Transcriptional activity in yeast strongly correlates with promoter occupancy by general factors such as TATA binding protein (TBP), TFIIA, and TFIIB, but not with occupancy by TBP-associated factors (TAFs). Thus, TBP exists in at least two transcriptionally active forms in vivo. The TAF-containing form corresponds to the TFIID complex, whereas the form lacking TAFs corresponds to TBP itself or to some other TBP complex. Heat shock treatment altered the relative utilization of these TBP forms, with TFIID being favored. Promoter-specific variations in the association of these distinct forms of TBP may explain why only some yeast genes require TFIID for transcriptional activity in vivo.

The fungus Cryptococcus is a major cause of meningoencephalitis in HIV-infected as well as HIV-uninfected individuals with mortalities in developed countries of 20% and 30%, respectively. In HIV-related disease, defects in T-cell immunity are paramount, whereas there is little understanding of mechanisms of susceptibility in non-HIV related disease, especially that occurring in previously healthy adults. The present description is the first detailed immunological study of non-HIV-infected patients including those with severe central nervous system (s-CNS) disease to 1) identify mechanisms of susceptibility as well as 2) understand mechanisms underlying severe disease. Despite the expectation that, as in HIV, T-cell immunity would be deficient in such patients, cerebrospinal fluid (CSF) immunophenotyping, T-cell activation studies, soluble cytokine mapping and tissue cellular phenotyping demonstrated that patients with s-CNS disease had effective microbiological control, but displayed strong intrathecal expansion and activation of cells of both the innate and adaptive immunity including HLA-DR+ CD4+ and CD8+ cells and NK cells. These expanded CSF T cells were enriched for cryptococcal-antigen specific CD4+ cells and expressed high levels of IFN-γ as well as a lack of elevated CSF levels of typical T-cell specific Th2 cytokines -- IL-4 and IL-13. This inflammatory response was accompanied by elevated levels of CSF NFL, a marker of axonal damage, consistent with ongoing neurological damage. However, while tissue macrophage recruitment to the site of infection was intact, polarization studies of brain biopsy and autopsy specimens demonstrated an M2 macrophage polarization and poor phagocytosis of fungal cells. These studies thus expand the paradigm for cryptococcal disease susceptibility to include a prominent role for macrophage activation defects and suggest a spectrum of disease whereby severe neurological disease is characterized by immune-mediated host cell damage.

Objective The management of complex patients with neuroimmunological diseases is hindered by an inability to reliably measure intrathecal inflammation. Currently implemented laboratory tests developed >40 years ago either are not dynamic or fail to capture low levels of central nervous system (CNS) inflammation. Therefore, we aimed to identify and validate biomarkers of CNS inflammation in 2 blinded, prospectively acquired cohorts of untreated patients with neuroimmunological diseases and embedded controls, with the ultimate goal of developing clinically useful tools. Methods Because biomarkers with maximum utility reflect immune phenotypes, we included an assessment of cell specificity in purified primary immune cells. Biomarkers were quantified by optimized electrochemiluminescent immunoassays. Results Among markers with cell-specific secretion, soluble CD27 is a validated biomarker of intrathecal T-cell activation, with an area under the receiver operating characteristic curve of 0.97. Comparing the quantities of cerebrospinal fluid (CSF) immune cells and their respective cell-specific soluble biomarkers (released by CSF cells as well as their counterparts in CNS tissue) provided invaluable information about stationary CNS immune responses, previously attainable via brain biopsy only. Unexpectedly, progressive and relapsing–remitting multiple sclerosis (MS) patients have comparable numbers of activated intrathecal T and B cells, which are preferentially embedded in CNS tissue in the former group. Interpretation The cell-specific biomarkers of intrathecal inflammation may improve diagnosis and management of neuroimmunological diseases and provide pharmacodynamic markers for future therapeutic developments in patients with intrathecal inflammation that is not captured by imaging, such as in progressive MS.

