Efficacy, safety, tolerability and pharmacokinetics of a novel human immune globulin subcutaneous, 20%: a Phase 2/3 study in Europe in patients with primary immunodeficiencies.

A highly concentrated (20%) immunoglobulin (Ig)G preparation for subcutaneous administration (IGSC 20%), would offer a new option for antibody replacement therapy in patients with primary immunodeficiency diseases (PIDD). The efficacy, safety, tolerability and pharmacokinetics of IGSC 20% were evaluated in a prospective trial in Europe in 49 patients with PIDD aged 2-67 years. Over a median of 358 days, patients received 2349 IGSC 20% infusions at monthly doses equivalent to those administered for previous intravenous or subcutaneous IgG treatment. The rate of validated acute bacterial infections (VASBIs) was significantly lower than 1 per year (0·022/patient-year, P < 0·0001); the rate of all infections was 4·38/patient-year. Median trough IgG concentrations were ≥ 8 g/l. There was no serious adverse event (AE) deemed related to IGSC 20% treatment; related non-serious AEs occurred at a rate of 0·101 event/infusion. The incidence of local related AEs was 0·069 event/infusion (0·036 event/infusion, when excluding a 13-year-old patient who reported 79 of 162 total related local AEs). The incidence of related systemic AEs was 0·032 event/infusion. Most related AEs were mild, none were severe. For 64·6% of patients and in 94·8% of IGSC 20% infusions, no local related AE occurred. The median infusion duration was 0·95 (range = 0·3-4·1) h using mainly one to two administration sites [median = 2 sites (range = 1-5)]. Almost all infusions (99·8%) were administered without interruption/stopping or rate reduction. These results demonstrate that IGSC 20% provides an effective and well-tolerated therapy for patients previously on intravenous or subcutaneous treatment, without the need for dose adjustment.

Efficacy and safety of OBI-1, an antihaemophilic factor VIII (recombinant), porcine sequence, in subjects with acquired haemophilia A.

Acquired haemophilia A (AHA) is a rare bleeding disorder caused by autoantibodies against human factor VIII (hFVIII). OBI-1 is an investigational, B-domain deleted, recombinant FVIII, porcine sequence, with low cross-reactivity to anti-hFVIII antibodies. Efficacy can be monitored with FVIII activity levels in addition to clinical assessments. This prospective, open label, phase 2/3 study was designed to evaluate the efficacy of OBI-1 treatment for bleeding episodes in subjects with AHA. After an initial dose of 200 U kg(-1) , OBI-1 was titrated to maintain target FVIII activity levels, in correlation with clinical assessments, throughout the treatment phase. All 28 subjects with AHA had a positive response to OBI-1 treatment 24 h after initiation despite inhibition of FVIII activity levels immediately after infusion in 10 subjects with baseline anti-porcine FVIII inhibitors. Control of the qualifying bleed was ultimately achieved in 24 of 28 subjects. No related serious adverse events, thrombotic events, allergic reactions or thrombocytopaenia occurred. The results of this study indicate that OBI-1 is safe and effective in treating bleeding episodes in subjects with AHA. The ability to safely and effectively titrate dosing based on FVIII activity levels in this study demonstrates that OBI-1 fulfils the unmet medical need to monitor the key coagulation parameter in AHA patients.

Identification of a bidirectional splicing enhancer: differential involvement of SR proteins in 5' or 3' splice site activation.

