Primary analysis of a prospective, randomized, single-blinded phase II trial evaluating the HER2 peptide AE37 vaccine in breast cancer patients to prevent recurrence.

BACKGROUND: AE37 is the Ii-Key hybrid of the MHC class II peptide, AE36 (HER2 aa:776-790). Phase I studies showed AE37 administered with granulocyte macrophage colony-stimulating factor (GM-CSF) to be safe and highly immunogenic. A prospective, randomized, multicenter phase II adjuvant trial was conducted to evaluate the vaccine's efficacy. METHODS: Clinically disease-free node-positive and high-risk node-negative breast cancer patients with tumors expressing any degree of HER2 [immunohistochemistry (IHC) 1-3+] were enrolled. Patients were randomized to AE37 + GM-CSF versus GM-CSF alone. Toxicity was monitored. Clinical recurrences were documented and disease-free survival (DFS) analyzed. RESULTS: The trial enrolled 298 patients; 153 received AE37 + GM-CSF and 145 received GM-CSF alone. The groups were well matched for clinicopathologic characteristics. Toxicities have been minimal. At the time of the primary analysis, the recurrence rate in the vaccinated group was 12.4% versus 13.8% in the control group [relative risk reduction 12%, HR 0.885, 95% confidence interval (CI) 0.472-1.659, P = 0.70]. The Kaplan-Meier estimated 5-year DFS rate was 80.8% in vaccinated versus 79.5% in control patients. In planned subset analyses of patients with IHC 1+/2+ HER2-expressing tumors, 5-year DFS was 77.2% in vaccinated patients (n = 76) versus 65.7% in control patients (n = 78) (P = 0.21). In patients with triple-negative breast cancer (HER2 IHC 1+/2+ and hormone receptor negative) DFS was 77.7% in vaccinated patients (n = 25) versus 49.0% in control patients (n = 25) (P = 0.12). CONCLUSION: The overall intention-to-treat analysis demonstrates no benefit to vaccination. However, the results confirm that the vaccine is safe and suggest that vaccination may have clinical benefit in patients with low HER2-expressing tumors, specifically TNBC. Further evaluation in a randomized trial enrolling TNBC patients is warranted.

Final report of the phase I/II clinical trial of the E75 (nelipepimut-S) vaccine with booster inoculations to prevent disease recurrence in high-risk breast cancer patients.

BACKGROUND: E75 (nelipepimut-S) is a human leukocyte antigen (HLA)-A2/A3-restricted immunogenic peptide derived from the HER2 protein. We have conducted phase I/II clinical trials vaccinating breast cancer patients with nelipepimut-S and granulocyte-macrophage colony-stimulating factor (GM-CSF) in the adjuvant setting to prevent disease recurrence. All patients have completed 60 months follow-up, and here, we report the final analyses. PATIENTS AND METHODS: The studies were conducted as dose escalation/schedule optimization trials enrolling node-positive and high-risk node-negative patients with tumors expressing any degree of HER2 (immunohistochemistry 1-3+). HLA-A2/3+ patients were vaccinated; others were followed prospectively as controls. Local and systemic toxicity was monitored. Clinical recurrences were documented, and disease-free survival (DFS) was analyzed by Kaplan-Meier curves; groups were compared using log-rank tests. RESULTS: Of 195 enrolled patients, 187 were assessable: 108 (57.8%) in the vaccinated group (VG) and 79 (42.2%) in the control group (CG). The groups were well matched for clinicopathologic characteristics. Toxicities were minimal. Five-year DFS was 89.7% in the VG versus 80.2% in the CG (P = 0.08). Due to trial design, 65% of patients received less than the optimal vaccine dose. Five-year DFS was 94.6% in optimally dosed patients (P = 0.05 versus the CG) and 87.1% in suboptimally dosed patients. A voluntary booster program was initiated, and among the 21 patients that were optimally boosted, there was only one recurrence (DFS = 95.2%). CONCLUSION: The E75 vaccine is safe and appears to have clinical efficacy. A phase III trial evaluating the optimal dose and including booster inoculations has been initiated. CLINICAL TRIALS: NCT00841399, NCT00584789.

An eleven-vertex metallaborane with tetracapped pentagonal bipyramidal geometry.

