Effects of tofersen treatment in patients with SOD1-ALS in a "real-world" setting - a 12-month multicenter cohort study from the German early access program.

Background: In April 2023, the antisense oligonucleotide tofersen was approved by the U.S. Food and Drug Administration (FDA) for treatment of SOD1-amyotrophic lateral sclerosis (ALS), after a decrease of neurofilament light chain (NfL) levels had been demonstrated. Methods: Between 03/2022 and 04/2023, 24 patients with SOD1-ALS from ten German ALS reference centers were followed-up until the cut-off date for ALS functional rating scale revised (ALSFRS-R), progression rate (loss of ALSFRS-R/month), NfL, phosphorylated neurofilament heavy chain (pNfH) in cerebrospinal fluid (CSF), and adverse events. Findings: During the observation period, median ALSFRS-R decreased from 38.0 (IQR 32.0-42.0) to 35.0 (IQR 29.0-42.0), corresponding to a median progression rate of 0.11 (IQR -0.09 to 0.32) points of ALSFRS-R lost per month. Median serum NfL declined from 78.0 pg/ml (IQR 37.0-147.0 pg/ml; n = 23) to 36.0 pg/ml (IQR 22.0-65.0 pg/ml; n = 23; p = 0.02), median pNfH in CSF from 2226 pg/ml (IQR 1061-6138 pg/ml; n = 18) to 1151 pg/ml (IQR 521-2360 pg/ml; n = 18; p = 0.02). In the CSF, we detected a pleocytosis in 73% of patients (11 of 15) and an intrathecal immunoglobulin synthesis (IgG, IgM, or IgA) in 9 out of 10 patients. Two drug-related serious adverse events were reported. Interpretation: Consistent with the VALOR study and its Open Label Extension (OLE), our results confirm a reduction of NfL serum levels, and moreover show a reduction of pNfH in CSF. The therapy was safe, as no persistent symptoms were observed. Pleocytosis and Ig synthesis in CSF with clinical symptoms related to myeloradiculitis in two patients, indicate the potential of an autoimmune reaction. Funding: No funding was received towards this study.

Clinical and genetic features of amyotrophic lateral sclerosis patients with C9orf72 mutations.

An expansion of the GGGGCC hexanucleotide in the non-coding region of C9orf72 represents the most common cause of familial amyotrophic lateral sclerosis. The objective was to describe and analyse the clinical and genetic features of amyotrophic lateral sclerosis patients with C9orf72 mutations in a large population. Between November 2011 and December 2020, clinical and genetic characteristics of n = 248 patients with amyotrophic lateral sclerosis carrying C9orf72 mutations were collected from the clinical and scientific network of German motoneuron disease centres. Clinical parameters included age of onset, diagnostic delay, family history, neuropsychological examination, progression rate, phosphorylated neurofilament heavy chain levels in CSF and survival. The number of repeats was correlated with the clinical phenotype. The clinical phenotype was compared to n = 84 patients with SOD1 mutations and n = 2178 sporadic patients without any known disease-related mutations. Patients with C9orf72 featured an almost balanced sex ratio with 48.4% (n = 120) women and 51.6% (n = 128) men. The rate of 33.9% patients (n = 63) with bulbar onset was significantly higher compared to sporadic (23.4%, P = 0.002) and SOD1 patients (3.1%, P < 0.001). Of note, 56.3% (n = 138) of C9orf72, but only 16.1% of SOD1 patients reported a negative family history (P < 0.001). The GGGGCC hexanucleotide repeat length did not influence the clinical phenotypes. Age of onset (58.0, interquartile range 52.0-63.8) was later compared to SOD1 (50.0, interquartile range 41.0-58.0; P < 0.001), but earlier compared to sporadic patients (61.0, interquartile range 52.0-69.0; P = 0.01). Median survival was shorter (38.0 months) compared to SOD1 (198.0 months, hazard ratio 1.97, 95% confidence interval 1.34-2.88; P < 0.001) and sporadic patients (76.0 months, hazard ratio 2.34, 95% confidence interval 1.64-3.34; P < 0.001). Phosphorylated neurofilament heavy chain levels in CSF (2880, interquartile range 1632-4638 pg/ml) were higher compared to sporadic patients (1382, interquartile range 458-2839 pg/ml; P < 0.001). In neuropsychological screening, C9orf72 patients displayed abnormal results in memory, verbal fluency and executive functions, showing generally worse performances compared to SOD1 and sporadic patients and a higher share with suspected frontotemporal dementia. In summary, clinical features of patients with C9orf72 mutations differ significantly from SOD1 and sporadic patients. Specifically, they feature a more frequent bulbar onset, a higher share of female patients and shorter survival. Interestingly, we found a high proportion of patients with negative family history and no evidence of a relationship between repeat lengths and disease severity.

