma cells lines.

Kinetic and genetic bases for the heteroclitic recognition of mouse cytochrome c by mouse anti-pigeon cytochrome c monoclonal antibodies.

The B lymphocyte response to pigeon cytochrome c (CYT) in BALB/c mice was previously shown to initiate as a heteroclitic response specific for the self antigen mouse CYT. As the immune response progressed, the mAb that were produced became less heteroclitic and often bound pigeon CYT with higher affinity than they bound mouse CYT [Minnerath, J. et al., 1995. Proc. Natl. Acad. Sci. USA 92, 12379-12383]. To study the basis for heterocliticity and its loss in this system, the H and L chain amino acid sequences of anti-pigeon CYT mAb obtained from the primary and secondary Ab responses were first compared. The most frequent somatic mutations and Ig gene joints were then introduced into an engineered single-chain Fv (scFv) that expressed the germline-encoded V(H) and V(L) amino acid sequences. The effects of those changes on the on- rate, off-rate, and affinity constants in binding both mouse and pigeon CYT were determined by surface plasmon resonance. In this system, the heterocliticity of primary mAb was largely due to a decreased on-rate constant for binding pigeon CYT relative to mouse CYT. Various combinations of the three frequently occurring H chain somatic mutations (H31, H56, and H58) increased the on-rate constant to maximal levels. Only one of the mutations (H58) decreased the off-rate constant when in combination with the other mutations and the effect of H58 was greater for scFv binding mouse CYT than pigeon CYT. Consequently, the mutated scFv and many secondary mAb remained heteroclitic, although their affinities for pigeon CYT increased. Secondary mAb that were no longer heteroclitic expressed non-canonical amino acid sequences in the V(H)-D-J(H) joint in the context of the canonical V genes or expressed different V genes. In addition to providing insight into the molecular basis for heterocliticity, our findings confirm that both faster on-rate and slower off-rate constants are favored during affinity maturation of the Ab response.

Spontaneous rupture of the urinary bladder in the alcoholic patient.

A case of a young man with an acute abdominal condition and hematuria is presented. BUN and SCr levels were markedly elevated. Retrograde cystography revealed intraperitoneal extravasation of contrast material. At exploration, a large intraperitoneal bladder perforation was noted and repaired in two layers. Recovery was uneventful. The presentation, diagnosis, and treatment of spontaneous rupture of the urinary bladder are discussed.

Praise for intelligence can undermine children's motivation and performance.

Praise for ability is commonly considered to have beneficial effects on motivation. Contrary to this popular belief, six studies demonstrated that praise for intelligence had more negative consequences for students' achievement motivation than praise for effort. Fifth graders praised for intelligence were found to care more about performance goals relative to learning goals than children praised for effort. After failure, they also displayed less task persistence, less task enjoyment, more low-ability attributions, and worse task performance than children praised for effort. Finally, children praised for intelligence described it as a fixed trait more than children praised for hard work, who believed it to be subject to improvement. These findings have important implications for how achievement is best encouraged, as well as for more theoretical issues, such as the potential cost of performance goals and the socialization of contingent self-worth.

B lymphocyte recognition of the self antigen mouse cytochrome C in different mouse strains: targeting of the same dominant epitope by naturally-occurring cells expressing distinct VH genes.

Previously we reported that, early in the antibody response of BALB/c mice to several cytochromes c (CYT) coupled to ovalbumin (OVA), B cells responding to the self antigen mouse CYT recognized a single site on mouse CYT and were in much higher frequency than B cells responding to foreign CYT. In the present study these B cells were shown by in vitro activation of primary splenocytes to be present in naive BALB/c mice, i.e. prior to exposure to exogenous CYT. The higher frequency of B cells responsive to self versus foreign CYT was also shown in this study to occur in the early antibody response to CYT-OVA in C57BL/6 mice. The same dominant site was recognized in BALB/c mice (IgHa), C57BL/6 mice (IgHb) and the congenic strains BC-17 (IgHa on the C57BL/6 background) and CB-20 (IgHb on the BALB/c background). However, anti-mouse CYT mAbs produced in IgHb mice were shown to derive from the VH gene 5.54.4 while mAbs in IgHa mice derive from the VH gene 19.1.2. The polypeptides encoded by these VH genes, which differ by only five amino acid residues, paired with polypeptides encoded by the same Vk genes (R9 and 2G5). In both VH 19.1.2- and VH 5.54.4-derived mAbs H3 and the Vk-Jk join were variable. The affinity for mouse CYT was reduced in the VH gene 5.54.4-derived mAb due to a faster off-rate constant. This difference in affinity may relate to the lower frequency of B cells responding to mouse CYT in C57BL/6 mice than in BALB/c mice. The results show that naturally-occurring CYT-specific autoreactive B cells occur normally in more than one mouse strain and that self antigen recognition by those cells appears to be atypical involving mostly the immunoglobulin V gene-encoded segments.

