A novel anti-CD19 monoclonal antibody (GBR 401) with high killing activity against B cell malignancies.

BACKGROUND: CD19 is a B cell lineage specific surface receptor whose broad expression, from pro-B cells to early plasma cells, makes it an attractive target for the immunotherapy of B cell malignancies. In this study we present the generation of a novel humanized anti-CD19 monoclonal antibody (mAb), GBR 401, and investigate its therapeutic potential on human B cell malignancies. METHODS: GBR 401 was partially defucosylated in order to enhance its cytotoxic function. We analyzed the in vitro depleting effects of GBR 401 against B cell lines and primary malignant B cells from patients in the presence or in absence of purified NK cells isolated from healthy donors. In vivo, the antibody dependent cellular cytotoxicity (ADCC) efficacy of GBR 401 was assessed in a B cell depletion model consisting of SCID mice injected with healthy human donor PBMC, and a malignant B cell depletion model where SCID mice are xenografted with both primary human B-CLL tumors and heterologous human NK cells. Furthermore, the anti-tumor activity of GBR 401 was also evaluated in a xenochimeric mouse model of human Burkitt lymphoma using mice xenografted intravenously with Raji cells. Pharmacological inhibition tests were used to characterize the mechanism of the cell death induced by GBR 401. RESULTS: GBR 401 exerts a potent in vitro and in vivo cytotoxic activity against primary samples from patients representing various B-cell malignancies. GBR 401 elicits a markedly higher level of ADCC on primary malignant B cells when compared to fucosylated similar mAb and to Rituximab, the current anti-CD20 mAb standard immunotherapeutic treatment for B cell malignancies, showing killing at 500 times lower concentrations. Of interest, GBR 401 also exhibits a potent direct killing effect in different malignant B cell lines that involves homotypic aggregation mediated by actin relocalization. CONCLUSION: These results contribute to consolidate clinical interest in developing GBR 401 for treatment of hematopoietic B cell malignancies, particularly for patients refractory to anti-CD20 mAb therapies.

Heritability, determinants and reference values of renal length: a family-based population study.

OBJECTIVES: In this population-based study, reference values were generated for renal length, and the heritability and factors associated with kidney length were assessed. METHODS: Anthropometric parameters and renal ultrasound measurements were assessed in randomly selected nuclear families of European ancestry (Switzerland). The adjusted narrow sense heritability of kidney size parameters was estimated by maximum likelihood assuming multivariate normality after power transformation. Gender-specific reference centiles were generated for renal length according to body height in the subset of non-diabetic non-obese participants with normal renal function. RESULTS: We included 374 men and 419 women (mean ± SD, age 47 ± 18 and 48 ± 17 years, BMI 26.2 ± 4 and 24.5 ± 5 kg/m(2), respectively) from 205 families. Renal length was 11.4 ± 0.8 cm in men and 10.7 ± 0.8 cm in women; there was no difference between right and left renal length. Body height, weight and estimated glomerular filtration rate (eGFR) were positively associated with renal length, kidney function negatively, age quadratically, whereas gender and hypertension were not. The adjusted heritability estimates of renal length and volume were 47.3 ± 8.5 % and 45.5 ± 8.8 %, respectively (P < 0.001). CONCLUSION: The significant heritability of renal length and volume highlights the familial aggregation of this trait, independently of age and body size. Population-based references for renal length provide a useful guide for clinicians. KEY POINTS: • Renal length and volume are heritable traits, independent of age and size. • Based on a European population, gender-specific reference values/percentiles are provided for renal length. • Renal length correlates positively with body length and weight. • There was no difference between right and left renal lengths in this study. • This negates general teaching that the left kidney is larger and longer.

Altered NKG2D function in NK cells induced by chronic exposure to NKG2D ligand-expressing tumor cells.

