Expression of nicotinic acetylcholine receptor subunits in HEp-2 cells for immunodetection of autoantibody specificities in sera from Myasthenia gravis patients.

Myasthenia gravis (MG) is an autoimmune disease characterized by the formation of pathogenic autoantibodies mostly targeting the nicotinic acetylcholine receptor (AChR). The AChR is composed of two alpha subunits and one subunit of each beta, delta and gamma (fetal AChR), or epsilon (adult AChR), respectively. Serological diagnostics is commonly done by radioimmunoassay (RIA). Here we used an indirect immunofluorescence assay with MG patient sera on transiently transfected HEp-2 cells expressing selected components of the AChR. Our data show that already 12 out of 13 MG patient sera showed autoantibody binding to HEp-2 cells transfected to express the alpha subunit solely. Interestingly, 11 out of 13 patient sera reacted positive with cells transfected to reconstitute the complete fetal AChR, but only 6 out of 13 sera showed positive signals with cells expressing the components of adult AChR. Moreover, there was no strict correlation of the serum concentration required to obtain clear-cut fluorescence signals to the antibody titer as measured by RIA. It will be an interesting topic to further investigate if the optimal serum dilution for indirect immunofluorescence as well as the autoantibody binding preferences to defined AChR subunits and to the adult versus the fetal receptor variant could provide additional predictive value in MG diagnostics.

On-line estimation of biomass concentration using a neural network and information about metabolic state.

This paper deals with the design of a neural network-based biomass concentration estimation system. This system is enhanced by the incorporation of information about the actual metabolism of the microorganism cultivated, which is taken from an on-line knowledge-based system. Two different design approaches have been investigated using the fed-batch cultivation of baker's yeast as the model process. In the first, metabolic state (MS) data were passed as additional input to the neural network; in the second, these data were used to select a neural network suitable for the specific MS. Two neural network types--feed-forward (Levenberg-Marquardt) and cascade correlation--were applied to this system and tested, and the performances of these neural networks were compared.

Chronic hypertension and the risk for adverse pregnancy outcome after superimposed pre-eclampsia.

OBJECTIVE: To determine the risk factors and pregnancy outcome of patients with chronic hypertension during pregnancy after controlling for superimposed preeclampsia. METHOD: A comparison of all singleton term (>36 weeks) deliveries occurring between 1988 and 1999, with and without chronic hypertension, was performed. Stratified analyses, using the Mantel-Haenszel technique, and a multiple logistic regression model were performed to control for confounders. RESULTS: Chronic hypertension complicated 1.6% (n=1807) of all deliveries included in the study (n=113156). Using a multivariable analysis, the following factors were found to be independently associated with chronic hypertension: maternal age >40 years (OR=3.1; 95% CI 2.7-3.6), diabetes mellitus (OR=3.6; 95% CI 3.3-4.1), recurrent abortions (OR=1.5; 95% CI 1.3-1.8), infertility treatment (OR=2.9; 95% CI 2.3-3.7), and previous cesarean delivery (CD; OR=1.8 CI 1.6-2.0). After adjustment for superimposed preeclampsia, using the Mantel-Haenszel technique, pregnancies complicated with chronic hypertension had higher rates of CD (OR=2.7; 95% CI 2.4-3.0), intra uterine growth restriction (OR=1.7; 95% CI 1.3-2.2), perinatal mortality (OR=1.6; 95% CI 1.01-2.6) and post-partum hemorrhage (OR=2.2; 95% CI 1.4-3.7). CONCLUSION: Chronic hypertension is associated with adverse pregnancy outcome, regardless of superimposed preeclampsia.

Far-infrared sensor for cirrus (FIRSC): an aircraft-based Fourier-transform spectrometer to measure cloud radiance.

We describe an aircraft-based Fourier-transform spectrometer (FTS) designed to measure the Earth outgoing radiance spectrum in the far-infrared-submillimeter spectral range. The instrument features include a rapid-scan FTS to obtain high spatial resolution from a moving aircraft platform, a sensitive two-channel detector, and a CCD camera for recording the nadir cloud scene with each scan record. Such measurements demonstrate the sensitivity of Earth radiance to high clouds and provide spectral data for improving techniques for remote sensing and retrieval of atmospheric and cloud properties.

The kinetics of association and phosphorylation of IkappaB isoforms by IkappaB kinase 2 correlate with their cellular regulation in human endothelial cells.