Development and Characterization of a 96-Well Exposure System for Safety Assessment of Nanomaterials.

In this study, a 96-well exposure system for safety assessment of nanomaterials is developed and characterized using an air-liquid interface lung epithelial model. This system is designed for sequential nebulization. Distribution studies verify the reproducible distribution over all 96 wells, with lower insert-to-insert variability compared to non-sequential application. With a first set of chemicals (TritonX), drugs (Bortezomib), and nanomaterials (silver nanoparticles and (non-)fluorescent crystalline nanocellulose), sequential exposure studies are performed with human lung epithelial cells followed by quantification of the deposited mass and of cell viability. The developed exposure system offers for the first time the possibility of exposing an air-liquid interface model in a 96-well format, resulting in high-throughput rates, combined with the feature for sequential dosing. This exposure system allows the possibility of creating dose-response curves resulting in the generation of more reliable cell-based assay data for many types of applications, such as safety analysis. In addition to chemicals and drugs, nanomaterials with spherical shapes, but also morphologically more complex nanostructures can be exposed sequentially with high efficiency. This allows new perspectives on in vivo-like and animal-free approaches for chemical and pharmaceutical safety assessment, in line with the 3R principle of replacing and reducing animal experiments.

Metabolic alterations precede neurofilament changes in presymptomatic ALS gene carriers.

BACKGROUND: The emergence of potentially effective new therapies for genetic forms of amyotrophic lateral sclerosis (ALS) necessitates the identification of biomarkers to facilitate early treatment, prior to the onset of motor symptoms. Here, we sought to investigate whether metabolic alterations are detectable in presymptomatic ALS gene mutation carriers, and whether such alterations precede neurofilament light chain (NfL) changes in serum. METHODS: Between 02/2014 and 11/2021, we prospectively studied 60 presymptomatic ALS gene mutation carriers (40% male, age 48.7 ± 14.9; 28 C9orf72, 22 SOD1, 10 other) compared to 73 individuals from the same families (47% male, age 47.4 ± 12.9) without pathogenic mutations as controls. Bioimpedance analysis (BIA) and indirect calorimetry were performed, and Body Mass Index (BMI), Fat Mass (FM), Body Fat Percentage, Body Water (BW), Lean Body Mass (LBM), Extracellular Mass (ECM), Body Cell Mass (BCM), ECM/BCM ratio, Cells Percentage, Phase Angle, Resting Metabolic Rate (RMR), Metabolic Ratio (MR), and NfL were measured. Participants and evaluators were blinded regarding gene carrier status. FINDINGS: Presymptomatic ALS gene carriers showed reduced LBM (p = 0.02), BCM (p = 0.004), Cells Percentage (p = 0.04), BW (p = 0.02), Phase Angle (p = 0.04), and increased ECM/BCM ratio (p = 0.04), consistently indicating a loss of metabolically active body cells. While in C9orf72 mutation carriers all tissue masses were reduced, only metabolically active tissue was affected in SOD1 mutation carriers. Unexpectedly, RMR (p = 0.009) and MR (p = 0.01) were lower in presymptomatic ALS gene carriers compared to non-carriers. NfL serum levels were similar in mutation carriers and non-carriers (p = 0.60). INTERPRETATION: The observed metabolic phenomena might reflect reduced physical activity and/or preemptive, insufficient compensatory mechanisms to prepare for the later hypermetabolic state. As pre-symptomatic biomarkers we propose ECM/BCM ratio and Phase Angle for SOD1, and a 4-compartment affection in BIA for C9orf72 mutation carriers. FUNDING: This work was an investigator-initiated trial. On the German side, there was no institutional or industrial funding. On the Swedish side, this work was supported by grants from the Swedish Brain Foundation (grants nr. 2013-0279, 2016-0303, 2018-0310, 2020-0353), the Swedish Research Council (grants nr. 2012-3167, 2017-03100), the Knut and Alice Wallenberg Foundation (grants nr. 2012.0091, 2014.0305, 2020.0232), the Ulla-Carin Lindquist Foundation, Umeå University (223-2808-12, 223-1881-13, 2.1.12-1605-14) and the Västerbotten County Council (grants nr 56103-7002829), King Gustaf V:s and Queen Victoria's Freemason's Foundation.