NKG2D is an activation receptor that allows natural killer (NK) cells to detect diseased host cells. The engagement of NKG2D with corresponding ligand results in surface modulation of the receptor and reduced function upon subsequent receptor engagement. However, it is not clear whether in addition to modulation the NKG2D receptor complex and/or its signaling capacity is preserved. We show here that the prolonged encounter with tumor cell-bound, but not soluble, ligand can completely uncouple the NKG2D receptor from the intracellular mobilization of calcium and the exertion of cell-mediated cytolysis. However, cytolytic effector function is intact since NKG2D ligand-exposed NK cells can be activated via the Ly49D receptor. While NKG2D-dependent cytotoxicity is impaired, prolonged ligand exposure results in constitutive interferon gamma (IFNgamma) production, suggesting sustained signaling. The functional changes are associated with a reduced presence of the relevant signal transducing adaptors DNAX-activating protein of 10 kDa (DAP-10) and killer cell activating receptor-associated protein/DNAX-activating protein of 12 kDa (KARAP/DAP-12). That is likely the consequence of constitutive NKG2D engagement and signaling, since NKG2D function and adaptor expression is restored to normal when the stimulating tumor cells are removed. Thus, the chronic exposure to tumor cells expressing NKG2D ligand alters NKG2D signaling and may facilitate the evasion of tumor cells from NK cell reactions.

Differential effects of two anti-apo-cytochrome c-specific monoclonal antibodies on the function of apo-cytochrome c-specific murine T cell clones.

A murine monoclonal antibody (SJL 2-4) specific for the antigen apo-cytochrome c was shown to inhibit both antigen-induced proliferation and lymphokine secretion by an apo-cytochrome c-specific BALB/c helper T cell clone. The inhibition was specific because additional apo-cytochrome c-specific T cell clones were not inhibited by the same monoclonal antibody. Time course studies of the inhibition indicated that the initial 8 hr of contact between T cell clones and antigen-presenting cells were critical for activation of the T cell clones. Inhibition of T cell functions by antigen-specific antibodies appeared to correlate with the antibody-antigen binding constant because a second monoclonal antibody (Cyt-1-59), with identical specificity but with a lower affinity constant for apo-cytochrome c, had very little inhibitory effect on the proliferation or lymphokine secretion of apo-cytochrome c-specific T cell clones.

Structure-function relationships of the alpha1b-adrenergic receptor.

The alpha1b-adrenergic receptor (AR) is a member of the large superfamily of seven transmembrane domain (TMD) G protein-coupled receptors (GPCR). Combining site-directed mutagenesis of the alpha1b-AR with computational simulations of receptor dynamics, we have explored the conformational changes underlying the process of receptor activation, i.e. the transition between the inactive and active states. Our findings suggest that the structural constraint stabilizing the alpha1b-AR in the inactive form is a network of H-bonding interactions amongst conserved residues forming a polar pocket and R143 of the DRY sequence at the end of TMDIII. We have recently reported that point mutations of D142, of the DRY sequence and of A293 in the distal portion of the third intracellular loop resulted in ligand-independent (constitutive) activation of the alpha1b-AR. These constitutively activating mutations could induce perturbations resulting in the shift of R143 out of the polar pocket. The main role of R143 may be to mediate receptor activation by triggering the exposure of several basic amino acids of the intracellular loops towards the G protein. Our investigation has been extended also to the biochemical events involved in the desensitization process of alpha1b-AR. Our results indicate that immediately following agonist-induced activation, the alpha1b-AR can undergo rapid agonist-induced phosphorylation and desensitization. Different members of the G protein coupled receptor kinase family can play a role in agonist-induced regulation of the alpha1b-AR. In addition, constitutively active alpha1b-AR mutants display different phosphorylation and internalization features. The future goal is to further elucidate the molecular mechanism underlying the complex equilibrium between activation and inactivation of the alpha1b-AR and its regulation by pharmacological substances. These findings can help to elucidate the mechanism of action of various agents displaying properties of agonists or inverse agonists at the adrenergic system.

Connexin-36 contributes to control function of insulin-producing cells.

Connexin-36 (Cx36) is a gap junction protein expressed by the insulin-producing beta-cells. We investigated the contribution of this protein in normal beta-cell function by using a viral gene transfer approach to alter Cx36 content in the insulin-producing line of INS-1E cells and rat pancreatic islets. Transcripts for Cx43, Cx45, and Cx36 were detected by reverse transcriptase-PCR in freshly isolated pancreatic islets, whereas only a transcript for Cx36 was detected in INS-1E cells. After infection with a sense viral vector, which induced de novo Cx36 expression in the Cx-defective HeLa cells we used to control the transgene expression, Western blot, immunofluorescence, and freeze-fracture analysis showed a large increase of Cx36 within INS-1E cell membranes. In contrast, after infection with an antisense vector, Cx36 content was decreased by 80%. Glucose-induced insulin release and insulin content were decreased, whether infected INS-1E cells expressed Cx36 levels that were largely higher or lower than those observed in wild-type control cells. In both cases, basal insulin secretion was unaffected. Comparable observations on basal secretion and insulin content were made in freshly isolated rat pancreatic islets. The data indicate that large changes in Cx36 alter insulin content and, at least in INS-1E cells, also affect glucose-induced insulin release.