A metallaborane of novel structure, [(Cp*Mo)(2)B(3)H(3)Se(2){Fe(CO)(2)}(2){Fe(CO)(3)}(2)] (2; Cp* = η(5)-C(5)Me(5)), with tetracapped pentagonal bipyramidal geometry, isolated from the reaction of [(Cp*Mo)(2)B(4)H(4)Se(2)], 1 with [Fe(2)(CO)(9)]; the title compound exhibit an 11-vertex closo-cage geometry, having eight skeletal electron pairs (sep) and 98 valence electrons, appropriate for its geometric structure.

Monitoring circulating tumor cells in cancer vaccine trials.

The presence of circulating tumor cells (CTC) from various cancers has provided a wealth of information and possibilities. As the role of CTC detection in the treatment assessment of metastatic breast cancer becomes standard, there is interest in applying this tool in cancer vaccine development and clinical trial monitoring. Since we lack a proven immunologic assay that correlates with clinical response, CTC detection, quantification and phenotypic characterization may be a useful surrogate for clinical outcome. The Cancer Vaccine Development Program is involved in the development of HER2/neu peptide based vaccine development for the prevention of recurrence in HER2/neu expressing cancers like breast cancer. The CellSearch System (Veridex, LLC Warren, NJ) has been used by our lab in conjunction with in vivo and/or in vitro immunologic measurements to define a monitoring tool that could predict clinical response. Once validated, this assay could significantly shorten clinical trials and lead to more efficient assessment of potentially promising cancer vaccines.

PSA is a candidate self-antigen in autoimmune chronic prostatitis/chronic pelvic pain syndrome.

BACKGROUND: Previous studies demonstrated that recognition of seminal plasma antigens can occur in patients with chronic prostatitis/chronic pelvic pain syndrome. This suggests that an autoimmune component may contribute to symptoms in some men. To determine if any of the principal secretory proteins of the prostate could be candidate antigens in autoimmune prostatitis, we examined the recall proliferative response of purified CD4 T cells in patients with chronic prostatitis and in normal volunteers using purified seminal plasma antigens and autologous dendritic cells. METHODS: Peripheral blood mononuclear cells were harvested from 14 patients with chronic prostatitis and 12 normal volunteers by density gradient centrifugation. The stimulating cells were irradiated autologous dendritic cells produced by culture of monocyte-enriched fractions with IL-4 and Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF). Purified CD4 T cells were the responding population. Recall proliferation assays were performed, using purified seminal plasma proteins as antigens. RESULTS: In 14 patients with chronic prostatitis, we detected a greater than 2-fold increase in proliferative response to PSA compared to control in 5 patients (36%). No response to Prostatic Acid Phosphatase (PAP) or beta-microseminoprotein was observed in these 14 patients. In 12 normal volunteer donors with no history of genitourinary disease or symptoms, no proliferative response above background was observed for any prostatic antigen. CONCLUSIONS: The data suggest that some men with symptoms of chronic prostatitis have evidence of a proliferative CD4 T-cell response to PSA. PSA is a candidate antigen in chronic prostatitis/chronic pelvic pain syndrome and may be an appropriate target for immunotherapy for prostatic cancer.

B cell development and activation defects resulting in xid-like immunodeficiency in BLNK/SLP-65-deficient mice.

Engagement of the B cell receptor (BCR) leads to the activation of tyrosine kinases and other signaling molecules that ultimately determine the type and magnitude of the B lymphocyte's cellular response. The adaptor protein BLNK/SLP-65 plays a pivotal role in BCR signal transduction by coupling Syk activation to downstream elements such as Grb2, phospholipase C-gamma, Vav and Nck. We have generated BLNK(-/-) mice to determine the physiological role of this protein in B cell development and activation. BLNK(-/-) mice exhibit an incomplete block in B cell development with a severe inhibition of pro-B to pre-B cell differentiation. BLNK(-/-) sIgM(+) cells can develop, seed the peripheral lymphoid tissues and accumulate in numbers overtime. However, these mutant B cells failed to mature and are non-responsive to BCR cross-linking in terms of proliferation and up-regulation of activation markers such as CD69 and CD86 (B7-2). In addition, the CD5(+) subset of B cells is absent. The immune response to T cell-independent antigen but not T cell-dependent antigen is also impaired. Overall, the phenotype of BLNK(-/-) mice bears a striking resemblance to that of xid mice which is the murine model of human XLA that has a mutation in Bruton's tyrosine kinase. This raises the interesting possibility that mutation in BLNK/SLP-65 may be responsible for certain human immunodeficiencies.