Activation of the transcription factor NF-kappaB depends on the specific dual phosphorylation of its inhibitor protein IkappaB by the homologous cytokine-inducible IkappaB kinases 1 and 2 (IKK1/2). Various IkappaB isoforms exist: IkappaBalpha, IkappaBbeta1/2 (two alternative splice variants), and IkappaBepsilon. However, the individual relevance and the specific regulation of these isoforms is not well-understood. We have studied the direct interaction of recombinant IkappaBalpha, IkappaBbeta1, IkappaBbeta2, and IkappaBepsilon with the recombinant homodimeric IKK2. Fluorescence-based active site titration revealed that each IKK2 dimer contains two binding sites for IkappaB. By using surface plasmon resonance analysis, we found that all IkappaB proteins interact with the IKK2 dimer following a noncooperative binding mechanism. Further, the four IkappaB proteins bind to the kinase with equilibrium dissociation constants (KD) in the range of 50-300 nM; the association rate constants for all IkappaB isoforms with IKK2 were between 6.0 x 10(3) and 22.5 x 10(3) M-1 s-1, and the dissociation rate constants were between 1.25 x 10(-3) and 1.75 x 10(-3) s-1. This high-affinity binding suggests that the previously observed preassociation of all analyzed IkappaB proteins with the biochemically purified 700 kDa IkappaB kinase (IKK) complex is based on a direct enzyme-substrate association between the various IkappaB isoforms and the IKK proteins. The apparent catalytic efficiencies (kcat/KM) of IKK2 for IkappaBalpha, IkappaBbeta1, IkappaBbeta2, and IkappaBepsilon were 22 x 10(3), 10 x 10(3), 5.4 x 10(3), and 8.5 x 10(3) s-1 M-1, respectively, with KM values ranging between 1.7 x 10(-6) and 3.2 x 10(-6) M and kcat values ranging between 1.5 x 10(-2) and 3.7 x 10(-2) s-1. The relative affinities and catalytic efficiencies of IKK2 for the IkappaB isoforms were also reflected by the kinetics observed for the TNF-induced, phosphorylation-dependent degradation of the alpha, beta1, beta2, and epsilon isoforms of IkappaB in human umbilical vein endothelial cells. Therefore, differential regulation of the IkappaB isoforms in some cell types is not a direct result of the IKK activity, but appears to be due to parallel events.

Is 'routine' laboratory testing a thing of the past? Current recommendations regarding screening.

A 55-year-old man comes to you for a routine physical examination. He is a nonsmoker who takes no medications and has no signs of acute or chronic disease, and he has not seen a doctor in years. What blood work should you order for this patient? The authors of this article help you answer this question in light of recent advances in technology, restrictions in healthcare reimbursement, and increased sophistication in cost-benefit analysis for laboratory testing.

Activation of protein kinase C subtypes alpha, gamma, delta, epsilon, zeta, and eta by tumor-promoting and nontumor-promoting agents.

Protein kinase C (PKC) subtypes alpha, gamma, delta, epsilon, zeta, and eta have been expressed using the baculovirus expression system. The partially purified PKC subtypes have been studied for their substrate specificities and phospholipid-independent activation by various chemically different nontumor- and tumor-promoting agents, as well as their inhibition of kinase activity by staurosporine and two related compounds. An endogenous PKC-like kinase activity of Sf9 cells was detected and analyzed for cofactor requirements and inhibition. Protamine sulfate was most efficiently phosphorylated by all of the PKC subtypes tested, although this phosphorylation was independent of phosphatidylserine (PS) and diacylglycerol (DAG) or 12-O-tetradecanoylphorbol 13-acetate (TPA). Except for PKC-zeta, all subtypes tested phosphorylated myelin basic protein (MBP), histone, or a peptide derived from the pseudosubstrate region of PKC-alpha in a PS/DAG-dependent manner but to varying extents. Among the various agents tested, TPA most efficiently stimulated the kinase activities of the PKC subtypes in a phospholipid-dependent manner. Phorbol 12,13-dibutyrate (PDBu) was less effective than TPA but displayed no major difference among the subtypes. Activation of PKC-alpha by bryostatin-1 reached only half of the TPA response whereas the other subtypes were activated more effectively. The weak tumor promoter resiniferonol 9,13,14-orthophenyl acetate (ROPA) mainly stimulated PKC-alpha and PKC-gamma at 1 microM concentration, whereas PKC-epsilon and PKC-eta were much less activated. Sapintoxin D, mezerein, indolactam V, and resiniferatoxin at concentrations of 1-100 nM preferentially activated PKC-alpha in a DAG-like manner, whereas at 1 microM other subtypes were activated as well. Preferential activation of PKC-alpha was also noted for tinyatoxin and thapsigargin, but their mode of activation is unclear because these two compounds did not compete for the phorbol ester binding of the PKC subtypes as the other agents did. Of the three PKC inhibitors tested, staurosporine most efficiently inhibited kinase activity of the PKC subtypes, whereas K252a and CGP 41251 were at least 10 times less effective. However, K252a showed certain specificity for inhibition of PKC-alpha, and CGP 41251 failed to inhibit PKC-epsilon and PKC-zeta. Given the different substrate specificities and modes of activation by various tumor-promoting and nontumor-promoting agents, as well as the different sensitivities towards different inhibitors, our results indicate a divergence of individual PKC subtypes in signal transduction.