Pharyngeal electrical stimulation in amyotrophic lateral sclerosis: a pilot study.

BACKGROUND: Patients with amyotrophic lateral sclerosis (ALS) suffer from dysphagia that increases the risk for aspiration, pneumonia and weight loss. Pharyngeal electrical stimulation (PES) is a therapeutic technique that applies electric stimuli to the patient's pharynx in order to improve swallowing based on the principle of cortical plasticity and reorganization. Previous studies have demonstrated positive effects in patients with various neurological diseases. OBJECTIVE: This study was initiated to investigate the effect of PES on swallowing function in patients with ALS. METHODS: In all, 20 ALS patients with severe dysphagia [characterized by a Penetration Aspiration Scale (PAS) of at least 4 in thin liquid] were randomized to receive either PES for 10 min at 3 consecutive days in addition to Standard Logopaedic Therapy (SLT) or SLT alone. Swallowing function was evaluated by Fiberoptic Endoscopic Evaluation of Swallowing (FEES) at five timepoints: at baseline, 1 day, 4 days, 3 weeks and 3 months after treatment. Primary endpoint was the severity of penetrations or aspirations as classified by PAS. Secondary endpoints were adverse events, dysphagia-related quality of life, Swallowing Quality of Life (SWAL-QOL), Dysphagia Severity Rating Scale (DSRS), residues, leaking, ALS Functional Rating Scale Revised (ALSFRS-R), and the performance in Clinical Evaluation of Swallowing (CES). The trial is registered under the name of 'Pharyngeal Electrical Stimulation in Amyotrophic Lateral Sclerosis' with ClinialTrials.gov, number NCT03481348 (https://clinicaltrials.gov/ct2/show/NCT03481348). RESULTS: Both groups combined showed a significant improvement (p = 0.003) of median Total-PAS from 3.6 [interquartile range (IQR) = 2.9-5.0] at baseline to 2.3 (IQR = 1.8-4.0) 1 day after treatment. During subsequent study visits, PAS increased again but remained below baseline. PES and control group did not differ significantly 1 day after intervention (p = 0.32). Similar effects were found in the majority of secondary endpoints. INTERPRETATION: The findings suggest that PES may not provide an additional positive effect on swallowing function in ALS. SLT seems to yield at least short-term positive effects on swallowing function and swallowing-specific life quality in ALS.Registration: ClinialTrials.gov: NCT03481348.

How shape influences uptake: interactions of anisotropic polymer nanoparticles and human mesenchymal stem cells.

