Real-World Treatment Patterns, Outcomes, Resource Utilization and Costs in Treatment-Resistant Major Depressive Disorder: PATTERN, a Retrospective Cohort Study in Belgium.

OBJECTIVE: Treatment-resistant depression (TRD), a subgroup of major depressive disorder (MDD) that does not adequately respond to treatment, has a substantial impact on the quality of life of patients and is associated with higher medical and mental health care costs. This study aimed to report real-world treatment patterns, outcomes, resource utilization, and costs in the management of TRD by psychiatrists in Belgium. METHODS: We conducted a retrospective, non-interventional cohort study of patients ≥ 18 years, with diagnosed MDD who are treatment-resistant, defined as not responding to two different antidepressant treatments in the current moderate to severe major depressive episode (MDE). Data obtained from medical records of patients included patient health state (MDE, response, remission, and recovery) and resource use (number of consultations and emergency room visits, non-drug and drug interventions, and hospitalizations). RESULTS: One hundred and twenty-five patients were enrolled in nine sites, with an average observation period of 34 months. During the MDE, 89.7% of patients were treated with selective serotonin reuptake inhibitors, 63.2% with serotonin-norepinephrine reuptake inhibitors, and 60.8% with anti-psychotics. Twenty-four percent of patients did not respond to any treatment; 76% responded, of whom 61% experienced a relapse; 28% of patients reached recovery, of whom 31.4% experienced recurrence. The average yearly direct cost of a TRD patient is €9012, mainly driven by hospitalization in the MDE. The observed absenteeism relates to a high indirect cost, representing 70% of the total MDE cost. CONCLUSION: TRD is associated with a high unmet need and economic burden for patients and society, with highest costs in the MDE health state driven by absenteeism.

The functional gamma-secretase inhibitor prevents production of amyloid beta 1-34 in human and murine cell lines.

The amyloid precursor protein (APP) undergoes two consecutive cleavages by different proteases, beta-secretase and gamma-secretase, leading to the release of an amyloidogenic 4 kDa fragment called amyloid beta (Abeta). Combining immunoprecipitation and mass spectrometry, we characterized soluble Abeta in cultured cell media of mouse neuroblastoma N2a cells and double hAPP/hBACE-1 transfected HEK293. The major Abeta isoforms detected were Abeta11-34, Abeta1-34, Abeta11-40 and Abeta1-40. In this study, we demonstrate that overexpression of human beta-secretase (BACE-1) in HEK293 cells resulted in predominant Abeta cleavage at position Glu(11) rather than Asp(1), as well as increased production of Abeta(x)-34, but not Abeta(x)-40. Incubation of cells with a specific gamma-secretase inhibitor suggests that cleavage of APP at Leu(34) could be mediated by gamma-secretase itself or by a gamma-secretase dependent process.

NF-kappaB is involved in the regulation of CD154 (CD40 ligand) expression in primary human T cells.

Cognate interactions between CD154 (CD40 ligand, CD40L) on activated T cells and its receptor CD40 on various antigen-presenting cells are involved in thymus-dependent humoral immune responses and multiple other cell-mediated immune responses. We have studied the regulation of CD154 expression in human T cells after activation with anti-CD3 and anti-CD28 antibodies or after pharmacological activation of protein kinase C with phorbol 12-myristate 13-acetate, and the calcium ionophore ionomycin. Under these conditions, transcription of the CD154 gene was rapidly induced without requiring de novo protein synthesis. Pharmacological inhibitors of NF-kappaB activation down-regulated CD154 mRNA and protein levels. Cyclosporin A, an inhibitor of NF-AT activation, acted similarly, and the effects of both inhibitors were additive. A potential NF-kappaB binding site is present within the CD154 promoter at positions -1190 to - 1181. In electrophoretic mobility shift assays, this sequence was specifically bound by NF-kappaB present in nuclear extracts from activated T cells. Furthermore, in transient co-transfection of Jurkat T cells, p65 activated the transcription of a reporter construct containing a multimer of this NF-kappaB binding site. These observations demonstrate a role of NF-kappaB transcription factors in the regulation of CD40L expression in activated primary human T cells.

TTRAP, a novel protein that associates with CD40, tumor necrosis factor (TNF) receptor-75 and TNF receptor-associated factors (TRAFs), and that inhibits nuclear factor-kappa B activation.