Molecular cloning of a human melanoma-associated chondroitin sulfate proteoglycan.

A human melanoma-associated chondroitin sulfate proteoglycan (MCSP), recognized by mAb 9.2.27, plays a role in stabilizing cell-substratum interactions during early events of melanoma cell spreading on endothelial basement membranes. We report here the molecular cloning and nucleotide sequencing of cDNA encoding the entire core protein of human MCSP and provide its deduced amino acid sequence. This core protein contains an open reading frame of 2322 aa, encompassing a large extracellular domain, a hydrophobic transmembrane region, and a relatively short cytoplasmic tail. Northern blot analysis indicated that MCSP cDNA probes detect a single 8.0-kb RNA species expressed in human melanoma cell lines. In situ hybridization experiments with a segment of the MCSP coding sequence localized MCSP mRNA in biopsies prepared from melanoma skin metastases. Multiple human Northern blots with an MCSP-specific probe revealed a strong hybridization signal only with melanoma cells and not with other human cancer cells or a variety of human fetal and adult tissues. These data indicate that MCSP represents an integral membrane chondroitin sulfate proteoglycan expressed by human malignant melanoma cells. The availability of cDNAs encoding MCSP should facilitate studies designed to establish correlations between structure and function of this molecule and help to establish its role in the progression of human malignant melanoma.

Coupling of the C5a receptor to Gi in U-937 cells and in cells transfected with C5a receptor cDNA.

The signaling properties of the receptor for the chemoattractant C5a (C5aR) were investigated in differentiated U-937 cells and in NIH/3T3 cells transfected with the C5aR. In both U-937 cells and transfected cells (2A3 cells), C5a induced the mobilization of intracellular calcium, phosphoinositide breakdown, and activation of mitogen-activated protein kinase. In addition, in 2A3 cells C5a induced the inhibition of forskolin-stimulated cAMP generation. Pretreatment with pertussis toxin suppressed all C5a-mediated signal transduction in both cell lines. In the presence of cholera toxin, C5a induced the ribosylation of a 39-40-kDa protein in membranes of both U-937 cells and 2A3 cells. Similar phenomena have been described in other systems, whereby Gi alpha subunits are substrates for cholera toxin-induced ribosylation in the presence of receptor agonists. Moreover, the C5a-induced ribosylation was eliminated in membranes of cells that had been pretreated with pertussis toxin. The G protein alpha subunit G alpha 16, which is insensitive to pertussis toxin, has been reported to couple selectively to C5aR in cells co-transfected with C5aR and G alpha 16 cDNAs. G alpha 16 expression was not detected in U-937 cells or in 2A3 cells, either by reverse transcription-polymerase chain reaction or by immunoblotting. Because pertussis toxin modifies only G alpha subunits of the Gi/o family and all signaling by C5aR was abolished by pertussis toxin pretreatment, the results strongly suggest that, in U-937 and 2A3 cells, C5a-mediated responses can be accounted for entirely through coupling with G proteins of the Gi subtype.

Cloning and tissue distribution of subunits C, D, and E of the human vacuolar H(+)-ATPase.

The vacuolar proton ATPase (V-ATPase) translocates protons into intracellular organelles or across the plasma membrane of specialised cells such as osteoclast and renal intercalated cells. The catalytic site of the V-ATPase consists of a hexamer of three A subunits and three B subunits which bind and hydrolyse ATP and are regulated by accessory subunits C, D and E. cDNAs encoding subunits C, D, and E were cloned from human osteoclastoma, a tissue highly enriched in osteoclasts, as a first step in the characterisation of the V-ATPase used by the osteoclast. By Northern blot analysis only one mRNA species were detected for each of these subunits, which is consistent the constant transcription level in all tissues irrespective of the presence of specialised cells highly enriched in V-ATPases.