Use of a human-like low-grade bacteremia model of experimental endocarditis to study the role of Staphylococcus aureus adhesins and platelet aggregation in early endocarditis.

Animal models of infective endocarditis (IE) induced by high-grade bacteremia revealed the pathogenic roles of Staphylococcus aureus surface adhesins and platelet aggregation in the infection process. In humans, however, S. aureus IE possibly occurs through repeated bouts of low-grade bacteremia from a colonized site or intravenous device. Here we used a rat model of IE induced by continuous low-grade bacteremia to explore further the contributions of S. aureus virulence factors to the initiation of IE. Rats with aortic vegetations were inoculated by continuous intravenous infusion (0.0017 ml/min over 10 h) with 10(6) CFU of Lactococcus lactis pIL253 or a recombinant L. lactis strain expressing an individual S. aureus surface protein (ClfA, FnbpA, BCD, or SdrE) conferring a particular adhesive or platelet aggregation property. Vegetation infection was assessed 24 h later. Plasma was collected at 0, 2, and 6 h postinoculation to quantify the expression of tumor necrosis factor (TNF), interleukin 1α (IL-1α), IL-1β, IL-6, and IL-10. The percentage of vegetation infection relative to that with strain pIL253 (11%) increased when binding to fibrinogen was conferred on L. lactis (ClfA strain) (52%; P = 0.007) and increased further with adhesion to fibronectin (FnbpA strain) (75%; P < 0.001). Expression of fibronectin binding alone was not sufficient to induce IE (BCD strain) (10% of infection). Platelet aggregation increased the risk of vegetation infection (SdrE strain) (30%). Conferring adhesion to fibrinogen and fibronectin favored IL-1β and IL-6 production. Our results, with a model of IE induced by low-grade bacteremia, resembling human disease, extend the essential role of fibrinogen binding in the initiation of S. aureus IE. Triggering of platelet aggregation or an inflammatory response may contribute to or promote the development of IE.

In vitro engineering of human stratified urothelium: analysis of its morphology and function.

PURPOSE: Gastric or intestinal patches, commonly used for reconstructive cystoplasty, may induce severe metabolic complications. The use of bladder tissues reconstructed in vitro could avoid these complications. We compared cellular differentiation and permeability characteristics of human native with in vitro cultured stratified urothelium. MATERIALS AND METHODS: Human stratified urothelium was induced in vitro. Morphology was studied with light and electron microscopy and expression of key cellular proteins was assessed using immunohistochemistry. Permeability coefficients were determined by measuring water, urea, ammonia and proton fluxes across the urothelium. RESULTS: As in native urothelium the stratified urothelial construct consisted of basal membrane and basal, intermediate and superficial cell layers. The apical membrane of superficial cells formed villi and glycocalices, and tight junctions and desmosomes were developed. Immunohistochemistry showed similarities and differences in the expression of cytokeratins, integrin and cellular adhesion proteins. In the cultured urothelium cytokeratin 20 and integrin subunits alpha6 and beta4 were absent, and symplekin was expressed diffusely in all layers. Uroplakins were clearly expressed in the superficial umbrella cells of the urothelial constructs, however, they were also present in intermediate and basal cells. Symplekin and uroplakins were expressed only in the superficial cells of native bladder tissue. The urothelial constructs showed excellent viability, and functionally their permeabilities for water, urea and ammonia were no different from those measured in native human urothelium. Proton permeability was even lower in the constructs compared to that of native urothelium. CONCLUSIONS: Although the in vitro cultured human stratified urothelium did not show complete terminal differentiation of its superficial cells, it retained the same barrier characteristics against the principal urine components. These results indicate that such in vitro cultured urothelium, after being grown on a compliant degradable support or in coculture with smooth muscle cells, is suitable for reconstructive cystoplasty.

IV thrombolysis and renal function.