Developing dendritic cell polynucleotide vaccination for prostate cancer immunotherapy.

Immunotherapy has been successfully used to treat some human malignancies, principally melanoma and renal cell carcinoma. Genetic-based cancer immunotherapies were proposed which prime T lymphocyte recognition of unique neo-antigens arising from specific mutations. Genetic immunization (polynucleotide vaccination, DNA vaccines) is a process whereby gene therapy methods are used to create vaccines and immunotherapies. Recent findings indicate that genetic immunization works indirectly via a bone marrow derived cell, probably a type of dendritic antigen presenting cell (APC). Direct targeting of genetic vaccines to these cells may provide an efficient method for stimulating cellular and humoral immune responses to infectious agents and tumor antigens. Initial studies have provided monocytic-derived dendritic cell (DC) isolation and culture techniques, simple methods for delivering genes into these cells, and have also uncovered potential obstacles to effective cancer immunotherapy which may restrict the utility of this paradigm to a subset of patients.

Targeted disruption of the tyrosine phosphatase PTPalpha leads to constitutive downregulation of the kinases Src and Fyn.

A role for the receptor-like protein tyrosine phosphatase alpha (PTPalpha) in regulating the kinase activity of Src family members has been proposed because ectopic expression of PTPalpha enhances the dephosphorylation and activation of Src and Fyn [1] [2] [3]. We have generated mice lacking catalytically active PTPalpha to address the question of whether PTPalpha is a physiological activator of Src and Fyn, and to investigate its other potential functions in the context of the whole animal. Mice homozygous for the targeted PTPalpha allele (PTPalpha-/-) and lacking detectable PTPalpha protein exhibited no gross phenotypic defects. The kinase activities of Src and Fyn were significantly reduced in PTPalpha-/- mouse brain and primary embryonic fibroblasts, and this correlated with enhanced phosphorylation of the carboxy-terminal regulatory Tyr527 of Src in PTPalpha-/- mice. Thus, PTPalpha is a physiological positive regulator of the tyrosine kinases Src and Fyn. Increased tyrosine phosphorylation of several unidentified proteins was also apparent in PTPalpha-/- mouse brain lysates. These may be PTPalpha substrates or downstream signaling proteins. Taken together, the results indicate that PTPalpha has a dual function as a positive and negative regulator of tyrosine phosphorylation events, increasing phosphotyrosyl proteins through activation of Src and Fyn, and directly or indirectly removing tyrosine phosphate from other unidentified proteins.

Elevated levels of proinflammatory cytokines in the semen of patients with chronic prostatitis/chronic pelvic pain syndrome.

OBJECTIVES: Chronic prostatitis/chronic pelvic pain syndrome is a common diagnosis, but the disease is poorly understood. The diagnosis is based only on symptoms, and no measurable parameter can help in defining the presence of the disease, its severity, or its cause. Cytokines are soluble proteins secreted by cells of the immune system that principally regulate inflammatory and immune responses. To provide an objective measure of inflammation in the genital tract, we measured levels of the proinflammatory cytokines, tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) in the semen of men with chronic prostatitis/chronic pelvic pain syndrome and compared these with levels in normal men. METHODS: We obtained semen samples from 18 men with chronic prostatitis/chronic pelvic pain syndrome and from 8 normal male volunteers. Cytokine levels were measured in the seminal plasma by two-antibody enzyme-linked immunosorbent assay. RESULTS: Men with prostatitis had higher mean levels of IL-1 beta and TNF-alpha in seminal plasma (mean +/-SEM) than normal men: TNF-alpha 98+/-39 versus 17+/-8; IL-1 beta 246+/-63 versus 27+/-10, respectively; P <0.05. There was a strong correlation between the levels of TNF-alpha and IL-1 beta in the semen of men with chronic prostatitis/chronic pelvic pain syndrome. There was no correlation between either TNF-alpha or IL-1 beta levels and the number of leukocytes per high power field in expressed prostatic secretions in patients. CONCLUSIONS: Some men with chronic prostatitis/chronic pelvic pain syndrome have elevated levels of TNF-alpha and IL-1 beta in the semen. This suggests that inflammation of the genital tract is a feature of this disease, irrespective of the presence or absence of leukocytes in the expressed prostatic secretions. Seminal cytokine levels may provide an objective measure of disease in these patients and suggest specific therapeutic strategies to treat chronic prostatitis/chronic pelvic pain syndrome in such patients.

