Calcineurin B1 is essential for positive but not negative selection during thymocyte development

During development, discrete cell fates often result from variation in the intensity of a particular signal. The mechanisms underlying these seemingly analog-to-digital switches are not understood. In developing T lymphocytes, low-intensity signals through the antigen receptor result in positive selection while more intense signals give rise to negative selection. By deleting the genetic locus encoding the regulatory B1 subunit of calcineurin specifically in thymocytes, we found an absolute requirement for calcineurin in positive selection. In contrast, calcineurin activity was dispensable in several models of negative selection. Unexpectedly, we found that removal of calcineurin activity from thymocytes results in inefficient ERK activation at the double-positive stage of thymocyte development, when selection occurs. These studies clarify the mechanism by which graded signals are converted to discrete outcomes in T cell development and further indicate that the developmental roles of calcineurin likely contribute to immunosuppression by calcineurin inhibitors.     

Interleukin-1 beta, but not interleukin-1 alpha, is required for T-cell-dependent antibody production

Interleukin-1 (IL-1) consists of two molecules, IL-1 alpha and IL-1 beta, and IL-1 receptor antagonist (IL-1Ra) is a natural inhibitor of these molecules. Although the adjuvant effects of exogenously administered IL-1 in the humoral immune response are well known, the roles of endogenous IL-1 and the functional discrimination between IL-1 alpha and IL-1 beta have not been elucidated completely. In this report, we investigated the role of IL-1 in the humoral immune response using gene-targeted mice. Both primary and secondary antibody production against T-dependent antigen, sheep red blood cells (SRBC), was significantly reduced in IL-1 alpha/beta-/- mice, and was enhanced in IL-1Ra-/- mice. The intrinsic functions of B cells, such as antibody production against type 1 T-independent antigen, trinitrophenyl-lipopolysaccharide and proliferative responses against mitogenic stimuli, were normal in IL-1 alpha/beta-/- mice. The proliferative response of T cells and cytokine production upon stimulation with anti-CD3 monoclonal antibody were also normal, as was the phagocytotic ability of antigen-presenting cells (APCs). However, SRBC-specific proliferative response and cytokine production of T cells through the interaction with APCs were markedly impaired in IL-1 alpha/beta-/- mice, and enhanced in IL-1Ra-/- mice. Moreover, we show that SRBC-specific antibody production was reduced in IL-1 beta-/- mice, but not in IL-1 alpha-/- mice. These results show that endogenous IL-1 beta, but not IL-1 alpha, is involved in T-cell-dependent antibody production, and IL-1 promotes the antigen-specific T-cell helper function through the T-cell-APC interaction.     

Parvalbumin-positive CA1 interneurons are required for spatial working but not for reference memory

Parvalbumin-positive GABAergic interneurons in cortical circuits are hypothesized to control cognitive function. To test this idea directly, we functionally removed parvalbumin-positive interneurons selectively from hippocampal CA1 in mice. We found that parvalbumin-positive interneurons are dispensable for spatial reference, but are essential for spatial working memory.     

Sedative but not anxiolytic properties of benzodiazepines are mediated by the GABA(A) receptor alpha1 subtype

Inhibitory neurotransmission in the brain is largely mediated by GABA(A) receptors. Potentiation of GABA receptor activation through an allosteric benzodiazepine (BZ) site produces the sedative, anxiolytic, muscle relaxant, anticonvulsant and cognition-impairing effects of clinically used BZs such as diazepam. We created genetically modified mice (alpha1 H101R) with a diazepam-insensitive alpha1 subtype and a selective BZ site ligand, L-838,417, to explore GABA(A) receptor subtypes mediating specific physiological effects. These two complimentary approaches revealed that the alpha1 subtype mediated the sedative, but not the anxiolytic effects of benzodiazepines. This finding suggests ways to improve anxiolytics and to develop drugs for other neurological disorders based on their specificity for GABA(A) receptor subtypes in distinct neuronal circuits.     