CD40 belongs to the tumor necrosis factor (TNF) receptor family. CD40 signaling involves the recruitment of TNF receptor-associated factors (TRAFs) to its cytoplasmic domain. We have identified a novel intracellular CD40-binding protein termed TRAF and TNF receptor-associated protein (TTRAP) that also interacts with TNF-R75 and CD30. The region of the CD40 cytoplasmic domain that is required for TTRAP association overlaps with the TRAF6 recognition motif. Association of TTRAP with CD40 increases profoundly in response to treatment of cells with CD40L. Interestingly, TTRAP also associates with TRAFs, with the highest affinity for TRAF6. In transfected cells, TTRAP inhibits in a dose-dependent manner the transcriptional activation of a nuclear factor-kappaB (NF-kappaB)-dependent reporter mediated by CD40, TNF-R75 or Phorbol 12-myristate 13-acetate (PMA) and to a lesser extent by TRAF2, TRAF6, TNF-alpha, or interleukin-1beta (IL-1beta). TTRAP does not affect stimulation of NF-kappaB induced by overexpression of the NF-kappaB-inducing kinase (NIK), the IkappaB kinase alpha (IKKalpha), or the NF-kappaB subunit P65/RelA, suggesting it acts upstream of the latter proteins. Our results indicate that we have isolated a novel regulatory factor that is involved in signal transduction by distinct members of the TNF receptor family.

Alzheimer's disease associated presenilin 1 interacts with HC5 and ZETA, subunits of the catalytic 20S proteasome.

Proteolytic processing and degradation tightly regulate the amount of stable, functional presenilin 1 (PSEN1) in the cell. The approximately 46-kDa PSEN1 holoprotein is cleaved into a approximately 30-kDa N-terminal fragment (NTF) and a approximately 20-kDa C-terminal fragment (CTF) by an unknown protease. The fragments are stabilized in a high molecular weight complex and nonincorporated fragments and excess holoprotein are degraded by the 26S proteasome. The tight balance between, on the one hand, processing and incorporation into the stable complex and, on the other hand, proteolytic degradation of excess PSEN1, indicates that minor changes in one of these two processes could be pathologically relevant. Here we demonstrate the direct physical interaction between PSEN1 and two subunits, HC5 and ZETA, of the 20S proteasome. These interactions were identified using an interaction trap screening and were further established in an in vitro binding assay. Furthermore, we were able to coimmunoprecipitate the transfected binding partners, as well as the endogenous PSEN1 and ZETA proteins from HEK 293T cells. Finally, degradation of ubiquitinated wild-type and mutant PSEN1 by the 26S proteasome was demonstrated. In conclusion, we report a direct interaction between PSEN1 and subunits of the 20S catalytic particle of the 26S proteasome, further establishing the involvement of proteasomal degradation in the regulation of PSEN1 turnover.

A novel expression cloning method to isolate mammalian transcription factors in Schizosaccharomyces pombe.

We describe a novel expression cloning strategy in the fission yeast for the isolation of mammalian transcription factors using a mammalian promoter as target. This strategy is possible because of the conservation between mammalian cells and Schizosaccharomyces pombe of the mechanism that leads to the selection of the transcription start site. It also opens new perspectives to investigate the transcriptional regulation of genes for which detailed promoter analysis is difficult.

Identification of a new caspase homologue: caspase-14.

Caspases are cysteinyl aspartate-specific proteinases, many of which play a central role in apoptosis. Here, we report the identification of a new murine caspase homologue, viz. caspase-14. It is most related to human/murine caspase-2 and human caspase-9, possesses all the typical amino acid residues of the caspases involved in catalysis, including the QACRG box, and contains no or only a very short prodomain. Murine caspase-14 shows 83% similarity to human caspase-14. Human caspase-14 is assigned to chromosome 19p13.1. Northern blot analysis revealed that mRNA expression of caspase-14 is undetectable in all mouse adult tissues examined except for skin, while it is abundantly expressed in mouse embryos. In contrast to many other caspase family members, murine caspase-14 is not cleaved by granzyme B, caspase-1, caspase-2, caspase-3, caspase-6, caspase-7 or caspase-11, but is weakly processed into p18 and p11 subunits by murine caspase-8. No aspartase activity of murine caspase-14 could be generated by bacterial or yeast expression. Transient overexpression of murine caspase-14 in mammalian cells did not elicit cell death and did not interfere with caspase-8-induced apoptosis. In conclusion, caspase-14 is a member of the caspase family but no proteolytic or biological activities have been identified so far. The high constitutive expression levels in embryos and specific expression in adult skin suggest a role in ontogenesis and skin physiology.