The adenovirus E1A pre-mRNA undergoes alternative splicing whose modulation occurs during infection, through the use of three different 5' splice sites and of one major or one minor 3' splice site. Although this pre-mRNA has been extensively used as a model to compare the transactivation properties of SR proteins, no cis-acting element has been identified in the transcript sequence. Here we describe the identification and the characterization of a purine-rich splicing enhancer, located just upstream of the 12S 5' splice site, which is formed from two contiguous 9-nucleotide (nt) purine motifs (Pu1 and Pu2). We demonstrate that this sequence is a bidirectional splicing enhancer (BSE) in vivo and in vitro, because it activates both the downstream 12S 5' splice site through the Pu1 motif and the upstream 216-nt intervening sequence (IVS) 3' splice site through both motifs. UV cross-linking and immunoprecipitation experiments indicate that the BSE interacts with several SR proteins specifically, among them 9G8 and ASF/SF2, which bind preferentially to the Pu1 and Pu2 motifs, respectively. Interestingly, we show by in vitro complementation assays that SR proteins have distinct transactivatory properties. In particular, 9G8, but not ASF/SF2 or SC35, is able to strongly activate the recognition of the 12S 5' splice site in a BSE-dependent manner in wild-type E1A or in a heterologous context, whereas ASF/SF2 or SC35, but not 9G8, activates the upstream 216-nt IVS splicing. Thus, our results identify a novel exonic BSE and the SR proteins which are involved in its differential activity.

The RNA-binding protein TIA-1 is a novel mammalian splicing regulator acting through intron sequences adjacent to a 5' splice site.

Splicing of the K-SAM alternative exon of the fibroblast growth factor receptor 2 gene is heavily dependent on the U-rich sequence IAS1 lying immediately downstream from its 5' splice site. We show that IAS1 can activate the use of several heterologous 5' splice sites in vitro. Addition of the RNA-binding protein TIA-1 to splicing extracts preferentially enhances the use of 5' splice sites linked to IAS1. TIA-1 can provoke a switch to use of such sites on pre-mRNAs with competing 5' splice sites, only one of which is adjacent to IAS1. Using a combination of UV cross-linking and specific immunoprecipitation steps, we show that TIA-1 binds to IAS1 in cell extracts. This binding is stronger if IAS1 is adjacent to a 5' splice site and is U1 snRNP dependent. Overexpression of TIA-1 in cultured cells activates K-SAM exon splicing in an IAS1-dependent manner. If IAS1 is replaced with a bacteriophage MS2 operator, splicing of the K-SAM exon can no longer be activated by TIA-1. Splicing can, however, be activated by a TIA-1-MS2 coat protein fusion, provided that the operator is close to the 5' splice site. Our results identify TIA-1 as a novel splicing regulator, which acts by binding to intron sequences immediately downstream from a 5' splice site in a U1 snRNP-dependent fashion. TIA-1 is distantly related to the yeast U1 snRNP protein Nam8p, and the functional similarities between the two proteins are discussed.

Metabolic response to insulin and glucose infusions in starved tumor-bearing rats.

Tumor-induced alterations in insulin sensitivity and glucose metabolism were investigated by examining the effect of glucose and insulin infusions in 72-h-starved tumor-bearing (TB) rats. Following glucose infusion, the rate of glucose disappearance from the blood was similar in TB and non-tumor-bearing (NTB) rats, even though insulin concentrations were lower in TB rats. Blood lactate was increased in TB rats prior to treatment and increased immediately following glucose infusion. Insulin alone decreased blood glucose in NTB but not TB rats. When insulin was infused together with glucose, the rate of glucose disappearance increased similarly in both TB and NTB rats. The immediate increase in blood lactate seen in TB rats following glucose infusion was not apparent in the TB rats receiving insulin and glucose. TB rats infused with glucose and insulin showed a greater rise in blood alanine concentrations, compared with all other infusion regimens. While ketone body concentrations decreased in both TB and NTB rats in response to the different infusion regimens, plasma free fatty acids in TB rats were not decreased by insulin and glucose treatments. TB rats therefore not only have decreased insulin release, but adipose tissue is also less sensitive to insulin action. In vivo studies using 2-deoxy[U-14C]glucose showed that glucose uptake by the muscle and adipose tissue, but not the tumor, was significantly increased by the infusion of insulin, thereby demonstrating one of the mechanisms by which insulin may act to conserve host tissue.

Protein tyrosine kinases in activation signal of human basophils through the immunoglobulin E receptor type l.

