Assay for the specificity of monoclonal antibodies in crossed immunoelectrophoresis

A method is described based on crossed immunoelectrophoresis of a complex antigen mixture in agarose gel followed by incubation of the gel with the monoclonal antibody. The bound monoclonal antibody is detected by the use of a secondary enzyme-labelled antibody.Using this technique we have been able to identify the precipitate arc in crossed immunoelectrophoresis of major histocompatibility complex (MHC) class I molecules in a mixture of all detergent solubilized cell membrane molecules by means of a monoclonal antibody, the specificity of which was known independently to be against MHC class I molecules. In other experiments using the same technique we demonstrated the reaction of a monoclonal antibody specific for chicken Ig light chains.     

Chlorotetracycline Induces calcium mediated shape changes in human erythrocytes

Calcium was localized in the red cell membrane by light microscopy using chlorotetracycline hydrochloride (CTC) as chelate probe. Treating human erythrocytes with CTC dissolved in saline free of divalent cations, leads to a 530 nm fluorescence emission in the cell border and to characteristic cell shape changes which were evaluated to assess intramembrane calcium distribution. CTC prevented and reverted erythrocyte crenation induced either by washing or superfusing the cells with saline. The ionophore A23187, EGTA and glucose depletion depressed the shape modifying effect of CTC. Thus, CTC appears to act on red cell shape by complex formation with membrane associated calcium. This is further confirmed by the failure of degraded CTC, devoid of metal binding capacity, to modify the crenated shape. The CTC effect can be reverted by superfusing the erythrocytes with CTC-free medium. Thus, calcium binds more tightly to the membrane than to CTC and is not displaced by the antibiotic. If the bilayer couple hypothesis [Sheetz, M.P., Singer, S.J., Proc. Natl. Acad. Sci. USA 71, 4457-4461 (1974)]applies, crenation is reverted by expansion of the inner membrane half relative to the outer membrane half. Expansion of the inner membrane half results from intercalation of CTC which binds to calcium. Thus, calcium in the red cell membrane preferentially occupies the inner leaflet of the bilayer.     

A unique exonic splice enhancer mutation in a family with X-linked mental retardation and epilepsy points to a novel role of the renin receptor

The renin-angiotensin system (RAS) is essential for blood pressure control and water-electrolyte balance. Until the discovery of the renin receptor, renin was believed to be mainly a circulating enzyme with a unique function, the cleavage of angiotensinogen. We report a unique mutation in the renin receptor gene (ATP6AP2) present in patients with X-linked mental retardation and epilepsy (OMIM no. 300423), but absent in 1200 control X-chromosomes. A silent mutation (c.321C>T, p.D107D) residing in a putative exonic splicing enhancer site resulted in inefficient inclusion of exon 4 in 50% of renin receptor mRNA, as demonstrated by quantitative RT-PCR. Analysis of membrane associated-receptor molecular forms showed the presence of full-length and truncated proteins in the patient. Functional analysis demonstrated that the mutated receptor could bind renin and increase renin catalytic activity, similar to the wild-type receptor, but resulted in a modest and reproducible impairment of ERK1/2 activation. Thus, our findings confirm the importance of the RAS in cognitive processes and indicate a novel specific role for the renin receptor in cognitive functions and brain development.     

Site-independent prognostic value of chromosome 9q loss in primary gastrointestinal stromal tumours

Although the significance of tumour site for estimating malignant potential in gastrointestinal stromal tumours (GISTs) has recently been recognized, site-specific genetic patterns have not to date been defined. This study examined 52 c-kit-positive primary GISTs (with a mean follow-up of 42.3 months in 51 cases) from three different locations (35 gastric, 12 small intestinal, and five colorectal) using comparative genomic hybridization (CGH). In general, tumour site correlated with key prognostic factors, including tumour size, mitotic rate, proliferative activity, and probable malignant potential. Furthermore, several DNA copy number changes showed a site-dependent pattern. These included losses at 14q (gastric 83%, intestinal 35%; p = 0.001), losses at 22q (gastric 46%, intestinal 82%; p = 0.02), losses at 1p (gastric 23%, intestinal 88%; p = 1 x 10(-5)), losses at 15q (gastric 14%, intestinal 59%; p = 0.002), losses at 9q (gastric 14%, intestinal 53%; p = 0.006), and gains at 5p (gastric 11%, intestinal 53%; p = 0.002). These data demonstrate strong site-dependent genetic heterogeneity in GISTs that may form a basis for subclassification. Prognostic evaluation of DNA copy number changes identified losses at 9q as a site-independent prognostic marker associated with shorter disease-free survival (p = 0.03) and overall survival (p = 0.002). Furthermore, 9q loss also appeared to carry prognostic value in predicting overall survival for patients with advanced or progressive GISTs (p = 0.003).     