A pericellular proteolytic pathway initiated by the transmembrane serine protease matriptase plays a critical role in the terminal differentiation of epidermal tissues. Matriptase is constitutively expressed in multiple other epithelia, suggesting a putative role of this membrane serine protease in general epithelial homeostasis. Here we generated mice with conditional deletion of the St14 gene, encoding matriptase, and show that matriptase indeed is essential for the maintenance of multiple types of epithelia in the mouse. Thus, embryonic or postnatal ablation of St14 in epithelial tissues of diverse origin and function caused severe organ dysfunction, which was often associated with increased permeability, loss of tight junction function, mislocation of tight junction-associated proteins, and generalized epithelial demise. The study reveals that the homeostasis of multiple simple and stratified epithelia is matriptase-dependent, and provides an important animal model for the exploration of this membrane serine protease in a range of physiological and pathological processes.

Archaea possess a basal transcriptional apparatus that resembles that of eukaryotes. Here we report the 2.1-Å crystal structure of the archaeal transcription factor complex formed by the TATA-box-binding protein (TBP), the transcription factor IIB homolog, and a DNA target, all from the hyperthermophile Pyrococcus woesei. The overall fold of these two basal transcription factors is essentially the same as that of their eukaryotic counterparts. However, in comparison with the eukaryotic complexes, the archaeal TBP-DNA interface is more symmetrical, and in this structure the orientation of the preinitiation complex assembly on the promoter is inverted with respect to that seen in all crystal structures of comparable eukaryotic systems. This study of the structural details of an archaeal transcription factor complex presents the opportunity to examine the evolution of the basal eukaryotic transcriptional apparatus from a stereochemical viewpoint and to extend our understanding of the physical biochemistry of transcriptional initiation.The recent sequencing of the first archaeal genome has confirmed the designation of the archaea as a third kingdom of life, distinct from eubacteria and eukaryotes (1). Recent discoveries indicate that the basal components of transcription in archaea resemble those in the eukaryotic RNA polymerase II transcriptional system (2). Archaeal homologs to the transcription factors TATA-box binding protein (TBP) and transcription factor IIB (TFIIB) have been reported (3-6). As in eukaryotic polymerase II transcription, archaeal transcription is initiated by an AϩT-rich TATA-like segment known as the "boxA" sequence, containing a consensus T C TTA T A ANN (hereafter referred to as the boxA͞TATA element) (7,8). Interestingly, one of the common archaeal promoter sequences contains the boxA͞TATA element, TTTATATA, which is an inverted canonical eukaryotic TATA box (8). Like eukaryotic TBP, archaeal TBP recognizes the boxA͞TATA element, and transcription factor B (TFB) from Pyrococcus woesei (pwTFB) binds to the TBP-DNA complex (3). Homologs to other eukaryotic basal transcription factors, or TBP-associated factors (TAFs), however, are not present in the archaeal genome. In fact, promoter-specific transcription can occur in a purified reconstituted archaeal system, with only TBP, TFB, and polymerase (9).We present here the 2.1-Å crystal structure of the ternary archaeal transcription complex, illustrating the interaction of TBP, TFB, and a promoter fragment. The components are all from P. woesei, a hyperthermophilic archaeon which exhibits an optimal growth temperature of 105°C (10). The amino acid sequence of pwTBP is 36-41% identical to the conserved C-terminal core of its eukaryotic counterparts (3), and, as expected from this degree of homology, we have recently found that the general features of its three-dimensional structure are the same as those of eukaryotic TBP (11). pwTFB is 28-32% identical to full-length eukaryotic TFIIBs, and 25% identical to the C-terminal domain of the yeas...