Evidence for positive selection driving the evolution of HIV-1 env under potent antiviral therapy

In HIV-infected individuals treated with potent antiretroviral therapy, viable virus can be isolated from latently infected cells several years into therapy, due to the long life of these cells, ongoing replication replenishing this population, or both. We have analysed the V3 region of the HIV-1 env gene isolated from six patients who have undergone 2 years of potent antiretroviral therapy without frank failure of viral suppression. We show that in two (and possibly three) patients, the sequence changes between baseline virus and virus isolated from infected cells persisting 2 years into infection result from positive selection driving adaptive evolution, occurring either prior to or during therapy. Our analyses suggest low-level replication despite absence of drug resistance due to drug sanctuary sites, or to low-level ongoing replication in the presence of alterations in the selective environment during therapy, perhaps due to a decline in HIV-specific immune responsiveness or changes in target cell pools. In one patient, genetic divergence between baseline plasma and infected cells isolated during therapy may reflect the long half-life of some of these persistent cell populations and the divergence of viral subpopulations that occurred prior to therapy.     

Intraepidermal but not dermal T lymphocytes are positive for a cell-cycle-associated antigen (Ki-67) in mycosis fungoides.

The Ki-67 antibody, which reacts with nuclei of actively proliferating cells, was used in an immunohistochemical study to determine if there was any difference between T cells located in the epidermis rather than the dermis, in mycosis fungoides. In 12 of 14 cases of patch/plaque stage mycosis fungoides, the epidermal T cells were Ki-67 positive, while the dermal T cells were Ki-67 negative in all cases. Both epidermal and dermal T cells belonged primarily to the memory-versus-naive subset. The intraepidermal Ki-67-positive T cells were slightly larger than the dermal Ki-67-negative cells and could be easily distinguished from occasional basal keratinocytes that were also Ki-67 positive. We conclude that dermal T cells, despite expressing HLA-DR and a memory phenotype, are essentially in a resting (Go or noncycling state) in mycosis fungoides. Furthermore, it appears that the movement of T cells into the epidermal compartment is associated with activation and entry into the cell cycle. Such intraepidermal activation may lead to lymphokine release, and play an important pathophysiologic role in mycosis fungoides.     

Universally conserved and yeast-specific U1 snRNA sequences are important but not essential for U1 snRNP function

To study the contribution of the large, 568-nucleotide yeast (Saccharomyces cerevisiae) U1 snRNA to pre-mRNA splicing, we generated mutations in two regions of the molecule and introduced each mutant gene back into yeast as the sole copy of the U1 snRNA gene. We mutagenized the "A loop," a subregion highly conserved in primary sequence in all U1 snRNA molecules analyzed to date. We also mutagenized a portion of the yeast core subdomain, a region conserved in primary and secondary structure among several yeast species but absent from the much smaller metazoan U1 molecule. Surprisingly, mutations in these two regions had little or no effect on growth rate, yet several of them affected an inefficiently spliced reporter gene construct. In addition, combinations of mutants in both regions gave rise to reduced growth rates. Using the latter assay, we confirmed some of the proposed secondary structure of the yeast core domain. The experiments indicate that both regions contribute to U1 snRNP activity but that mutations in a single region do not have a substantial effect on growth rate because U1 snRNP activity is not rate-limiting for growth.     

Kinase suppressor of Ras 1 is required for full ERK activation in thymocytes but not for thymocyte selection

The scaffold protein kinase suppressor of Ras 1 (KSR1) is critical for efficient activation of ERK in a number of cell types. Consistent with this, we observed a defect in ERK activation in thymocytes that lack KSR1. Interestingly, we found that the defect was much greater after PMA stimulation than by CD3 activation. Since ERK activation is believed to be important for thymocyte development, we analyzed thymocyte selection in KSR1‐deficient (KSR1^?/?) mice. We found that positive selection in two different TCR transgenic models, HY and AND, was normal. On the other hand, negative selection in the HY model was slightly impaired in KSR1^?/? mice. However, a defect in negative selection was not apparent in the AND TCR model system or in an endogenous superantigen‐mediated model of negative selection. These results suggest that, despite a requirement for KSR1 for full ERK activation in thymocytes, full and efficient ERK activation is not essential for the majority of thymocyte selection events.     