Human basophils activated through high-affinity immunoglobulin E (IgE) receptors (Fc epsilon RI) are involved in the late phase of the allergic reaction. To investigate the possible involvement of protein-tyrosine kinases in this activation we used human acute basophilic leukemia (ABL) cells in culture as well as a pure population of normal basophils in vitro-derived from human bone marrow precursor cells (HBMB). ABL cells were 50-80% basophils at various stages of maturation as assessed by staining, morphology, ultrastructure, and flow cytometry analysis, and only basophils in ABL cells expressed Fc epsilon RI. Aggregation of Fc epsilon RI by IgE and anti-IgE, IgE and antigen, or anti-Fc epsilon RI monoclonal antibodies on ABL cells or on HBMB, led to increased tyrosine phosphorylation of 120-, 100-, 80-, 72-, 50- to 65-, and 38-kDa substrates. Tyrosine phosphorylations in ABL cells were in basophils because 1) they were detected after a 5-s stimulation, 2) they were observed under conditions where mediator release is minimal, i.e., in the absence of extracellular calcium, 3) hapten addition during antigen stimulation resulted in almost total disappearance of tyrosine phosphorylations within 30 s. There was correlation between histamine release and tyrosine phosphorylation in anti-IgE dose-responses and in dose-responses of the tyrosine kinase inhibitor genistein. The tyrosine kinase p72syk was detected in the cells. Stimulation of ABL cells for 1 min resulted in extracellular calcium-independent tyrosine phosphorylation and activation of p72syk. Therefore, tyrosine kinases are involved in the early steps of human Fc epsilon RI signaling in basophils. Tyrosine kinases and their substrates could represent new potential therapeutic targets to prevent the development of the allergic reaction.

A CpG-rich RNA and an RNA helicase tightly associated with the DNA demethylation complex are present mainly in dividing chick embryo cells.

In the developing chicken embryo, active DNA demethylation requires both RNA and proteins (Nucleic Acids Res. 25, 2375-2380, 1997; ibid. 25, 4545-4550, 1997, FEBS Lett. 449, 251-254, 1999a). In vitro assays indicate that in the 5- and 12-day-old embryos the highest specific activity of 5-methylcytosine DNA glycosylase is found in the brain, the eyes and the skin. In situ hybridization with antisense CpG-rich RNA tightly associated to the DNA demethylation complex shows a restricted expression pattern only in proliferating tissues such as the neuroepithelia of the brain in 5-day-old embryos. The RNA is absent in differentiated tissues like the skeletal and heart muscle, liver and the crystallin-producing cells in the lens. The CpG-rich RNA is transcribed in a developmental stage-specific rather than in a cell-specific manner. In contrast transcripts of DNA methyltransferase are found in dividing and quiescent cells. In situ hybridization with a probe of a RNA helicase which is also associated with the DNA demethylation complex shows a very similar localization in mitotically active tissues as the CpG-rich RNA. The content of 5-methylcytosine in individual cells was determined with a specific monoclonal antibody and cytometric analysis on tissue sections. The results indicate that proliferating cells have on the average 15% more methylated cytosines than non-dividing cells. This represents roughly 3x10(6) more methylation sites per haploid genome.

Endothelin-1 and ETA receptor expression in vascular smooth muscle cells from human placenta: a new ETA receptor messenger ribonucleic acid is generated by alternative splicing of exon 3.

Endothelin-1 (ET-1) is a potent vasoactive peptide in stem villi vessels, which are considered to be the major sites of placental vascular resistance. To investigate the influence of pregnancy-specific hormonal environment on ET and ET receptor (ET-R) expression, we first developed and characterized a culture of vascular smooth muscle cells from stem villi vessels. Secondly, we investigated whether the muscular layer of stem villi vessels could be a site of the ET expression described in the placenta, and we examined this expression in placental vascular smooth muscle cells (PVSMCs). Prepro-ET-1 and prepro-ET-3 messenger ribonucleic acid (mRNA) were identified in stem villi vessels, whereas only prepro-ET-1 mRNA was observed in PVSMCs. Third, with the goal of using PVSMCs as ET target cells, we characterized the ET-R expressed by these cells in comparison with the muscular layer of stem villi vessels. Whereas both ETA-R and ETB-R are present in stem villi vessels, we found that PVSMCs express exclusively ETA-R. In addition to the previously reported ETA-R spliced transcripts, we described a new ETA-R transcript, ETA-R delta 3, generated by exclusion of exon 3 in stem villi vessels and PVSMCs. Alternative splicing mechanisms of ETA-R mRNA could constitute a control of the abundance of active ETA-R in terms of contractility. PVSMCs will be a useful model to study the environmental stimuli involved in the regulation of ET and ET-R expression in the muscular layer of feto-placental vasculature.