Nitric oxide inhibition simulates the enhancement of alpha 1 agonist-induced vasoconstriction in diabetes.

We have previously reported that endothelium-dependent, nitric oxide (NO)-mediated vasorelaxation is impaired in diabetic mesenteric arteries. We hypothesized that vasoconstrictor responses should therefore be enhanced. The purpose of this study was to determine whether diabetic mesenteric arteries exhibit increased vasoconstrictor responses, and to investigate if these changes are receptor and/or NO mediated. Thirty age-matched male Sprague-Dawley rats were divided into control (C) and diabetic (D, streptozotocin: 60 mg/kg) groups and studied after 4 weeks. Terminal branches of ileal mesenteric arteries (300 +/- 9 microns) were isolated, pressurized, and superfused with modified Krebs solution. Changes in vessel internal diameter were measured and dose-response curves (DRC) for each vasoactive agent were determined. Each vessel was initially constricted with 40 mM of KC1 to determine maximal vasoconstriction. Phenylephrine (Phe, 10(-8)-10(-4) M) and UK14304 (10(-9)-10(-5) M) were used to determine alpha 1- and alpha 2-receptor responses, respectively. Similar studies were performed in the presence of N omega-nitro-L-arginine methyl ester (L-NAME, 10(-4) M), a competitive inhibitor of NO synthase. Maximal response (Max), area under the curve (AUC), and vessel sensitivity (ED50) for each DRC were calculated. Comparisons among groups were made using analysis of variance and Student's t test with Bonferroni correction. There were no differences in vasoconstrictor responses induced by KCl (C: 82 +/- 2% vs D: 80 +/- 1%). alpha 1-vasoconstrictor responses to Phe were enhanced in diabetes with significantly higher Max (96 +/- 2% vs 83 +/- 3%), and AUC (1.92 +/- 0.09 vs 1.56 +/- 0.08), but no difference in ED50. The addition of L-NAME enhanced only Phe-induced vasoconstrictor response significantly in control rats. Thus, differences in Phe-induced vasoconstrictor responses between C and D were abolished in the presence of L-NAME. alpha 2-vasodilator responses induced by UK14304 were similar between C and D and unaffected by L-NAME. alpha 1-, but not alpha 2-, vasoconstrictor responses are enhanced in streptozotocin-induced diabetic rats. These enhanced responses can be duplicated by treatment of control vessels with L-NAME.

Maturation of the antibody response to the major epitope on the self antigen mouse cytochrome c. Restricted V gene usage, selected mutations, and increased affinity.

Most B lymphocytes recognizing the major epitope on the self Ag mouse cytochrome c (CYT) express one particular VH gene (19.1.2) in combination with one of two Vkappa genes (R9 or 2G5). This restriction made it possible to observe changes in the primary structure of mouse CYT-specific mAb as the B cell response to mouse CYT (coupled to OVA) progressed. Thus, somatic mutations at positions 31 and 58 in the VH gene complementarity-determining regions (CDR) 1 and 2, respectively, and tyrosine at position 96 in the Vkappa-Jkappa join were frequently selected during the response. The affinity constant (ka), which increased as much as 90-fold due to a decrease in the off-rate constant, correlated with the number of somatic mutations but not strictly with the commonly selected changes. There appears to be little selection for a particular H chain CDR3 in the mouse CYT-specific mAb since it is extremely variable, in sequence and length, throughout the response. This variability may reflect that mouse CYT is a self Ag. Otherwise, the Ab response to this protein Ag is consistent with the paradigm for affinity maturation that is well established for Ab responses to haptens.