Cryosurgical ablation of the normal ventral prostate plus adjuvant does not protect Copenhagen rats from Dunning prostatic adenocarcinoma challenge.

PURPOSE: We wished to determine if cryosurgical ablation of the normal ventral prostate of Copenhagen rats confers protective immunity against a subsequent challenge with Dunning R3327 MatLyLu prostatic adenocarcinoma. In human melanoma, tumor antigens have been characterized as normal cellular proteins. We reasoned that cryosurgical ablation of the normal prostate along with immunostimulatory adjuvants might release prostatic antigens to the immune system engendering an immune response and rendering rats immune to prostatic cancer cells. MATERIALS AND METHODS: On day 0, Copenhagen rats underwent cryosurgical ablation of the normal ventral prostate, cryosurgery and intraprostatic injection of Complete Freund's Adjuvant (CFA), CFA injection alone, or laparotomy alone. On day 21, animals received a subcutaneous challenge of MatLyLu tumor cells. Tumor dimensions were recorded at regular intervals by a single blinded investigator. RESULTS: Animals receiving cryosurgical ablation of the normal ventral prostate or intraprostatic CFA developed tumors more frequently than animals receiving laparotomy alone and the effect was statistically significant if animals received both cryosurgical ablation of the prostate and intraprostatic CFA (3 experiments, 1 x 10(4) MatLyLu cells), total number with tumors/total number challenged: laparotomy alone 3/17, cryosurgical ablation 7/17, cryosurgery plus CFA 10/16 (p = 0.013 versus laparotomy, Fisher's exact test), CFA alone 9/17. CONCLUSIONS: Cryosurgical ablation of the normal rat ventral prostate and intraprostatic CFA does not protect against and can enhance the tumorigenicity of MatLyLu prostatic cancer cells at distant sites. This could be occurring through specific immunologic effects or non-specific mechanisms induced by cryosurgery and CFA.

In vitro and in vivo promoter analyses of the mouse phospholamban gene.

To determine the mechanisms responsible for regulation of the phospholamban (PLB) gene expression, a critical regulatory phosphoprotein in cardiac muscle, the mouse PLB gene was isolated and promoter analysis was performed in vitro and in vivo. The PLB gene consists of two exons separated by a single large intron. Deletion analysis revealed that a 7-kb 5' flanking fragment (including exon 1, the entire intron and part of exon 2) was necessary for maximal transcriptional activity in H9c2 and L6 cell lines. Interestingly, deletion of a 2.4-kb intronic region, which contained repetitive elements, caused a dramatic increase in CAT activity in both these cell lines. In vivo analysis indicated that the PLB fusion gene containing 7 kb of the 5'-flanking region was capable of cardiac specific gene expression in transgenic mice. Furthermore, these mice exhibited 3-fold higher levels of CAT activity in the ventricles compared with the atria, mimicking endogenous PLB mRNA expression. Our findings suggest that: (a) PLB gene expression may be regulated by the interplay of cis-acting regulatory elements located within the 5' flanking and intronic regions; and (b) the 7-kb upstream region is capable of directing cardiac-specific and compartment-specific expression in vivo.

Autoimmune prostatitis: evidence of T cell reactivity with normal prostatic proteins.