Mutations in NSD1 are responsible for Sotos syndrome, but are not a frequent finding in other overgrowth phenotypes

Recently, deletions encompassing the nuclear receptor binding SET-Domain 1 (NSD1) gene have been described as the major cause of Japanese patients with the Sotos syndrome, whereas point mutations have been identified in the majority of European Sotos syndrome patients. In order to investigate a possible phenotype-genotype correlation and to further define the predictive value of NSD1 mutations, we performed mutational analysis of the NSD1 gene in 20 patients and one familial case with Sotos syndrome, five patients with Weaver syndrome, six patients with unclassified overgrowth/mental retardation, and six patients with macrocephaly/mental retardation. We were able to identify mutations within the NSD1 gene in 18 patients and the familial case with Sotos syndrome (90%). The mutations (six nonsense, eight frame shifts, three splice site, one missense, one in-frame deletion) are expected to result in an impairment of NSD1 function. The best correlation between clinical assessment and molecular results was obtained for the Sotos facial gestalt in conjunction with overgrowth, macrocephaly, and developmental delay. In contrast to the high mutation detection rate in Sotos syndrome, none of the patients with Weaver syndrome, unclassified overgrowth/mental retardation and macrocephaly/mental retardation, harbored NSD1 mutations. We tested for large deletions by FISH analysis but were not able to identify any deletion cases. The results indicate that the great majority of patients with Sotos syndrome are caused by mutations in NSD1. Deletions covering the NSD1 locus were not found in the patients analyzed here.     

Linkage of human cytomegalovirus glycoprotein gO variant groups identified from worldwide clinical isolates with gN genotypes, implications for disease associations and evidence for N-terminal sites of positive selection

Previously, we identified the glycoprotein gO gene, UL74, as a hypervariable locus in the human cytomegalovirus (HCMV) genome [Virology 293 (2002) 281]. Here, we analyze gO from 50 isolates from congenitally infected newborns, transplant recipients, and HIV/AIDS patients from Italy, Australia, and UK. These are compared to four gO groups described from USA transplantation patients [J. Virol. 76 (2002) 10841]. Phylogenetic analyses identified seven genotypes. Divergence between genotypes was up to 55% and within 3%. Discrete linkage was shown between seven hypervariable gO and gN genotypes, but not with gB. This suggests interactions, while gN and gO are known to form complexes with distinct conserved glycoproteins gM, gH/gL, respectively, both are involved in fusogenic entry and exit. Codon-based maximum likelihood models showed evidence for sites of positive selection. Further analyses of disease relationships should take into account these newly defined gO/gN groups.     

TNF receptor 1 (TNFR1) and CD95 are not required for T cell deletion after virus infection but contribute to peptide-induced deletion under limited conditions

Deletion of mature T cells maintains cellular homeostasis and is involved in the maintenance of self tolerance to some peripheral self antigens. Previous studies have presented conflicting evidence for a role of the tumor necrosis factor receptor (TNFR) family member CD95 (Fas) in peripheral T cell deletion using CD95-deficient mice. To evaluate cooperation between CD95 and another TNFR family molecule, TNFR1, we generated mice deficient for both CD95 and TNFR1. We showed that TNFR1 and CD95 do not contribute to the decline of antigen-specific cytotoxic T lymphocytes after virus infection. Using TNFR1 / CD95-deficient mice expressing the P14 TCR specific for a lymphocytic choriomeningitis virus-derived peptide (p33) we showed that deletion of p33-specific CD8(+) T cells following high dose p33 administration is also normal. However, in non-TCR-transgenic TNFR1 / CD95-deficient mice treated with the same p33 regimen, tolerance induction was defective. These data indicate that TNFR1 and CD95 are dispensable for deletion of antigen-specific T cells after viral infection. However, under certain conditions, both TNFR1 and CD95 appear to cooperate in CD8(+) T cell deletion.     

TLR4, TLR9 and MyD88 are not required for the positive selection of autoreactive B cells into the primary repertoire

Toll-like receptors (TLR) have been shown to play an essential role in the generation of autoantibodies in mouse models of autoimmunity, but the timing and context of these effects are poorly understood. One hypothesis is that TLR ligands assist in the positive selection of self-reactive B cells into the primary repertoire and, in this way, distinguish between immunogenic and tolerogenic forms of self-antigen. To explore this idea we generated hen egg lysozyme-specific immunoglobulin (Ig(HEL)) and isotype class-switching anti-HEL mice deficient in MyD88, TLR4 or TLR9 signalling and studied B cell development and autoantibody secretion in the presence or absence of an intracellular form of self-antigen HEL that positively selects B1 cells. Our findings show that TLR4, TLR9 and MyD88 are not required for the positive selection of autoreactive B cells in the primary B cell repertoire, nor is MyD88 required for the generation of isotype-switched antibodies in the absence of antigen. These results suggest that the significant effects of TLR on autoimmunity occur in the established repertoire and not during B cell development.     