Effects of diabetes and uremia on mesenteric vascular reactivity.

BACKGROUND: Diabetes and uremia are comorbid conditions that have significant effects on cardiovascular physiology. These studies were designed to examine the effects of diabetes and uremia on vascular reactivity. METHODS: Sprague-Dawley rats were divided into control (C), diabetic (D), uremic (U), and diabetic/uremic (D + U) groups. Diabetes (D, D + U groups) was induced with an injection of streptozotocin. Uremic (U, D + U groups) was produced by seven-eighths nephrectomy. Serum glucose, blood urea nitrogen, creatinine, creatinine clearance, and protein excretion were measured at baseline and before microvascular studies at 4 or 8 weeks after injection. Vascular reactivity was studied in isolated, pressurized, and superfused segments of mesenteric arterioles (300 microns). Changes in internal vessel diameter were measured in response to phenylephrine (10(-8) to 10(-4) mol/L), acetylcholine (10(-9) to 10(-5) mol/L), and nitroprusside (10(-9) to 10(-2) mol/L). RESULTS: Results at 4 and 8 weeks were similar in all groups. Vasoconstrictor responses to phenylephrine and endothelium-independent vasodilator responses to nitroprusside were not altered in any experimental group. Endothelium-dependent vasodilator responses to acetylcholine were significantly depressed in both diabetic groups (D and D + U, p < 0.01 versus control), and there were no differences between the two diabetic groups. CONCLUSIONS: Streptozotocin-induced diabetes results in impairment of endothelial-dependent (nitric oxide mediated) vasodilator responses in mesenteric resistance vessels, which are unaffected by coexisting uremia. Uremia has little effect on mesenteric vascular reactivity in this model.

Order writing for parenteral nutrition by registered dietitians.

OBJECTIVES: The purpose of the study was to describe the involvement of registered dietitians in parenteral nutrition (PN) orders, to identify characteristics of those who write orders; and to rank education, training, and support elements that dietitians believe facilitate PN order-writing skills. DESIGN: Mail survey method. SUBJECTS: Usable returned questionnaires (n = 266) represented randomly sampled membership of the Dietitians in Nutrition Support (a dietetic practice group of The American Dietetic Association) and registered dietitian membership of the American Society of Parenteral and Enteral Nutrition. STATISTICAL ANALYSIS: Data were analyzed using basic frequency displays on all questionnaire items, Pearson correlations between the quantitative variables, and one-way analysis of variance with each of the categorical variables and the quantitative variables. Detail exploration among variables was done by chi(2) analyses and two-tailed t tests. RESULTS: "Recommend" was the most frequent response to a query about participation in PN orders. An average of 37% of respondents sometimes or always wrote orders for nutrients. Specialists were more likely to write orders than clinicians and managers. No relationship was found between those who wrote orders and educational level, and writers had been registered for a shorter length of time. Agreement on the training and support needed for order-writing skills was nearly unanimous. APPLICATIONS: The PN skills of respondents who were specialists distinguished them from managers and clinicians. Specialists did not fit criteria for advanced practice, and specialty practice in metabolic nutrition care may be too broad to define their practice. Educators must recognize the importance of including PN training in didactic and clinical training programs. Registered dietitians with PN skills will be well positioned to play an active role in the evolving health care system.

Ambulatory monitoring of respiration in anxiety.

An ambulatory monitor, body suit, and calibration procedure were developed to compare the respiration of seven patients with panic disorder and twelve normal volunteers. Subjects wore a body suit with Respitrace bands, connected to a portable respiratory monitor for a period of 24 hours. Breath by breath values for respiratory rate and tidal volume were computed every two minutes. There was a significant difference between patients and controls in their patterns of minute ventilation during sleep. Tidal volume, rather that respiratory rate increases characterized the periods of anxiety and limited symptom attacks.

B lymphocyte recognition of cytochrome c: higher frequency of cells specific for self versus foreign antigen early in the immune response and V gene usage in the response to self antigen.