OBJECTIVES: To determine whether men with chronic prostatitis/chronic pelvic pain syndrome have evidence of an autoimmune response to prostatic proteins. We examined men with a history of chronic prostatitis/ chronic pelvic pain syndrome for evidence of T lymphocyte reactivity to seminal plasma. METHODS: Patients underwent automated leukopheresis to obtain peripheral blood mononuclear cells. We performed a recall antigen proliferation assay to detect specific proliferation of peripheral helper T lymphocytes in men. with chronic prostatitis/chronic pelvic pain syndrome and compared the results with those of normal men. The antigen for these studies consisted of seminal plasma from normal donors and men with seminal vesicle atresia. RESULTS: A specific recall proliferative response to seminal plasma was observed in 3 of 10 men with a history of chronic prostatitis/chronic pelvic pain syndrome compared with none of 15 normal men. The CD4 T cell proliferative response to seminal plasma was statistically significant when compared with medium alone in men with a history of chronic prostatitis/chronic pelvic pain syndrome but it was not statistically significant in normal men. The recall responses of both the chronic prostatitis/chronic pelvic pain syndrome group and normal subjects to the recall antigens tetanus toxoid and Candida extract were equivalent. CONCLUSIONS: The data represent the first direct evidence that some men with chronic prostatitis/chronic pelvic pain syndrome have an autoimmune component to their disease. Autoimmunity is a potential etiology for chronic nonbacterial prostatitis.

Selective response of gamma delta T-cell hybridomas to orthomyxovirus-infected cells.

A gamma delta T-cell hybridoma established from influenza virus-infected mice responded to a reproducible way when cultured with influenza virus-infected stimulators. Subclones of this line responded to cells infected with influenza viruses A/PR/8/34 (H1N1), X-31 (H3N2), and B/HK/8/73 but not to cells infected with vaccinia virus or Sendai virus. This spectrum of response to both type A and type B orthomyxoviruses has never been recognized for the alpha beta T-cell receptor-positive subsets. There was no response to cells infected with a panel of recombinant vaccinia viruses expressing all individual influenza virus proteins, and so it is unlikely that the stimulating antigen is of viral origin. The alternative is that the antigen is a cellular molecule induced in influenza virus-infected cells. Infectious virus was required for stimulation, and immunofluorescence studies showed increased expression of heat shock protein 60 (Hsp60) in influenza virus- but not Sendai virus- or vaccinia virus-infected cells. Both the hybridoma generated from influenza virus-infected mice and an established hybridoma which uses the same gamma delta T-cell receptor combination responded to recombinant Hsp60. Furthermore, the Hsp60-reactive hybridoma, which was obtained from an uninfected mouse, also responded to influenza virus-infected cells, indicating that Hsp60 may indeed be the target antigen.

Cardiac-specific overexpression of phospholamban alters calcium kinetics and resultant cardiomyocyte mechanics in transgenic mice.

Phospholamban is the regulator of the cardiac sarcoplasmic reticulum (SR) Ca(2+)-ATPase activity and an important modulator of basal contractility in the heart. To determine whether all the SR Ca(2+)-ATPase enzymes are subject to regulation by phospholamban in vivo, transgenic mice were generated which overexpressed phospholamban in the heart, driven by the cardiac-specific alpha-myosin heavy chain promoter. Quantitative immunoblotting revealed a twofold increase in the phospholamban protein levels in transgenic hearts compared to wild type littermate hearts. The transgenic mice showed no phenotypic alterations and no changes in heart/body weight, heart/lung weight, and cardiomyocyte size. Isolated unloaded cardiac myocytes from transgenic mice exhibited diminished shortening fraction (63%) and decreased rates of shortening (64%) and relengthening (55%) compared to wild type (100%) cardiomyocytes. The decreases in contractile parameters of transgenic cardiomyocytes reflected decreases in the amplitude (83%) of the Ca2+ signal and prolongation (131%) in the time for decay of the Ca2+ signal, which was associated with a decrease in the apparent affinity of the SR Ca(2+)-ATPase for Ca2+ (56%), compared to wild type (100%) cardiomyocytes. In vivo analysis of left ventricular systolic function using M mode and pulsed-wave Doppler echocardiography revealed decreases in fractional shortening (79%) and the normalized mean velocity of circumferential shortening (67%) in transgenic mice compared to wild type (100%) mice. The differences in contractile parameters and Ca2+ kinetics in transgenic cardiomyocytes and the depressed left ventricular systolic function in transgenic mice were abolished upon isoproterenol stimulation. These findings indicate that a fraction of the Ca(2+)-ATPases in native SR is not under regulation by phospholamban. Expression of additional phospholamban molecules results in: (a) inhibition of SR Ca2+ transport; (b) decreases in systolic Ca2+ levels and contractile parameters in ventricular myocytes; and (c) depression of basal left ventricular systolic function in vivo.