Continuous positive airway pressure treatment for obstructive sleep apnoea reduces resting heart rate but does not affect dysrhythmias: a randomised controlled trial

Obstructive sleep apnoea (OSA) is associated with cardiovascular morbidity and may precipitate cardiac dysrhythmias. Uncontrolled reports suggest that continuous positive airway pressure (CPAP) may reduce dysrhythmia frequency and resting heart rate. We undertook a randomised controlled trial of therapeutic CPAP and compared with a subtherapeutic control which included an exploration of changes in dysrhythmia frequency and heart rate. Values are expressed as mean (SD). Eighty-three men [49.5 (9.6) years] with moderate–severe OSA [Oxygen Desaturation Index, 41.2 (24.3) dips per hour] underwent 3-channel 24-h electrocardiograms during normal daily activities, before and after 1 month of therapeutic ( n  = 43) or subtherapeutic ( n  = 40) CPAP. Recordings were manually analysed for mean heart rate, pauses, bradycardias, supraventricular and ventricular dysrhythmias. The two groups were well matched for age, body mass index, OSA severity, cardiovascular risk factors and history. Supraventricular ectopics and ventricular ectopics were frequently found in 95.2% and 85.5% of patients, respectively. Less common were sinus pauses (42.2%), episodes of bradycardia (12%) and ventricular tachycardias (4.8%). Compared with subtherapeutic control, CPAP reduced mean 24-h heart rate from 83.0 (11.5) to 79.7 (9.8) ( P  < 0.002) in the CPAP group compared with a non-significant rise ( P  = 0.18) from 79.0 (10.4) to 79.9 (10.4) in the subtherapeutic group; this was also the case for the day period analysed separately. There was no significant change in the frequencies of dysrhythmias after CPAP. Four weeks of CPAP therapy reduces mean 24-h heart rate possibly due to reduced sympathetic activation but did not result in a significant decrease in dysrhythmia frequency.     

<Emphasis Type="Italic">Sarcocystis neurona</Emphasis> major surface antigen gene 1 (<Emphasis Type="Italic">SAG1</Emphasis>) shows evidence of having evolved under positive selection pressure

The major surface antigen gene 1 (SAG1) is conserved among members of Sarcocystidae and may play an important role in parasite pathogenesis. Additionally, generation and selection of different antigenic variants of SAG1 has the potential for inclusion in a subunit vaccine or in the development of a diagnostic assay. In this study, patterns of nucleotide polymorphism were used to test the hypothesis that natural selection promotes diversity in different parts of SAG1 of Sarcocystis neurona. Nucleotide and amino acid sequence analysis of SAG1 from multiple S. neurona isolates identified two alleles. Sequences were identical intra-allele and highly divergent inter-alleles. Also, phylogenetic reconstruction showed sequences clustering into two clades. Tajimas and Fu and Lis neutrality tests indicated that selection is more likely to be acting on SAG1. Moreover, a sliding window analysis based on the ratio of silent substitutions to amino acid replacements provided strong evidence that two short segments in the central and 3 domain of SAG1 have been under positive selection in the divergence of the two alleles, suggesting that it may be important for the evasion of host immune responses and would be a suitable target for vaccine development.This revised version was published online in November 2004. In the Abstract an the Results section 5 has been corrected to 3.     

IL-1 alpha, but not IL-1 beta, is required for contact-allergen-specific T cell activation during the sensitization phase in contact hypersensitivity.