Effect of low dose rate ionizing radiation on the division potential of cells in vitro. VIII. Cytogenetic analysis of human fibroblasts.

Human embryonic and adult cells were irradiated with fractionated doses of low dose rate ionizing radiation starting early during their lifespan. Adult cells were found to be more sensitive than fetal cells to ionizing radiation in terms of the number of cells produced during the lifespan of the control and the irradiated cultures. Phase-III adult control cells had fewer chromosomal aberrations than phase-III embryonic control cells. After irradiation there was an increase in chromosomal aberrations in adult cells but no increase in embryonic cells beyond those found in the control cultures. It is suggested that cells that have a higher potential for chromosomal rearrangements survive better after low dose rate ionizing radiation.

Expression of endothelin precursor genes in human trophoblast in culture.

We have shown previously the presence of immunoreactive endothelin in cultured trophoblastic cells from human term placenta as well as in the trophoblast-conditioned medium. To confirm whether or not the differentiated syncytiotrophoblast is a site for endothelin synthesis, we investigated, by reverse transcription and polymerase chain reaction, the expression of the three preproendothelin genes in 3-day cultured trophoblast. While no endothelin-2 precursor mRNA was detected, preproendothelin-1 mRNA was found to be expressed by the trophoblast. The endothelin-3 precursor gene was also expressed, but at low level and it was detected only after Southern blotting and oligonucleotide hybridization. The ability of trophoblast in culture to express the endothelin precursor genes supports the idea that, in human term placenta, villous syncytiotrophoblast that lines the intervillous space containing maternal blood acts as an endothelial layer.

AIDS-related primary brain lymphomas: histopathologic and immunohistochemical study of 51 cases. The French Study Group for HIV-Associated Tumors.

Fifty-one cases of acquired immunodeficiency syndrome (AIDS)-related primary brain lymphomas (AR-PBL) were investigated for clinical characteristics; human immunodeficiency virus (HIV)-associated disorders; histopathologic features; immunophenotype; Epstein-Barr virus (EBV) infection; and, when frozen tissue was available, oncogene rearrangements. AR-PBL occurred late in the course of AIDS and were usually associated with other systemic or cerebral disorders and with a low level of CD4 lymphocytes. All cases were high grade lymphomas according to the Working Formulation or updated Kiel classification, and often displayed a multifocal pattern. Thirty cases were classified as immunoblastic with plasmacytic differentiation, 18 cases were large cell lymphomas with an immunoblastic component or centroblastic polymorphic lymphomas, and 2 were small noncleaved non-Burkitt lymphomas (Working Formulation). This latter category is classified as Burkitt's-like lymphoma in the REAL nomenclature. One case could not be classified because of necrosis. AR-PBL showed a high level expression of activation and adhesion molecules. The presence of EBV was detected in most cases, and, when PCR was used, this was a constant finding. bcl-2 oncoprotein and latent membrane protein-1 (LMP-1) were strongly expressed. None of the tested cases expressed p53, or were rearranged for bcl-2 or c-myc oncogenes. This study confirms the immunophenotypic specificity of AR-PBL, which may reflect the special immune status of the brain.

Characterization of chromosome changes in two human prostatic carcinoma cell lines (PC-3 and DU145) using chromosome painting and comparative genomic hybridization.