Differential phospholamban gene expression in murine cardiac compartments. Molecular and physiological analyses.

Phospholamban, the regulator of the Ca2+ pump in cardiac sarcoplasmic reticulum, is differentially expressed between murine atrial and ventricular muscles. Quantitative analyses of RNA isolated from atrial flaps and ventricular apices indicated that the phospholamban gene transcript copy number is 2.5-fold higher in the ventricle compared with the atrium of the FVB/N mouse and 6-fold higher in the ventricle compared with the atrium of the B6D2/F1 mouse strain. These findings were corroborated by in situ hybridization studies of cardiopulmonary sections from both murine strains, and phospholamban transcripts were also observed in pulmonary myocardia of both strains. Analyses of phospholamban transcript levels relative to alpha-myosin heavy chain (alpha-MHC) revealed a 3-fold higher phospholamban abundance in the ventricle compared with the atrium of the FVB/N murine strain. However, the relative mRNA level of Ca(2+)-ATPase (ratio of sarcoplasmic reticulum Ca(2+)-ATPase [SERCA2] to alpha-MHC) in the ventricle was 80% of that in the atrium. Consequently, the relative ratio of phospholamban to SERCA2 mRNA was 4.2-fold lower in the atrium than in the ventricle. The lower transcript ratio of phospholamban to SERCA2 in the atrium was associated with significantly shortened times to half-relaxation (17.40 +/- 0.71 milliseconds for atrium versus 30.58 +/- 2.04 milliseconds for ventricle), assessed in isolated superfused cardiac tissue preparations recorded at maximum length tension. Contraction times, measured as times to peak tension, were also significantly shortened in atrial muscle (27.36 +/- 0.82 milliseconds) compared with ventricular muscle (44.60 +/- 2.55 milliseconds), assessed in the same tissue preparations. These findings suggest that phospholamban gene expression is differentially regulated in murine atrial and ventricular muscles and that this differential expression may be associated with differences in the contractile parameters of these cardiac compartments.

Expression of phospholamban in C2C12 cells and regulation of endogenous SERCA1 activity.

Phospholamban (PLB) is a regulator of the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2) expressed in cardiac, slow-twitch skeletal, and smooth muscles. Phospholamban is not expressed in the sarcoplasmic reticulum of fast-twitch skeletal muscle, but it can regulate the sarcoplasmic reticulum Ca(2+)-ATPase activity (SERCA1) expressed in this muscle, in vitro. To determine whether phospholamban can regulate SERCA1 activity in its native membrane environment, phospholamban was stably transfected into a cell line (C2C12) derived from murine fast-twitch skeletal muscle. Differentiation of C2C12 myoblasts to myotubes was associated with induction of SERCA1 expression, assessed by Western blotting analysis using Ca(2+)-ATPase isoform specific antibodies. The expressed phospholamban protein was localized in the microsomal fraction isolated from C2C12 myotubes. To determine the effect of phospholamban expression on SERCA1 activity, microsomes were isolated from transfected and nontransfected C2C12 cell myotubes, and the initial rates of 45Ca(2+)-uptake were determined over a wide range of Ca2+ concentrations (0.1-10 microM). Expression of phospholamban was associated with inhibition of the initial rates of Ca(2+)-uptake at low [Ca2+] and this resulted in a decrease in the affinity of SERCA1 for Ca2+ (0.27 +/- 0.02 microM in nontransfected vs. 0.41 +/- 0.03 microM in PLB transfected C2C12 cells). These findings indicate that phospholamban expression in C2C12 cells is associated with inhibition of the endogenous SERCA1 activity and provide evidence that phospholamban is capable of regulating this Ca(2+)-ATPase isoform in its native membrane environment.

Targeted ablation of the phospholamban gene is associated with markedly enhanced myocardial contractility and loss of beta-agonist stimulation.