Among several nanoparticle properties, shape is important for their interaction with cells and, therefore, relevant for uptake studies and applications. In order to further investigate such characteristics, fluorescently labeled spherical polymer nanoparticles are synthesized by free-radical polymerization via the miniemulsion process. The spherical nanoparticles are subsequently submitted to controlled mechanical deformation to yield quasi-ellipsoidal polymeric nanoparticles with different aspect ratios. The uptake behaviors of spherical and non-spherical particles with equal volume are investigated qualitatively and quantitatively by electron microscopy, confocal laser scanning microscopy, and flow cytometry measurements. Non-spherical particles show fewer uptake by cells than their spherical counterparts with a negative correlation between aspect ratio and uptake rate. This is attributed to the larger average curvature radius of adsorbed non-spherical particles experienced by the cells.

Fabrication of Polymer Ellipsoids by the Electrospinning of Swollen Nanoparticles.

Electrospinning is used to deform originally spherical polymer nanoparticles into ellipsoidal nanoparticles. The polymer nanoparticles are swollen and the dispersion is then electrospun. Under certain conditions, the stretching generated in the electrospinning jet is enough to generate elongated nanoparticles embedded in fibers. The formation of the anisotropic particles is observed by stimulated emission depletion (STED) microscopy performed on fluorescent nanoparticles and by electron microscopy measurements on the nanoparticles recovered after removal of the fiber matrix.

Synthesis of narrowly size-distributed metal salt/poly(HEMA) hybrid particles in inverse miniemulsion: versatility and mechanism.

Hybrid particles containing different hydrophilic metal salts such as tetrafluoroborates of iron(II), cobalt(II), nickel(II), copper(II), and zinc(II), and nitrates of cobalt(II), nickel(II), copper(II), zinc(II), and iron(III), and cobalt(II) chloride were synthesized via inverse miniemulsion polymerization of 2-hydroxyethyl methacrylate (HEMA). All salts delivered narrowly size-distributed hybrid particles with the exception of iron(III), where only the nitrate salt could be successfully employed. The size and size distribution of the hybrid particles were characterized by dynamic light scattering (DLS) and transmission electron microscopy (TEM). The particle morphology and the distribution of salt in the dried particles were observed by TEM. The influences of the type of metal salts and salt content on the particle size distribution were extensively investigated.

55K isoform of CDK9 associates with Ku70 and is involved in DNA repair.

Positive elongation factor b (P-TEFb) is a cellular protein kinase that is required for RNA polymerase II (RNAP II) transcriptional elongation of protein coding genes. P-TEFb is a set of different molecular complexes, each containing CDK9 as the catalytic subunit. There are two isoforms of the CDK9 protein - the major 42KDa CDK9 isoform and the minor 55KDa isoform that is translated from an in-frame mRNA that arises from an upstream transcriptional start site. We found that shRNA depletion of the 55K CDK9 protein in HeLa cells induces apoptosis and double-strand DNA breaks (DSBs). The levels of apoptosis and DSBs induced by the depletion were reduced by expression of a 55K CDK9 protein variant resistant to the shRNA, indicating that these phenotypes are the consequence of depletion of the 55K protein and not off-target effects. We also found that the 55K CDK9 protein, but not the 42K CDK9 protein, specifically associates with Ku70, a protein involved in DSB repair. Our findings suggest that the 55K CDK9 protein may function in repair of DNA through an association with Ku70.

Narrowly size-distributed cobalt salt containing poly(2-hydroxyethyl methacrylate) particles by inverse miniemulsion.