Contact hypersensitivity (CHS) is a T cell-mediated cellular immune response caused by epicutaneous exposure to contact allergens. In this reaction, after the first epicutaneous allergen sensitization, Langerhans cells (LC) catch allergens and migrate from the skin to draining lymph nodes (LN) and activate naive T cells. Although IL-1 is suggested to be involved in these processes, the mechanisms have not been elucidated completely. In this report, to elucidate roles of IL-1alpha and IL-1beta in CHS, we analyzed ear swelling in 2,4,6-trinitrochlorobenzene (TNCB)-induced CHS using gene-targeted mice. We found that ear swelling was suppressed in IL-1alpha-deficient (IL-1alpha(-/-)) mice but not in IL-1beta(-/-) mice. LC migration from the skin into LN was delayed in both IL-1alpha(-/-) and IL-1beta(-/-) mice, suggesting that this defect was not the direct cause for the reduced CHS in these mice. However, we found that the proliferative response of trinitrophenyl (TNP)-specific T cells after sensitization with TNCB was specifically reduced in IL-1alpha(-/-) mice. Furthermore, adoptive transfer of TNP-conjugated IL-1-deficient epidermal cells (EC) into wild-type mice indicated that only IL-1alpha, but not IL-1beta, produced by antigen-presenting cells in EC could prime allergen-specific T cells. These observations indicate that IL-1alpha, but not IL-1beta, plays a crucial role in TNCB-induced CHS by sensitizing TNP-specific T cells.     

Myocytes positive for in situ markers for DNA breaks in human hearts which are hypertrophic,but neither failed nor dilated: a manifestation of cardiac hypertrophy rather than failure

The significance of DNA breaks reported in failing hearts is controversial, although they may suggest myocyte apoptosis and may thus be responsible for the progression of heart failure. This study attempted to check the validity of the in situ markers for DNA breaks for detecting myocyte death and to evaluate separately two factors, failure or hypertrophy, crucial for DNA breaks in pathological human hearts. In the autopsy study, myocytes showed positivity for in situ nick end-labelling (TUNEL) and of Taq and Pfu polymerase-based in situ ligation assays not only in dilated cardiomyopathy (DCM, n = 9) with failure, but also in hypertrophic cardiomyopathy (HCM, n = 8) and hypertensive heart disease (HHD, n = 4) without failure. There was a significant correlation between each in situ marker and heart weight. The incidence of TUNEL-positive myocytes always exceeded that seen in in situ ligation assays. In addition, there were significant correlations between the in situ markers and the expression of the proliferating cell nuclear antigen (PCNA) and of the spliceosome component of 35 kD (SC-35). Similarly, in the left ventricular biopsy study using 23 DCM, 21 HCM, 11 HHD, and 13 non-hypertrophic hearts, the incidence of the in situ markers showed significant correlations with the left ventricular mass index and myocyte size, but not with cardiac function and dilatation. Positivity of myocytes for in situ markers for DNA breaks, such as TUNEL and in situ ligation assays, may be an epiphenomenon accompanying cardiac hypertrophy, but not myocyte death in pathological human hearts.     

Choline kinase‐α protein and phosphatidylcholine but not phosphocholine are required for breast cancer cell survival

High levels of total choline and phosphocholine (PC) are consistently observed in aggressive cancers. Choline kinase (Chk) catalyzes choline phosphorylation to produce PC in phosphatidylcholine (PtdCho) biosynthesis. PtdCho is the most abundant phospholipid in eukaryotic cell membranes and plays a dual role as the structural component of membranes and as a substrate to produce lipid second messengers such as phosphatidic acid and diacylglycerol. Chk‐α, but not Chk‐β, is overexpressed in various cancers, and is closely associated with tumor progression and invasiveness. We have previously shown that downregulation of mRNA using small interfering RNA (siRNA) against Chk‐α (siRNA‐Chk) or Chk short hairpin RNA, and the resultant decrease of Chk‐α protein levels, significantly reduced proliferation in breast cancer cells and tumors. A novel potent and selective small‐molecule Chk‐α inhibitor, V‐11‐0711, that inhibits the catalytic activity of Chk has recently been developed. Here, we used triple negative MDA‐MB‐231 and SUM149 breast cancer cells to further investigate the role of Chk‐α in cancer, by examining Chk‐α protein levels, cell viability/proliferation, choline phospholipid and lipid metabolism, lipid droplet formation, and apoptosis, following treatment with V‐11‐0711. Under the conditions used in this study, treatment with V‐11‐0711 significantly decreased PC levels but did not reduce cell viability as long as Chk‐α protein and PtdCho levels were not reduced, suggesting that Chk‐α protein and PtdCho, but not PC, may be crucial for breast cancer cell survival. These data also support the approach of antitumor strategies that destabilize Chk‐α protein or downregulate PtdCho in breast cancer treatment. Copyright © 2015 John Wiley & Sons, Ltd.