OBJECTIVE: To investigate the association of renal impairment on functional outcome and complications in stroke patients treated with IV thrombolysis (IVT). METHODS: In this observational study, we compared the estimated glomerular filtration rate (GFR) with poor 3-month outcome (modified Rankin Scale scores 3-6), death, and symptomatic intracranial hemorrhage (sICH) based on the criteria of the European Cooperative Acute Stroke Study II trial. Unadjusted and adjusted odds ratios (ORs) with 95% confidence intervals (CIs) were calculated. Patients without IVT treatment served as a comparison group. RESULTS: Among 4,780 IVT-treated patients, 1,217 (25.5%) had a low GFR (<60 mL/min/1.73 m(2)). A GFR decrease by 10 mL/min/1.73 m(2) increased the risk of poor outcome (OR [95% CI]): (ORunadjusted 1.20 [1.17-1.24]; ORadjusted 1.05 [1.01-1.09]), death (ORunadjusted 1.33 [1.28-1.38]; ORadjusted 1.18 [1.11-1.249]), and sICH (ORunadjusted 1.15 [1.01-1.22]; ORadjusted 1.11 [1.04-1.20]). Low GFR was independently associated with poor 3-month outcome (ORadjusted 1.32 [1.10-1.58]), death (ORadjusted 1.73 [1.39-2.14]), and sICH (ORadjusted 1.64 [1.21-2.23]) compared with normal GFR (60-120 mL/min/1.73 m(2)). Low GFR (ORadjusted 1.64 [1.21-2.23]) and stroke severity (ORadjusted 1.05 [1.03-1.07]) independently determined sICH. Compared with patients who did not receive IVT, treatment with IVT in patients with low GFR was associated with poor outcome (ORadjusted 1.79 [1.41-2.25]), and with favorable outcome in those with normal GFR (ORadjusted 0.77 [0.63-0.94]). CONCLUSION: Renal function significantly modified outcome and complication rates in IVT-treated stroke patients. Lower GFR might be a better risk indicator for sICH than age. A decrease of GFR by 10 mL/min/1.73 m(2) seems to have a similar impact on the risk of death or sICH as a 1-point-higher NIH Stroke Scale score measuring stroke severity.

Spatial modelling of biodiversity at the community level

1. Statistical modelling is often used to relate sparse biological survey data to remotely derived environmental predictors, thereby providing a basis for predictively mapping biodiversity across an entire region of interest. The most popular strategy for such modelling has been to model distributions of individual species one at a time. Spatial modelling of biodiversity at the community level may, however, confer significant benefits for applications involving very large numbers of species, particularly if many of these species are recorded infrequently. 2. Community-level modelling combines data from multiple species and produces information on spatial pattern in the distribution of biodiversity at a collective community level instead of, or in addition to, the level of individual species. Spatial outputs from community-level modelling include predictive mapping of community types (groups of locations with similar species composition), species groups (groups of species with similar distributions), axes or gradients of compositional variation, levels of compositional dissimilarity between pairs of locations, and various macro-ecological properties (e.g. species richness). 3. Three broad modelling strategies can be used to generate these outputs: (i) 'assemble first, predict later', in which biological survey data are first classified, ordinated or aggregated to produce community-level entities or attributes that are then modelled in relation to environmental predictors; (ii) 'predict first, assemble later', in which individual species are modelled one at a time as a function of environmental variables, to produce a stack of species distribution maps that is then subjected to classification, ordination or aggregation; and (iii) 'assemble and predict together', in which all species are modelled simultaneously, within a single integrated modelling process. These strategies each have particular strengths and weaknesses, depending on the intended purpose of modelling and the type, quality and quantity of data involved. 4. Synthesis and applications. The potential benefits of modelling large multispecies data sets using community-level, as opposed to species-level, approaches include faster processing, increased power to detect shared patterns of environmental response across rarely recorded species, and enhanced capacity to synthesize complex data into a form more readily interpretable by scientists and decision-makers. Community-level modelling therefore deserves to be considered more often, and more widely, as a potential alternative or supplement to modelling individual species.

Evidence for transcript networks composed of chimeric RNAs in human cells.