Cobalt-containing hybrid particles have been prepared through the encapsulation of cobalt tetrafluoroborate hexahydrate (CoTFB) via inverse miniemulsion polymerization of 2-hydroxyethyl methacrylate (HEMA). We systematically varied the amount and type of cosolvent (water, methanol, ethanol, ethylene glycol), apolar continuous phase (cyclohexane, isooctane, isopar M, hexadecane), amount of cobalt salt, and molecular weight of the polymeric surfactant. The influence of those parameters on the particle size, size distribution, and particle morphology were investigated. Narrowly size-distributed hybrid particles with good colloidal stability could be obtained in a wide range of cobalt content between 5.7 and 22.6 wt % salt relative to the monomer. The addition of a cosolvent such as water not only promotes the loading of metal salt but also has a positive influence on narrowing the particle size distribution. We assume that generally narrowly size-distributed particles can be obtained for a large variety of combinations of polar/apolar phase by adjusting the balance between osmotic and Laplace pressure via the solubility of the metal salt in the continuous phase and lowering the interfacial tension by adjusting the hydrophilic-lipophilic balance (HLB) value of the surfactant. The results show a significant advantage of the inverse miniemulsion over the direct system with respect to the variability and total amount of metal salt without losing the narrow particle size distribution and colloidal stability.

Effect of lumbar extensor fatigue on paraspinal muscle reflexes.

Low back disorders are a frequent medical problem. Altered neuromuscular control of the spine has been associated with low back pain, and may contribute to its occurrence. The purpose of this study was to investigate the effect of lumbar extensor fatigue on reflex delay and amplitude in the paraspinal muscles. Ten healthy males (20-22 years of age) were subjected to an anteriorly-directed perturbation applied at the inferior margin of the scapulae while standing quietly before and after a lumbar extensor fatiguing protocol. The fatiguing protocol consisted of multiple sets of back extensions and intermittent isometric maximum voluntary contraction on a Roman chair for 14 min until 60% of unfatigued lumbar extensor MVC was reached. Reflexes were recorded from the paraspinal muscles at the level of L4. Results indicated the mean reflex delay was 60+/-18 ms and was not affected by fatigue (p=0.278). Reflex amplitude increased 36+/-32% with fatigue (p=0.017). The increase in reflex amplitude may reflect an attempt to compensate for losses in muscle force capacity with fatigue in order to maintain sufficient spinal stability. However, additional studies are necessary to investigate the mechanisms of this fatigue-related change in paraspinal reflex.

Cyclin T1 but not cyclin T2a is induced by a post-transcriptional mechanism in PAMP-activated monocyte-derived macrophages.

Positive transcription elongation factor b (P-TEFb) is an RNA polymerase II elongation factor which exists as multiple complexes in human cells. These complexes contain cyclin-dependent kinase 9 as the catalytic subunit and different cyclin subunits-cyclin T1, T2a, T2b, or K. Cyclin T1 is targeted by the human immunodeficiency virus (HIV) Tat protein to activate transcription of the HIV provirus. Expression of this P-TEFb subunit is highly regulated in monocyte-derived macrophages (MDMs). Cyclin T1 is induced early during differentiation and is shut off later by proteasome-mediated proteolysis. Cyclin T1 can be reinduced by pathogen-associated molecular patterns (PAMPs) or HIV infection. In this study, we analyzed regulation of P-TEFb in MDMs by examining 7SK small nuclear RNA and the HEXIM1 protein; these factors associate with P-TEFb and are thought to regulate its function. 7SK and HEXIM1 were induced early during differentiation, and this correlates with increased overall transcription. 7SK expression remained high, but HEXIM1 was shut off later during differentiation by proteasome-mediated proteolysis. Significantly, the cyclin T2a subunit of P-TEFb was not shut off during differentiation, and it was not induced by activation. Induction of cyclin T1 by PAMPs was found to be a slow process and did not involve an increase in cyclin T1 mRNA levels. Treatment of MDMs with PAMPs or a proteasome inhibitor induced cyclin T1 to a level equivalent to treatment with both agents together, suggesting that PAMPs and proteasome inhibitors act at a similar rate-limiting step. It is therefore likely that cyclin T1 induction by PAMPs is the result of a reduction in proteasome-mediated proteolysis.

siRNA depletion of 7SK snRNA induces apoptosis but does not affect expression of the HIV-1 LTR or P-TEFb-dependent cellular genes.