To study immunoglobulin gene usage in the antibody response of mice to the self antigen (Ag) mouse cytochrome c (cyt), B cell hybridomas were prepared from splenic B cells of immunized BALB/c mice prior to the onset of somatic mutation, i.e. 3 days after injecting ovalbumin (OVA)-primed mice with mouse cyt coupled to OVA. Monoclonal antibodies (mAb) from all of the seven primary hybridomas we obtained were sensitive to a single amino acid substitution from aspartic acid to glutamic acid at position 62 in mouse cyt. This is the specificity of the vast majority of B cells responding to mouse cyt as determined from assays of B cells activated in splenic fragment cultures. Six of the mAb derive from the 19.1.2 J558 VH gene which is also used in the response to alpha (1-->6) dextran and three of these mAb derive from the R9 V kappa gene, a member of the V kappa Ox-1 family. The other mAb derive from distinct, although similar, V kappa genes. Attempts to obtain hybridomas secreting primary (unmutated) mAb specific for cyt foreign to mice have been hampered by the much lower frequency of B cells responding early to foreign cyt in comparison to the self Ag. This suggests that, contrary to expectation of tolerance mechanisms, in naive BALB/c mice B lymphocytes specific for a single epitope on self cyt are present in higher frequency than B lymphocytes specific for similar epitopes on foreign cyt. Possible explanations for this result include biased expression in the B cell repertoire of the particular combination of V genes encoding mouse cyt-specific mAb or to positive selection of developing B lymphocytes by endogenous Ag.

Anticoagulation with prostacyclin and heparin during continuous venovenous hemofiltration.

OBJECTIVES: To investigate anticoagulation with prostacyclin (prostaglandin I2 [PGI2]) and/or heparin during continuous venovenous hemofiltration, and the role of in vitro tests of primary hemostasis in controlling anticoagulation. DESIGN: Prospective, randomized, controlled trial. SETTING: Intensive care unit. PATIENTS: Forty-six consecutive, critically ill, mechanically ventilated patients with postoperative acute renal failure. INTERVENTIONS: Anticoagulation of the patient's blood was accomplished using heparin (6.0 +/- 0.3 IU/kg/hr for group 1), PGI2 (7.7 +/- 0.7 ng/kg/min for group 2), or both PGI2 and heparin (6.4 +/- 0.3 ng/kg/min, 5.0 +/- 0.4 IU/kg/hr, respectively, for group 3), administered into the extracorporeal line before the hemofilter during continuous venovenous hemofiltration. MEASUREMENTS AND MAIN RESULTS: After Ethics Committee approval and informed consent were obtained, tests of primary and secondary hemostasis, plasma concentrations of 6-ketoprostaglandin F1 alpha (by radioimmunoassay), and hemodynamic measurements were performed before hemofiltration and 24 hrs after hemofiltration. In groups 1 and 3, hemodynamic parameters remained stable, whereas in group 2 (the PGI2 group), there were significant reductions in systemic and pulmonary vascular resistances and mean arterial pressure. Platelet function was unchanged in group 1, and was inhibited in groups 2 and 3. Corresponding with the prolongation of in vitro bleeding time, the 6-ketoprostaglandin F1 alpha concentration was increased, indicating an effective inhibition of platelet aggregation within the hemofilter. Platelet counts remained stable in all patients. Plasma coagulation tests were stable in groups 2 and 3, and were prolonged in group 1. In all patients, no major bleeding complications were observed and there was no clinically important bleeding. Mean hemofilter duration lasted longest in group 3. Blood urea nitrogen and circulating creatinine concentrations decreased significantly in groups 2 and 3 within the study period. CONCLUSIONS: Patients receiving both PGI2 and heparin showed better hemodynamic profiles and enhanced hemofilter duration compared with the other groups and no bleeding complications were observed. Therefore, we recommend anticoagulation with PGI2 and heparin during continuous venovenous hemofiltration with close monitoring of platelet function, coagulation profile, and overall hemodynamics.

Cross section parameterizations for cosmic-ray nuclei. II. Double nucleon removal.

Parameterizations of double nucleon removal from the electromagnetic and strong interactions of cosmic rays with nuclei are presented. These parameterizations are an extension of previous single nucleon removal parameterizations and combined they represent the dominant part of the electromagnetic dissociation encountered by a cosmic ray on its traversal through matter. Such parameterizations should be very useful in studying cosmic-ray transport through the interstellar medium, the Earth's atmosphere, spacecraft walls, and extraterrestrial matter.