ObjectiveInaccessibility of the inflammation compartmentalized to the central nervous system (CNS) may underlie the lack of efficacy of immunomodulatory treatments in progressive multiple sclerosis (MS). The double blind combination of Rituximab by IntraVenous and IntraThecAl injection versus placebo in patients with Low‐Inflammatory SEcondary progressive MS (RIVITALISE; NCT01212094) trial was designed to answer: (1) Whether an induction dose of intravenous and intrathecal rituximab efficiently depletes CNS B cells? and (2) If so, whether this leads to global inhibition of CNS inflammation and slowing of CNS tissue destruction?MethodsPatients aged 18–65 years were randomly assigned to rituximab or placebo. Protocol‐stipulated interim analysis quantified the efficacy of B‐cell depletion.ResultsThe efficacy on cerebrospinal fluid (CSF) biomarkers failed to reach criteria for continuation of the trial. B‐cell‐related CSF biomarkers (sCD21 and B‐cell activating factor) changed only in the active‐treatment arm. While CSF B cells were killed robustly (median −79.71%, P = 0.0176), B cells in CNS tissue were depleted inadequately (~−10–20%, P < 0.0001). Consequently, the T‐cell‐specific CSF biomarker sCD27 decreased slightly (−10.97%, P = 0.0005), while axonal damage marker, neurofilament light chain did not change. Insufficient saturation of CD20, lack of lytic complement, and paucity of cytotoxic CD56dim NK cells contribute to decreased efficacy of rituximab in the CNS.InterpretationBiomarker studies reliably quantified complementary pharmacodynamic effects of rituximab in the CNS, exposed causes for poor efficacy and determined that RIVITALISE trial would be underpowered to measure efficacy on clinical outcomes. Identified mechanisms for poor efficacy are applicable to all CNS‐inflammation targeting monoclonal antibodies.

Hypomorphic mutations in the human SPINT2 gene cause a broad spectrum of abnormalities in organogenesis, including organ and digit duplications, atresia, fistulas, hypertelorism, cleft palate and hamartoma. SPINT2 encodes the transmembrane serine protease inhibitor HAI2 (placental bikunin), and the severe developmental effects of decreased HAI2 activity can be hypothesized to be a consequence of excess pericellular proteolytic activity. Indeed, we show here that HAI2 is a potent regulator of protease-guided cellular responses, including motogenic activity and transepithelial resistance of epithelial monolayers. Furthermore, we show that inhibition of the transmembrane serine protease matriptase (encoded by St14) is an essential function of HAI2 during tissue morphogenesis. Genetic inactivation of the mouse Spint2 gene led to defects in neural tube closure, abnormal placental labyrinth development associated with loss of epithelial cell polarity, and embryonic demise. Developmental defects observed in HAI2-deficient mice were caused by unregulated matriptase activity, as both placental development and embryonic survival in HAI2-deficient embryos were completely restored by the simultaneous genetic inactivation of matriptase. However, neural tube defects were detected in HAI2-deficient mice even in the absence of matriptase, although at lower frequency, indicating that the inhibition of additional serine protease(s) by HAI2 is required to complete neural development. Finally, by genetic complementation analysis, we uncovered a unique and complex functional interaction between HAI2 and the related HAI1 in the regulation of matriptase activity during development. This study indicates that unregulated matriptase-dependent cell surface proteolysis can cause a diverse array of abnormalities in mammalian development.

Loss of either hepatocyte growth factor activator inhibitor (HAI)-1 or -2 is associated with embryonic lethality in mice, which can be rescued by the simultaneous inactivation of the membrane-anchored serine protease, matriptase, thereby demonstrating that a matriptase-dependent proteolytic pathway is a critical developmental target for both protease inhibitors. Here, we performed a genetic epistasis analysis to identify additional components of this pathway by generating mice with combined deficiency in either HAI-1 or HAI-2, along with genes encoding developmentally co-expressed candidate matriptase targets, and screening for the rescue of embryonic development. Hypomorphic mutations in Prss8, encoding the GPI-anchored serine protease, prostasin (CAP1, PRSS8), restored placentation and normal development of HAI-1–deficient embryos and prevented early embryonic lethality, mid-gestation lethality due to placental labyrinth failure, and neural tube defects in HAI-2–deficient embryos. Inactivation of genes encoding c-Met, protease-activated receptor-2 (PAR-2), or the epithelial sodium channel (ENaC) alpha subunit all failed to rescue embryonic lethality, suggesting that deregulated matriptase-prostasin activity causes developmental failure independent of aberrant c-Met and PAR-2 signaling or impaired epithelial sodium transport. Furthermore, phenotypic analysis of PAR-1 and matriptase double-deficient embryos suggests that the protease may not be critical for focal proteolytic activation of PAR-2 during neural tube closure. Paradoxically, although matriptase auto-activates and is a well-established upstream epidermal activator of prostasin, biochemical analysis of matriptase- and prostasin-deficient placental tissues revealed a requirement of prostasin for conversion of the matriptase zymogen to active matriptase, whereas prostasin zymogen activation was matriptase-independent.