P-TEFb is a general transcriptional elongation factor composed of Cdk9 and either cyclin T1, T2, or K. A substantial portion of P-TEFb is associated with the 7SK small nuclear RNA (7SK) and the HEXIM1 or HEXIM2 proteins; this complex has reduced kinase activity in vitro relative to free P-TEFb. Here we report that 7SK and HEXIM1 levels are induced in activated lymphocytes concomitantly with increased P-TEFb activity and global transcription. We used siRNA-mediated depletion to probe the function of 7SK in HeLa cells. Depletion of 7SK caused a large reduction in the association of HEXIM1 with Cdk9 and cyclin T1, and greatly reduced the amount of the cyclin T1 present in the 7SK/HEXIM1/P-TEFb complex. Similar to previous studies, siRNA-mediated depletion of 7SK resulted in increased expression of several reporter plasmids tested, including a plasmid lacking promoter elements. However, in contrast to previous studies, which did not examine the effects of 7SK depletion on endogenous gene expression, depletion of 7SK did not appear to affect the expression of the corresponding endogenous genes. Moreover, 7SK depletion had no effect on expression from the integrated HIV-1 provirus or the c-myc and MCL-1 genes, three transcription units known to be highly dependent upon P-TEFb. Importantly, depletion of 7SK was found to cause apoptosis by 72 h post-transfection in HeLa cells. These results suggest that 7SK may provide an essential cellular function whose relation to P-TEFb function is unclear.

Differential localization and expression of the Cdk9 42k and 55k isoforms.

Cdk9, a member of the cyclin-dependent kinase family, is the catalytic subunit of P-TEFb, a protein kinase complex that stimulates transcriptional elongation. Cdk9, complexed with its regulatory partner cyclin T1, serves as the cellular mediator of the transactivation function of the HIV Tat protein. There are two known isoforms of Cdk9: a 42 kDa protein (42k, originally identified as PITALRE) and a more recently identified 55 kDa form (55k). To investigate possible functional differences between the two isoforms, we examined their kinase activities, their subcellular distributions, and their expression levels in primary cells relevant to HIV infection. Both isoforms were found to hyper-phosphorylate the carboxyl-terminal domain of the largest subunit of RNA polymerase II and displayed identical phosphorylation patterns with 144 peptide substrates. Epitope-tagged transiently-expressed Cdk9 42k localized diffusely in the nucleoplasm, while Cdk9 55k accumulated in the nucleolus. In primary undifferentiated monocytes, Cdk9 55k expression was not detected although 42k was present at high levels; however, 55k expression was induced upon macrophage differentiation. In primary lymphocytes, the levels of 55k decreased or remained steady following activation, while the levels of 42k increased. The promoter for 42k was significantly stronger than that of 55k in HeLa cells, and only the 42k promoter was responsive to activation signals in primary lymphocytes. These results indicate that expression of the 42k and 55k isoforms is differentially regulated and suggest that functional differences between the 42k and 55k isoforms of Cdk9 are likely to depend on access to substrates based on their differential subcellular localization and expression patterns.

Human immunodeficiency virus type 1 infection induces cyclin T1 expression in macrophages.

The Tat protein of human immunodeficiency virus type 1 (HIV-1) is essential for viral replication and activates RNA polymerase II transcriptional elongation through the association with a cellular protein kinase composed of Cdk9 and cyclin T1. Tat binds to this kinase complex through a direct protein-protein interaction with cyclin T1. Monocytes/macrophages are important targets of HIV-1 infection, and previous work has shown that cyclin T1 but not Cdk9 protein expression is low in monocytes isolated from blood. While Cdk9 expression is expressed at a high level during monocyte differentiation to macrophages in vitro, cyclin T1 expression is induced during the first few days of differentiation and is shut off after 1 to 2 weeks. We show here that the shutoff of cyclin T1 expression in late-differentiated macrophages involves proteasome-mediated proteolysis. We also show that cyclin T1 can be reinduced by a number of pathogen-associated molecular patterns that activate macrophages, indicating that up-regulation of cyclin T1 is part of an innate immune response. Furthermore, we found that HIV-1 infection early in macrophage differentiation results in sustained cyclin T1 expression, while infection at late times in differentiation results in the reinduction of cyclin T1. Expression of the viral Nef protein from an adenovirus vector suggests that Nef contributes to the HIV-1 induction of cyclin T1. These findings suggest that HIV-1 infection hijacks a component of the innate immune response in macrophages that results in enhancement rather than inhibition of viral replication.