Using chromosome painting, a study of chromosomal abnormalities has been performed in two prostatic carcinoma cell lines, PC-3 and DU145. In PC-3, this analysis revealed a highly rearranged hypotriploid karyotype with 54 to 61 chromosomes and numerous rearrangements of chromosomes 1, 3, 5, 8, 10, and 14. At passage 73, DU145 had a hypotriploid karyotype with few rearrangements of chromosomes 1, 3, 5, 12, 13, and 20, whereas at passage 153, this cell line showed a near-tetraploid karyotype with a great number of rearrangements involving chromosomes 3, 6, 8, 10, 12, and 17. A single rearrangement was shared by the 2 cell lines, an i(5)(p10). A comparative genomic hybridization study demonstrated a noticeable amplification of bands 10q22.3-q23 and 14q22-q24 in the PC-3 cell line. No amplification signal was detected for DU145.

DNA hypomethylation in breast cancer: an independent parameter of tumor progression?

The global DNA methylation status was investigated on a series of 59 breast cancers by Southern blotting, using methylation sensitive restriction enzymes. By comparison to control DNA, almost all tumor DNAs were found globally hypomethylated. However, the demethylation was variable from tumor to tumor. Compared to other biological parameters, the methylation did not correlate with chromosome alterations, steroid hormone receptor status, or histopathological grading. Tumors which appeared to be the most evolved for other parameters were only mildly hypomethylated, whereas tumors with strongly hypomethylated DNA corresponded to those with slight alterations of the other parameters. Thus, DNA hypomethylation is a consistent characteristic of breast cancer, but its variations may not correlate with tumor progression of most breast cancers.

Value of a ligase chain reaction assay for detection of ganciclovir resistance-related mutation 594 in UL97 gene of human cytomegalovirus.

Human cytomegalovirus (HCMV) isolates resistant to ganciclovir were found in patients undergoing therapy. Therefore, we have developed a new specific and sensitive method--a ligase chain reaction (LCR) assay--for detection of frequently encountered 594 mutated codon in ganciclovir (GCV) resistant virus. Previous studies characterized an alanine to valine change on codon 594 in resistant strains. A novel substitution in 594, alanine to glycine, is described which is also capable of conferring ganciclovir resistance. LCR products were analyzed on polyacrylamide gel- and the mutant was detected using a non radioactive method. The LCR product detection was then adapted to a microtitre plate format with a colorimetric detection. This method allowed the distinction of mutated GCV-resistant strains from sensitive strains with a high sensitivity, and the detection of a low percentage of mutated DNA in virus load. This assay could be useful in following the evolution of mutated DNA compared to viral infection.

Altered insulin response to glucose in weight-losing cancer patients.

Cancer cachexia and the underlying metabolic disturbances are due in part to either altered insulin release and action. Glucose intolerance in cancer patients is frequently observed but the nature of the insulin response is not usually described. The aim of this study was to investigate the insulin response in fasted, weigh-losing cancer patients following an oral glucose load (75 g). All cancer patients (n = 35) showed glucose intolerance. Three types of response were identified; those with an increased insulin: glucose ratio (I:G) at 60 min, (average 12.3, n = 13), those with a normal I:G (average 7.2 n = 7) and those with a decrease I:G (average 4.2, n = 15). Fasting plasma glucose concentrations were normal in all groups prior to the glucose tolerance test. However, patients with the lowest I:G also had the lowest fasting plasma insulin concentrations, the lowest plasma albumin concentrations and the highest plasma triglyceride concentrations. Those patients with an abnormal insulin response (either high or low I:G) had significantly greater weight loss (16% for low I:G group, 13% for the high I:G) compared to the normal responders (8%). Plasma fatty acid concentrations were increased in all cancer patients and decreased appropriately after glucose administration, indicating that lipolysis remained sensitive to the action of insulin. It is concluded that weight loss in cancer is associated with glucose intolerance and an abnormal insulin response, and that this response is indicative of either insulin resistance (high I:G) or decreased pancreatic function (low I:G). These findings suggest a role for insulin replacement therapy in the latter group of patients.