Biological and clinical significance of cytogenetic abnormalities in low-risk and high-risk gastrointestinal stromal tumors

We report cytogenetic findings in 19 c-Kit-positive gastrointestinal stromal tumors (GISTs) that represent a heterogenous group of mesenchymal neoplasms with respect to site, histology, and biologic behavior. All of the GISTs (5 low-risk, 11 high-risk, 3 recurrences) displayed clonal chromosomal aberrations; 15 were hypo- to near-diploid, and 4 were near-triploid and hypotetraploid. The most common abnormalities were loss of chromosomes 14 and/or 22, demonstrated in 14 GISTs irrespective of site or predominant phenotype. Ten cases (2 low-risk, 5 high-risk, 3 recurrences) were characterized by loss of both chromosomes 14 and 22, 2 cases (1 low-risk, 1 high- risk), by loss of chromosome 14; and 2 high-risk cases, by loss of chromosome 22. Additional chromosomal aberrations occurred preferentially in high-risk and recurrent GISTs, including loss of 9p and 1p in 8 cases each, loss of 15 in 6 cases, loss of 3p in 5 cases, loss of 13q and 10q in 4 cases each, loss of 19 in 3 cases, and complete or partial gains of chromosomes 5 and 4 in 2 cases each. More significantly, 5 of 6 patients with clinically aggressive GISTs, including 2 recurrences and 3 metastasing GISTs, were additionally characterized by loss of 9p; four of these had additional loss of chromosomes 1p and 15. The presented results herein indicate that loss of chromosome 14 and/or 22 is an early change in GIST tumorigenesis irrespective of site or differentiation, whereas malignant transformation and progression of GISTs appear to be associated with an increasing incidence of additional secondary aberrations.     

An integrative approach to CTL epitope prediction: a combined algorithm integrating MHC class I binding, TAP transport efficiency, and proteasomal cleavage predictions

Reverse immunogenetic approaches attempt to optimize the selection of candidate epitopes, and thus minimize the experimental effort needed to identify new epitopes. When predicting cytotoxic T cell epitopes, the main focus has been on the highly specific MHC class I binding event. Methods have also been developed for predicting the antigen-processing steps preceding MHC class I binding, including proteasomal cleavage and transporter associated with antigen processing (TAP) transport efficiency. Here, we use a dataset obtained from the SYFPEITHI database to show that a method integrating predictions of MHC class I binding affinity, TAP transport efficiency, and C-terminal proteasomal cleavage outperforms any of the individual methods. Using an independent evaluation dataset of HIV epitopes from the Los Alamos database, the validity of the integrated method is confirmed. The performance of the integrated method is found to be significantly higher than that of the two publicly available prediction methods BIMAS and SYFPEITHI. To identify 85% of the epitopes in the HIV dataset, 9% and 10% of all possible nonamers in the HIV proteins must be tested when using the BIMAS and SYFPEITHI methods, respectively, for the selection of candidate epitopes. This number is reduced to 7% when using the integrated method. In practical terms, this means that the experimental effort needed to identify an epitope in a hypothetical protein with 85% probability is reduced by 20-30% when using the integrated method.The method is available at http://www.cbs.dtu.dk/services/NetCTL. Supplementary material is available at http://www.cbs.dtu.dk/suppl/immunology/CTL.php.     

Restoration of visual orienting into a cortically blind hemifield by reversible deactivation of posterior parietal cortex or the superior colliculus