HIV-1 infection and regulation of Tat function in macrophages.

The macrophage is an important cell type in the pathophysiology of human immunodeficiency virus type 1 (HIV-1) infection. Macrophages both support viral replication and are capable of attracting and activating lymphocytes, thus rendering CD4+ T lymphocytes highly permissive for infection. The viral Tat protein, whose function is mediated by the cellular cyclin T1 protein complexed with CDK9, is required for efficient transcription of the integrated HIV-1 provirus by RNA polymerase II. Cyclin T1 expression is highly regulated during macrophage differentiation, and this has important implications for HIV-1 replication. In monocytes isolated from healthy blood donors, cyclin T1 protein expression is low and is induced to high levels within the first few days of differentiation by a post-transcriptional mechanism. After 1-2 weeks of macrophage differentiation, however, cyclin T1 expression is shut off. Treatment of macrophages with lipopolysaccharide (LPS) can re-induce cyclin T1, indicating that the activation status of macrophages can regulate cyclin T1 expression. Recent results indicate that HIV-1 infection is able to induce cyclin T1 expression in macrophages. Future studies of cyclin T1 regulation in macrophages may suggest means of manipulating expression of this crucial cellular co-factor for therapeutic benefit in HIV-1 infected individuals.

Increased association of 7SK snRNA with Tat cofactor P-TEFb following activation of peripheral blood lymphocytes.

OBJECTIVE: This study was undertaken to determine whether 7SK small nuclear RNA (snRNA), which has been proposed to function as an inhibitor of Tat cofactor P-TEFb, plays a role in transcriptional latency in T cells. DESIGN AND METHODS: The association of 7SK snRNA with P-TEFb was investigated in resting and activated peripheral blood lymphocytes (PBLs). Primary PBLs were isolated by standard methods and activated with phytohemagglutinin (PHA). Levels of 7SK snRNA were determined by Northern blotting and levels of the P-TEFb subunits cyclin-dependent kinase 9 and cyclin T1 were analyzed by immunoblotting. RESULTS: The association of 7SK snRNA with P-TEFb complexes was specific. Following activation of PBLs, the levels of 7SK snRNA increased in a manner similar to U1 and U6 snRNA, sn RNAs involved in positive aspects of cellular gene expression. Unexpectedly, the association of 7SK snRNA with P-TEFb increased dramatically following lymphocyte activation. CONCLUSION: Increased association of 7SK snRNA with P-TEFb in activated lymphocytes correlates with increased global transcription. This suggests that 7SK snRNA is unlikely to promote transcriptional latency in lymphocytes through an association with P-TEFb; it also suggests that the proposal that the association of 7SK snRNA with P-TEFb acts to inhibit transcriptional elongation needs to be re-evaluated.

Regulation of TAK/P-TEFb in CD4+ T lymphocytes and macrophages.