Modelling Bacillus cereus growth.

The aim of this study was to model the growth, in a model system, of toxigenic strains of Bacillus cereus as a function of temperature, pH and water activity. Optical density (OD) values were transformed into numbers of colony-forming units (CFU) by the use of a 'calibrating' relation. The growth curves were then fitted to the Gompertz function which allowed the estimation of the lag-time (lambda) and the growth rate (mu). These two parameters were then modelled according to the controlling factors which were pH and water activity at 20 and 30 degrees C.

Modelling the growth kinetics of Listeria as a function of temperature, pH and organic acid concentration.

The combined effects of temperature, pH and organic acids (lactic, acetic and propionic) on the growth kinetics of Listeria innocua ATCC 33090 were studied. First, a multiplicative model was built assuming independent effects of all environmental factors. Thus, the model was expanded by the inclusion of a novel term describing the effects of interactions on the growth/no growth limits. The proposed approach allows an accurate description of the boundary between growth and no growth of Listeria.

Embryo-maternal interactions at the implantation site: a delicate equilibrium.

Blastocyst implantation and successful establishment of pregnancy require delicate interactions between the embryo and the maternal environment. During preimplantation, maternal/embryo communication is mediated by the trophectoderm. In the late luteal phase, physiological changes occur in the endometrium to allow blastocyst implantation. The "window of implantation" represents the period of maximum uterine receptivity for implantation. In response to signals from the embryo, pregnancy-specific proteins are released in maternal serum and a series of morphological, biochemical and immunological changes occur in the uterine environment. These systemic and local modifications can be considered to constitute "the maternal recognition of pregnancy". The human hemochorial placenta arises primarily through proliferation, migration and invasion of the endometrium and its vasculature by the embryonic trophoblast. The complex invasive processes accompanying implantation of the embryo are controlled at the embryo-maternal interface by factors from decidualized endometrium and the trophoblast itself. An inflammatory reaction and a proper maternal immune response allow survival and development of the feto-placental unit. In this review, we focus on interactions between trophoblast and uterine tissues and on cellular mechanisms and molecular signals involved in the closely regulated process of implantation.

Centrifugation and addition of glycerol at 22 degres C instead of 4 degrees C improve post-thaw motility and fertility of stallion spermatozoa.

The aims of this study were to evaluate the effects of cooling rate to 4 degrees C and temperature at the time of centrifugation/glycerol-addition (freezing extender: INRA82 + 2% egg yolk + 2.5% glycerol) on postcentrifugation recovery rate, post-thaw motility and per-cycle fertility. When centrifugation/glycerol-addition was performed at 4 degrees C (14 ejaculates), a moderate cooling rate (37 degrees C to 4 degrees C in I h) resulted in higher post-thaw motility (45%) than when using a slow cooling rate (37 degrees C to 4 degrees C in 4 h) (39%; P<0.05). When centrifugation/glycerol-addition was performed at 22 degrees C (37 degrees C to 22 degrees C in 10 min) (10 ejaculates), post-thaw motility was lower when spermatozoa were frozen directly from 22 degrees C (23%) than when spermatozoa were cooled to 4 degrees C (22 degrees C to 4 degrees C in 1 h) before freezing (47%; P<0.0001). When centrifugation/glycerol-addition was performed at 22 degrees C (before cooling at a moderate rate), as opposed to 4 degrees C (after cooling at a moderate rate), a significant improvement of 1) recovery of spermatozoa after centrifugation (P<0,0001), 2) post-thaw motility of spermatozoa at thawing (40% vs 36% (n < or = 291 ejaculates/group), P<0.0001) and 3) per-cycle fertility (56% vs 42% (n > or = 190 cycles/group), P<0.01) was observed. In conclusion, centrifugation/glycerol-addition at 22 degrees C followed by cooling to 4 degrees C at a moderate rate results in an improvement of post-thaw motility, spermatozoa recovery rate and per cycle fertility.