Expression and partial characterization of rat protein kinase C-delta and protein kinase C-zeta in insect cells using recombinant baculovirus.

Expression of rat protein kinase C-delta (PKC-delta) and PKC-zeta in insect cells using recombinant baculovirus resulted in the production of proteins with a molecular size of approximately 76 kD and 78 kD, respectively, as determined by immunoblotting with subtype-specific antisera. Although the PKC-zeta cDNA encoded for 592 amino acids, a 76 kD protein was also generated by in vitro transcription/translation. Extracts of cells expressing PKC-delta were able to bind phorbol ester to levels comparable to extracts of cells expressing PKC-alpha. No phorbol ester binding was, however, detected in insect cell extracts expressing PKC-zeta. However, similar levels of protein kinase activity were detected in lysates of cells expressing PKC-delta or PKC-zeta when protamine sulfate was used as exogenous substrate. Compared to protamine sulfate, both, myelin basic protein (MBP) or histone, were poor substrates for PKC-delta and PKC-zeta. In contrast to PKC-zeta, the PKC-delta enzyme activity phosphorylated MBP or histone in a phosphatidylserine-(PS)/diacylglycerol(DG)-dependent manner, albeit not to the same extent as PKC-alpha. Lack of stimulation of the enzyme activity of PKC-zeta by PS/DG, was confirmed by endogenous phosphorylation of insect cell proteins by PKC-zeta, whereas several insect cell proteins were phosphorylated by PKC-delta in a PS/DG-dependent manner, including a protein of 78 kD. Our data demonstrate that the 76 kD PKC-zeta, in contrast to PKC-delta, is unable to bind phorbol esters and displays a protein kinase activity that is independent of PS or PS/DG. In addition, staurosporine was about 2-4 order of magnitudes less effective in inhibiting the protein kinase activities of PKC-delta and PKC-zeta when compared to PKC-alpha.

Multiple forms of prolactin receptor messenger ribonucleic acid are specifically expressed and regulated in murine tissues and the mammary cell line HC11.

The expression of PRL receptor mRNA in mouse tissues was studied. Seven PRL receptor transcripts of different sizes were found. The pattern of expression was tissue specific. The PRL receptor protein exists in isoforms of approximately 300 and 600 amino acids, which differ in the sequence and length of the cytoplasmic domain. Probes specific for the lower mol wt forms of the receptor hybridized to transcripts of 1.4, 2.4, 3.5, and 4.2 kilobases (kb), which were predominantly expressed in the liver and kidney. Among the three isoforms of the small form of the receptor, PR-3 was highly expressed, PR-2 was weakly expressed, and PR-1 was undetectable. Probes specific for the higher mol wt receptor form detected transcripts of 9 and 10 kb, expressed most strongly in the ovary, mammary gland, and kidney. An additional 8.3-kb transcript was expressed in the kidney. The PRL-responsive mouse mammary epithelial cell line HC11 expressed only the 9- and 10-kb receptor mRNAs, as did the mammary gland. The transcripts for the two PRL receptor forms were independently regulated during pregnancy and lactation in a tissue-specific manner. The expression of the small receptor form in the liver increased 7-fold during pregnancy and decreased during lactation. Its expression in the kidney remained constant. Expression of the larger receptor form increased 2.5-fold in the kidney during lactation, but remained constant in the mammary gland. In the ovary the expression of the large receptor form could be elevated 4-fold after induction of hyperovulation with FSH and hCG. Thus, in the mouse there are at least two forms of the PRL receptor regulated independently in different tissues.

Influence of panel-reactive antibody and lymphocytotoxic crossmatch on survival after heart transplantation.