A contralateral hemineglect of the visual field can be induced by unilateral cooling deactivation of posterior middle suprasylvian (pMS) sulcal cortex of the posterior parietal region, and this neglect can be reversed by additional cooling deactivation of pMS cortex in the opposite hemisphere. The purpose of the present study was to test whether an enduring hemianopia induced by removal of all contiguous visual cortical areas of one hemisphere could be reversed by local cooling of pMS cortex in the opposite hemisphere. Two cats sustained large unilateral ablations of the contiguous visual areas, and cooling loops were placed in the pMS sulcus, and in contact with adjacent area 7 or posterior ectosylvian (PE) cortex of the opposite hemisphere. In both instances cooling of pMS cortex, but neither area 7 nor PE, restored a virtually normal level of orienting performance to stimuli presented anywhere in the previously hemianopic field. The reversal was highly sensitive to the extent of cooling deactivation. In a third cat, cooling deactivation of the superficial layers of the contralateral superior colliculus also restored orienting performance to a cortical ablation-induced hemianopia. This reversal was graded from center-to-periphery in a temperature-dependent manner. Neither the cortical ablation nor any of the cooling deactivations had any impact on an auditory detection and orienting task. The deactivations were localized and confirmed by reduced uptake of radiolabeled 2-deoxyglucose to be limited to the immediate vicinity of each cooling loop. The results are discussed in terms of excitation and disinhibition of visual circuits.     

Scalp and limb defects with cutis marmorata telangiectatica congenita: Adams-Oliver syndrome?

We report on a boy with congenital scalp and limb defects, consistent with a diagnosis of Adams-Oliver syndrome (aplasia cutis congenita with terminal transverse limb defects). An additional finding present in this child and in his mother was cutis marmorata telangiectatica congenita. Although this boy fits the diagnostic criteria for Adams-Oliver syndrome, his mother does not. We discuss whether this condition is highly variable, or heterogeneous.     

Endotoxin Binding and Elimination by Monocytes: Secretion of Soluble CD14 Represents an Inducible Mechanism Counteracting Reduced Expression of Membrane CD14 in Patients with Sepsis and in a Patient with Paroxysmal Nocturnal Hemoglobinuria

Little is known about the role of peripheral blood mononuclear cells (PBMCs) in lipopolysaccharide (LPS) elimination. We studied the endotoxin elimination capacities (EEC) of PBMCs of 15 healthy volunteers, 13 patients with sepsis, and 1 patient suffering from paroxysmal nocturnal hemoglobinuria (PNH). Although expression of CD14, the best-characterized receptor for LPS to date, was reduced from 93.6% ± 0.8% in healthy subjects to 50.5% ± 6.5% in patients with sepsis and was 0.3% in a patient with septic PNH, EEC were found to be unchanged. There was no difference in the amount of tumor necrosis factor alpha (TNF-α) released by PBMCs of healthy donors and patients with sepsis. Anti-CD14 antibodies (MEM-18) completely suppressed EEC, binding of fluorescein isothiocyanate-labeled LPS to monocytes as determined by FACScan analysis, and TNF-α release in all three groups studied. The concentrations of soluble CD14 (sCD14) secreted by endotoxin-stimulated PBMCs from healthy donors and patients with sepsis amounted to 4.5 ± 0.4 and 20.1 ± 1.8 ng/ml, respectively. Based on our results, we suggest that PBMCs eliminate LPS by at least two different mechanisms; in healthy subjects, the membrane CD14 (mCD14) receptor is the most important factor for LPS elimination, while in patients with sepsis (including the septic state of PNH), increased sCD14 participates in LPS elimination. Secretion of sCD14 is strongly enhanced under conditions of low expression of mCD14 in order to counteract the reduction of mCD14 and maintain the function of monocytes. This sCD14 may substitute the role of mCD14 in LPS elimination during sepsis. The elimination of LPS by PBMCs correlates with the binding reaction and the secretion of TNF-α.     

Generation of the eosinophil chemotactic factor (ECF) from various cell types by melittin

The peptide melittin, the main constituent of bee venom is a potent stimulus for the generation of an eosinophil chemotactic factor (ECF) from human polymorphonuclear neutrophils, rat mast cells and rat peritoneal cells depleted in mast cells. Optimal EFC induction required a sublytic activation of the cells. With each cell type the kinetics of ECF generation were similar in that after an early rise in activity a steep fall off occurred at later times of incubation suggesting a mechanism of inactivation. The induction of ECF by melittin is increased in the presence of calcium. The polar portion of the melittin molecule (aminoacids 20–26) is responsible for the generation of the chemotactic activity. Other peptides of honey bee venom such as the mast cell degranulating peptide (MCD) or apamine do not initiate ECF release. It appears that melittin leads to ECF induction via the phospholipase A₂-arachidonic acid dependent pathway of cell activation. Our data suggests that the lipid mediator ECF can be obtained from phagocytes and mast cells thus indicating the interdependence of inflammatory reactions.