HIV replication occurs principally in activated CD4+ T cells and macrophages. The HIV-1 Tat protein is essential for HIV replication and requires a cellular protein kinase activity termed TAK/P-TEFb, composed of CDK9 and cyclin T1, for its transactivation function. This article reviews recent work indicating that under some circumstances TAK/P-TEFb is likely to be limiting for HIV replication in CD4+ T cells and macrophages, and discusses mechanisms of regulation of the TAK/P-TEFb subunits in these cell types. In resting CD4+ T lymphocytes, TAK/P-TEFb function is low. Following lymphocyte activation, even under conditions of minimal activation in which activation markers and cellular proliferation are not induced, both CDK9 and cyclin T1 mRNA and protein levels are increased, leading to an induction of TAK/P-TEFb kinase activity that correlates with increased viral replication. In macrophages, regulation of TAK/P-TEFb involves mechanisms distinct from those in lymphocytes. In freshly isolated monocytes, CDK9 protein levels are high, while cyclin T1 protein levels are low to undetectable. Cyclin T1 protein expression is up-regulated during early macrophage differentiation by a mechanism that involves post-transcriptional regulation. Later during differentiation, cyclin T1 expression becomes shut off by a post-transcriptional mechanism, and this correlates with a decrease in Tat transactivation. Interestingly, cyclin T1 can be re-induced with lipopolysaccharide (LPS). These findings suggest that changes in cyclin T1 expression can influence HIV-1 replication levels in monocytes and macrophages. Important areas for future research on Tat and TAK/P-TEFb function are discussed.

Transient induction of cyclin T1 during human macrophage differentiation regulates human immunodeficiency virus type 1 Tat transactivation function.

The human immunodeficiency virus type 1 (HIV-1) Tat protein is essential for viral replication and stimulates transcription of the integrated provirus by recruiting the kinase complex TAK/P-TEFb, composed of cyclin T1 (CycT1) and Cdk9, to the viral TAR RNA element. TAK/P-TEFb phosphorylates the RNA polymerase II complex and stimulates transcriptional elongation. In this report, we investigated the regulation of TAK/P-TEFb in primary human macrophages, a major target cell of HIV infection. While Cdk9 levels remained constant, CycT1 protein expression in freshly isolated monocytes was very low, increased early during macrophage differentiation, and, unexpectedly, decreased to very low levels after about 1 week in culture. The kinase activity of TAK/P-TEFb paralleled the changes in CycT1 protein expression. RNA analysis indicated that the transient induction of CycT1 protein expression involves a posttranscriptional mechanism. In transient transfection assays, the ability of Tat to transactivate the HIV long terminal repeat (LTR) in the late differentiated macrophages was greatly diminished relative to its ability to transactivate the HIV LTR in early differentiated cells, strongly suggesting that CycT1 is limiting for Tat function in late differentiated macrophages. Interestingly, lipopolysaccharide, a component of the cell wall of gram-negative bacteria, reinduced CycT1 expression late in macrophage differentiation. These results raise the possibility that regulation of CycT1 expression may be involved in establishing latent infection in macrophages and that opportunistic infection may reactivate the virus by inducing CycT1 expression.

Apparent transgenerational effects of host plant in the leaf beetle Ophraella notulata (Coleoptera: Chrysomelidae).

The natural host of Ophraella notulata is Iva frutescens (Asteraceae); its close relative feeds on a related plant, Ambrosia artemisiifolia. We reared beetles on both plants, obtained progeny from the four possible crosses (two sexes X two parental hosts), and reared the progeny on both plant species. Survival to the imaginal stage of progeny reared on Iva varied with both maternal and paternal host. Hatchling feeding response to both plants showed a maternal host X paternal host interaction. Consumption of Ambrosia by adult beetles was, counter to expectation, higher for progeny of Iva-reared males than Ambrosia-reared males. Oviposition response, although based on too few data to be definitive, was peculiar: parental host did not affect oviposition on Ambrosia; on Iva daughters of Iva-reared males laid significantly more eggs than did daughters of Ambrosia-reared males, but only if they had been reared on Iva; those reared on Ambrosia displayed the reverse pattern. We discuss the possibility that nongenetic paternal transmission of host plant effects may explain these results, but offer a somewhat uncomfortable hypothesis of selection as a preferable explanation. An important outcome of the experiment is that it provided no evidence of maternal effects of host plant on offspring feeding or oviposition.