The classic organization of a gene structure has followed the Jacob and Monod bacterial gene model proposed more than 50 years ago. Since then, empirical determinations of the complexity of the transcriptomes found in yeast to human has blurred the definition and physical boundaries of genes. Using multiple analysis approaches we have characterized individual gene boundaries mapping on human chromosomes 21 and 22. Analyses of the locations of the 5' and 3' transcriptional termini of 492 protein coding genes revealed that for 85% of these genes the boundaries extend beyond the current annotated termini, most often connecting with exons of transcripts from other well annotated genes. The biological and evolutionary importance of these chimeric transcripts is underscored by (1) the non-random interconnections of genes involved, (2) the greater phylogenetic depth of the genes involved in many chimeric interactions, (3) the coordination of the expression of connected genes and (4) the close in vivo and three dimensional proximity of the genomic regions being transcribed and contributing to parts of the chimeric RNAs. The non-random nature of the connection of the genes involved suggest that chimeric transcripts should not be studied in isolation, but together, as an RNA network.

Long-term fluctuation of relative afferent pupillary defect in subjects with normal visual function.

PURPOSE: To determine whether the relative afferent pupillary defect (RAPD) remains constant over time in normal subjects. METHODS: Seventeen normal subjects were tested with infrared pupillography and automated perimetry in four sessions over 3 years. The changes in RAPD and visual field asymmetry between testing sessions were compared. RESULTS: The range of RAPD was 0.0 to 0.3 log unit, and the difference in the mean deviation between the eyes on automated static perimetry was 0 to 3 dB. Eight subjects repeatedly had an RAPD in the same eye. There was no correlation between the RAPD and the visual field asymmetry at the same visit. Changes in the magnitude of the RAPD between any two sessions were typically small (median, 0.08 log unit; 25th percentile, 0.04 log unit; 75th percentile, 0.15 log unit). CONCLUSIONS: Some normal subjects may show a persistent but small RAPD in the absence of detectable pathologic disease. Therefore, an isolated RAPD in the range of 0.3 log unit that is not associated with any other significant historical or clinical finding should probably be considered benign.

Regulation of blood pressure and renal function by NCC and ENaC: lessons from genetically engineered mice.

The activity of the thiazide-sensitive Na(+)/Cl(-) cotransporter (NCC) and of the amiloride-sensitive epithelial Na(+) channel (ENaC) is pivotal for blood pressure regulation. NCC is responsible for Na(+) reabsorption in the distal convoluted tubule (DCT) of the nephron, while ENaC reabsorbs the filtered Na(+) in the late DCT and in the cortical collecting ducts (CCD) providing the final renal adjustment to Na(+) balance. Here, we aim to highlight the recent advances made using transgenic mouse models towards the understanding of the regulation of NCC and ENaC function relevant to the control of sodium balance and blood pressure. We thus like to pave the way for common mechanisms regulating these two sodium-transporting proteins and their potential implication in structural remodeling of the nephron segments and Na(+) and Cl(-) reabsorption.

Identification of particular groups of microRNAs that positively or negatively impact on beta cell function in obese models of type 2 diabetes.

AIMS/HYPOTHESIS: MicroRNAs are key regulators of gene expression involved in health and disease. The goal of our study was to investigate the global changes in beta cell microRNA expression occurring in two models of obesity-associated type 2 diabetes and to assess their potential contribution to the development of the disease. METHODS: MicroRNA profiling of pancreatic islets isolated from prediabetic and diabetic db/db mice and from mice fed a high-fat diet was performed by microarray. The functional impact of the changes in microRNA expression was assessed by reproducing them in vitro in primary rat and human beta cells. RESULTS: MicroRNAs differentially expressed in both models of obesity-associated type 2 diabetes fall into two distinct categories. A group including miR-132, miR-184 and miR-338-3p displays expression changes occurring long before the onset of diabetes. Functional studies indicate that these expression changes have positive effects on beta cell activities and mass. In contrast, modifications in the levels of miR-34a, miR-146a, miR-199a-3p, miR-203, miR-210 and miR-383 primarily occur in diabetic mice and result in increased beta cell apoptosis. These results indicate that obesity and insulin resistance trigger adaptations in the levels of particular microRNAs to allow sustained beta cell function, and that additional microRNA deregulation negatively impacting on insulin-secreting cells may cause beta cell demise and diabetes manifestation. CONCLUSIONS/INTERPRETATION: We propose that maintenance of blood glucose homeostasis or progression toward glucose intolerance and type 2 diabetes may be determined by the balance between expression changes of particular microRNAs.