Phospholamban is the regulator of the Ca(2+)-ATPase in cardiac sarcoplasmic reticulum (SR), and it has been suggested to be an important determinant in the inotropic responses of the heart to beta-adrenergic stimulation. To determine the role of phospholamban in vivo, the gene coding for this protein was targeted in murine embryonic stem cells, and mice deficient in phospholamban were generated. The phospholamban-deficient mice showed no gross developmental abnormalities but exhibited enhanced myocardial performance without changes in heart rate. The time to peak pressure and the time to half-relaxation were significantly shorter in phospholamban-deficient mice compared with their wild-type homozygous littermates as assessed in work-performing mouse heart preparations under identical venous returns, afterloads, and heart rates. The first derivatives of intraventricular pressure (+/- dP/dt) were also significantly elevated, and this was associated with an increase in the affinity of the SR Ca(2+)-ATPase for Ca2+ in the phospholamban-deficient hearts. Baseline levels of these parameters in the phospholamban-deficient hearts were equal to those observed in hearts of wild-type littermates maximally stimulated with the beta-agonist isoproterenol. These findings indicate that phospholamban acts as a critical repressor of basal myocardial contractility and may be the key phosphoprotein in mediating the heart's contractile responses to beta-adrenergic agonists.

T-cell-independent stimulation of immunoglobulin secretion in resting human B lymphocytes by the mannose-specific adhesin of Escherichia coli type 1 fimbriae.

Purified Escherichia coli type 1 fimbriae have been shown previously to stimulate T-cell-independent proliferation of human B lymphocytes. The response is mediated by the mannose-specific, lectin-like adhesin protein FimH. Here we show that type 1 fimbriae also stimulate immunoglobulin (Ig) secretion by B cells. The response was maximal at three days of culture and consisted predominantly of the IgM isotype. It was independent of serum components, T lymphocytes, monocytes, and natural killer cells. Highly purified resting B cells were induced to proliferate and secrete Ig in response to the fimbriae. The role of FimH in the response was shown by the failure of FimH- type 1 fimbriae to stimulate and by inhibition of the response with alpha-methyl mannoside. In light of the fact that carbohydrate-binding adhesins have been found on a wide variety of microorganisms, these studies suggest the possibility that responses of other cell types to other microbial adhesins will be discovered.

Mouse phospholamban gene expression during development in vivo and in vitro.

To establish a murine model that may allow for definition of the precise role of phospholamban in myocardial contractility through selective perturbations in the phospholamban gene, we initiated studies on the role of phospholamban in the murine heart. Intact beating hearts were perfused in the absence or presence of isoproterenol, and quantitative measurements of cardiac performance were obtained. Isoproterenol stimulation was associated with increases in the affinity of the sarcoplasmic reticulum Ca2+ pump for Ca2+ that were due to phospholamban phosphorylation. To assess the regulation of phospholamban gene expression during murine development, Northern blot and polymerase chain reaction analyses were used. Phospholamban mRNA was first detected in murine embryos on the ninth day of development (the time when the cardiac tube begins to contract). In murine embryoid bodies, which have been shown to recapitulate several aspects of cardiogenesis, phospholamban mRNA was detected on the seventh day (the time when spontaneous contractions are first observed). Only those embryoid bodies that exhibited contractions expressed phospholamban transcripts, and these were accompanied by expression of the protein, as revealed by immunofluorescence microscopy. Sequence analysis of the cDNA encoding phospholamban in embryoid bodies indicated complete homology to that in adult hearts. The deduced amino acid sequence of murine phospholamban was identical to rabbit cardiac phospholamban but different from dog cardiac and human cardiac phospholamban by one amino acid. These data suggest that phospholamban, the regulator of the Ca(2+)-ATPase in cardiac sarcoplasmic reticulum, is present very early in murine cardiogenesis in utero and in vitro, and this may constitute an important determinant for proper development of myocardial contractility.

Intensive care experience in Guillain-Barre syndrome.

In a retrospective study of 34 patients with acute Guillain-Barre syndrome admitted to an intensive care unit (ICU) during a six year period from January 1984 to December 1989, for bulbar paresis or impending respiratory failure, 4 required endotracheal intubation to protect the airway, and 27 required mechanical ventilation. A high incidence of respiratory complications was noted. Two patients died of cardiovascular instability. Close monitoring of respiratory and cardiovascular status is essential to ensure survival.