Involvement of a tissue-specific RNA recognition motif protein in Drosophila spermatogenesis.

RNA binding proteins mediate posttranscriptional regulation of gene expression via their roles in nuclear and cytoplasmic mRNA metabolism. Many of the proteins involved in these processes have a common RNA binding domain, the RNA recognition motif (RRM). We have characterized the Testis-specific RRM protein gene (Tsr), which plays an important role in spermatogenesis in Drosophila melanogaster. Disruption of Tsr led to a dramatic reduction in male fertility due to the production of spermatids with abnormalities in mitochondrial morphogenesis. Tsr is located on the third chromosome at 87F, adjacent to the nuclear pre-mRNA binding protein gene Hrb87F. A 1.7-kb Tsr transcript was expressed exclusively in the male germ line. It encoded a protein containing two RRMs similar to those found in HRB87F as well as a unique C-terminal domain. TSR protein was located in the cytoplasm of spermatocytes and young spermatids but was absent from mature sperm. The cellular proteins expressed in premeiotic primary spermatocytes from Tsr mutant and wild-type males were assessed by two-dimensional gel electrophoresis. Lack of TSR resulted in the premature expression of a few proteins prior to meiosis; this was abolished by a transgenic copy of Tsr. These data demonstrate that TSR negatively regulated the expression of some testis proteins and, in combination with its expression pattern and subcellular localization, suggest that TSR regulates the stability or translatability of some mRNAs during spermatogenesis.

Tyrosine phosphorylation of a M(r) 38,000 A/B-type hnRNP protein selectively modulates its RNA binding.

The M(r) 38,000 RNA-binding protein (P38) is the major component of translationally repressed messenger ribonucleoproteins in cryptobiotic gastrulae of the brine shrimp Artemia. Partial elucidation of the amino acid sequence of P38 reveals that it is homologous to A/B-type hnRNP proteins. This was confirmed by immunodetection with antibodies specific for A/B-type hnRNP proteins from Drosophila melanogaster. P38 can be phosphorylated in vitro by a src-related protein tyrosine kinase on multiple tyrosine residues located predominantly in the glycine-rich domain. Tyrosine phosphorylated P38 can be efficiently dephosphorylated by a specific protein tyrosine phosphatase (1B-like) and by protein phosphatase 2A activated by the phosphotyrosyl phosphatase activator. Tyrosine phosphorylation of P38 slightly influences its subsequent phosphorylation by casein kinase II. The latter phosphorylation site is located in the glycine-rich domain of P38. Two-dimensional gel electrophoresis resolves P38 into multiple isoforms which shift to more acidic pI values after phosphorylation by protein tyrosine kinase or casein kinase II. From nitrocellulose filter binding and UV cross-linking analysis, evidence was obtained that tyrosine phosphorylation of P38 impairs its binding to poly(A) but not to poly(U). This demonstrates the involvement of tyrosine residues in polynucleotide-specific RNA binding that can be regulated by phosphorylation/dephosphorylation.

Inhibition of casein kinase II by dinucleoside polyphosphates.

In our search for potential inhibitors of casein kinase II (CKII) in Artemia, we have shown that dinucleoside polyphosphates are a novel class of effectors for this ubiquitous protein kinase. P1,P4-di(guanosine-5')-tetraphosphate (Gp4G) is a better CKII inhibitor than P1,P4-di(adenosine-5')-tetraphosphate (Ap4A). The inhibition by both effectors is more potent when GTP is used as phosphate donor instead of ATP. The inhibition of CKII increases with the number of phosphates linking the guanosine/adenosine moieties (for n = 2-6). Ap4A does not compete with the protein substrate and causes an increase in the apparent KmATP and a decrease in the apparent VmATP, indicating a mixed type of inhibition with respect to ATP.