Review of 463 heart transplants was undertaken to examine the relationship between level of panel-reactive antibody (PRA) and a standard donor-specific lymphocytotoxic crossmatch (LXM) on the incidence of death from hyperacute, acute, and chronic rejection. Death from chronic rejection was defined as being caused by graft atherosclerosis. Hyperacute rejection was diagnosed in 18 allografts, and only two recipients had PRA greater than 10% and another two a positive LXM. Five-year actuarial freedom from death caused by all forms of rejection correlated with PRA values as follows: PRA 0% to 10% (415 patients), 85%; PRA 11% to 25% (29 patients), 68%; PRA greater than 25% (19 patients), 57% (p less than 0.005). Additionally, there was a positive linear relationship between PRA and duration of acute rejection episodes in the first 3 months after transplantation. A positive retrospective donor-specific LXM was present in 42 of 401 patients; most of them (32 patients) were low positive (10% to 50% cell death), and none could be correlated with antibody specificity toward donor HLA antigens. Five-year actuarial freedom from death caused by rejection was 83% in those with a negative LXM, 74% in those with low-positive, and 79% in those with high-positive LXM (p = NS). Negative LXM result did not reduce the risk of death caused by rejection in any of the PRA subgroups. While PRA greater than 10% is a risk factor for rejection-related events, a negative LXM in patients with an elevated PRA does not reduce the risk of death resulting from acute or chronic rejection.

Multiscreen serum analysis of highly sensitized renal dialysis patients for antibodies toward public and private class I HLA determinants. Implications for computer-predicted acceptable and unacceptable donor mismatches in kidney transplantation.

A multiscreen serum analysis program has been developed that permits a determination of antibody specificity for the vast majority of highly sensitized patients awaiting transplantation. This program is based on a 2 x 2 table analysis of correlations between serum reactivity with an HLA-typed cell panel and incorporates two modifications. One implements the concept of public HLA determinants based on the serologic crossreactivity among class I HLA antigens. The other modification derives from the premise that most highly sensitized patients maintain the same PRA and antibody profiles over many months and even years. Monthly screening results for patients with persistent PRA values can therefore be combined for analysis. For 132 of 150 highly sensitized patients with greater than 50% PRA, this multiscreen serum analysis program yielded information about antibody specificity toward public and private class I HLA determinants. The vast majority of patients (108 of 112) with PRA values between 50 and 89% showed antibody specificity generally toward one, two, or three public markers and/or the more common private HLA-A,B antigens. For 24 of 38 patients with greater than 90% PRA, it was possible to define one or few HLA-specific antibodies. The primary objective of the multiscreen program was to develop an algorithm about computer-predicted acceptable and unacceptable donor HLA-A,B antigens for patients with preformed antibodies. A retrospective analysis of kidney transplants into 89 highly sensitized patients has demonstrated that allografts with unacceptable HLA-A,B mismatches had significantly lower actuarial survival rates than those with acceptable mismatches (P = 0.01). This was shown for both groups of 32 primary transplants (44% vs. 67% after 1 year) and 60 retransplants (50% vs. 68%). Also, serum creatinine levels were significantly higher in patients with unacceptable class I mismatches (3.0 vs. 8.4 mg% [P = 0.007] after 2 weeks; 3.9 vs. 9.1 mg% [P = 0.014] after 4 weeks). Histopathologic analysis of allograft tissue specimens from 47 transplant recipients revealed a significantly higher incidence of humoral rejection (P = 0.02), but not cellular rejection, in the unacceptable mismatch group. These results suggest that the multiscreen program can establish which donor HLA-A,B mismatches must be avoided in kidney transplantation for most highly sensitized patients. For 18 of 150 high PRA renal dialysis patients, the multiscreen program could not define HLA-specific antibody. Most patients had greater than 90% PRA, and many of their sera appeared to contain IgM type nonspecific lymphocytotoxins that could be inactivated by dithioerythreitol (DTE).(ABSTRACT TRUNCATED AT 400 WORDS)

Histocompatibility and liver transplant outcome. Does HLA exert a dualistic effect?

An analysis of more than 500 liver transplants has demonstrated that HLA compatibility is associated with diminished allograft survival. Liver transplants with zero mismatches for class I and/or class II HLA antigens have shown significantly lower actuarial survival rates than transplants with one or more mismatches for these loci. In a group of 119 failed liver allografts from patients undergoing retransplantation, a higher incidence of failure due to rejection correlated with a lower degree of HLA compatibility especially for HLA-DR. In contrast, the incidence of liver transplant failures due to primary nonfunction was relatively higher with HLA-DR compatible transplants. Considering the role of HLA as a restriction element in cellular interactions during the immune response, these findings suggest that HLA compatibility may have a dualistic effect on liver transplant outcome. On one hand, HLA compatibility reduced transplant rejection--and on the other hand, it may enhance other immunological mechanisms leading to allograft dysfunction, particularly in patients at risk of developing recurrent autoimmune diseases or infection.