An analysis of the requirements for human cytomegalovirus oriLyt-dependent DNA synthesis in the presence of the herpes simplex virus type 1 replication fork proteins

Activation of the human cytomegalovirus (HCMV) origin of replication (oriLyt) was previously demonstrated in transient transfection assays in permissive human fetal fibroblasts and nonpermissive Vero cells, and shown to require six viral proteins that function at the replication fork plus a number of HCMV products that perform auxiliary roles. The six replication fork proteins could be substituted by their Epstein-Barr virus homologues. In this paper we demonstrate that the corresponding herpes simplex virus type 1 replication fork proteins can similarly replace those of HCMV in Vero cells. Under these conditions the essential auxiliary functions were mapped to two plasmids: pSVH (containing the major immediate-early locus) and pZP8 (spanning genes UL32-UL38). Mutants of pSVH and pZP8 and cloned cDNAs encoding the IE1-p72 and IE2-p86 proteins were tested for their ability to support DNA synthesis. The results showed that IE2-p86 was necessary for activation of the origin, and that the UL37x1 and IE1-p72 products exerted strong stimulatory effects. In contrast to the previous work, omission of the UL84 protein had no effect upon oriLyt-dependent DNA synthesis.     

The demonstration of an essential role for macrophages in the in vivo generation of IgG2a antibodies.

BALB/c mice were depleted of macrophages by intravenous inoculation of dichloromethylene diphosphonate entrapped in liposomes 24 h before primary and 24 h before secondary sensitization intravenously with 100 micrograms bovine serum albumin (BSA) or ovalbumin (OVA). The effectiveness of macrophage depletion was confirmed by immunocytochemistry. Five days and 14 days after secondary challenge with BSA, plasma samples from these and control mice inoculated with empty liposomes were examined for the production of BSA-specific IgG1 and IgG2a antibodies. Macrophage depletion resulted in a significantly increased production of the Th2 lymphocyte-associated IgG1 isotype, while the production of specific IgG2a antibodies, produced under the influence of Th1 cells, was totally ablated. Similar results were obtained when OVA was used as the test antigen. Furthermore, analysis of interferon-gamma (IFN-gamma) production after antigen or concanavalin A (Con A) restimulation in vitro indicated that macrophage depletion in vivo significantly reduced production of this Th1 cell-associated cytokine. These results provide strong in vivo and in vitro evidence for the macrophage being the antigen-presenting cell population responsible for Th1 cell activation.     

IL-3 is an important differentiation factor for the development of prostaglandin E2-producing macrophages between C57BL/6 and BALB/c mice

We have previously reported that peritoneal and splenic macrophages from Th2-dominant BALB/c mice produced higher amounts of prostaglandin (PG) E2 than cells from C57BL/6 mice. In this study, we investigated how macrophages from BALB/c mice acquire the ability of enhanced PGE2 production, using bone marrow-derived macrophages differentiated by M-CSF, GM-CSF or IL-3. There is no strain difference in PGE2 production by GM-CSF- and M-CSF-differentiated macrophages; however, IL-3-differentiated macrophages from BALB/c mice produced higher amounts of PGE2 and lower amounts of type I cytokines than cells from C57BL/6 mice. IL-3-differentiated macrophages from BALB/c mice expressed larger amounts of mRNA of membrane-bound (microsomal) PGE synthase-1 (mPGES-1). The amounts of PGE2 produced by macrophages were significantly reduced in mPGES-1-deficient mice, and these mice displayed enhanced Th1 responses after Propionibacterium acnes treatment compared with wild-type mice. Microarray analysis revealed 63 genes that are differentially expressed more than fivefold in macrophages between C57BL/6 and BALB/c mice. These results indicate that mPGES-1-mediated PGE2 produced by macrophages regulates immune responses, and IL-3 is an important factor for the differentiation of macrophages that produce higher amounts of PGE2 through mPGES-1 activity in BALB/c mice.     

A role for gangliosides and β1‐integrin in the motility of olfactory ensheathing glia

Olfactory ensheathing glia (OEG) are found in the olfactory mucosa, nerve and bulb, and provide in vivo ensheathment for the unmyelinated olfactory axons within the central and peripheral nervous system domains. OEG cells are able to migrate long distances within the neuropil of the central nervous system. Because gangliosides such as 9‐O‐acetyl GD3 have crucial regulatory roles in neuronal migration during development, we analyzed whether OEG in organotypical cultures are revealed by anti‐9‐O‐acetyl GD3 and/or gangliosides are recognized by the A2B5 antibody (G‐A2B5), and whether these gangliosides are involved in OEG migration. Our results showed that all OEG migrating out of a section of olfactory bulb onto a laminin substrate bound to the 9‐O‐acetyl GD3 and A2B5 antibodies, and that 2′,3′‐cyclic nucleotide phosphodiesterase (CNPase) colocalized with 9‐O‐acetyl GD3 and with G‐A2B5. Additionally, we showed that the immune blockade of 9‐O‐acetyl GD3 or G‐A2B5 reduced the migration of OEG on laminin, and that 9‐O‐acetyl GD3 and G‐A2B5 colocalized with the β1‐integrin subunit. We also confirmed the phenotype of in‐vitro‐grown OEG cells derived from adult rats, showing that they express CNPase, and also α‐smooth muscle actin, which is not expressed by Schwann cells. Our data showed that the gangliosides 9‐O‐acetyl GD3 and G‐A2B5 participate in the migratory activity of OEG cells, and that the β1‐integrin subunit colocalizes with these gangliosides. These results suggest a new role for β1‐integrin and gangliosides in the polarized migration of OEG cells, and provide new information on the molecules controlling OEG motility and behavior.     

Lung neuroendocrine tumours: deep sequencing of the four World Health Organization histotypes reveals chromatin‐remodelling genes as major players and a prognostic role for TERT,RB1, MEN1 and KMT2D

Next‐generation sequencing (NGS) was applied to 148 lung neuroendocrine tumours (LNETs) comprising the four World Health Organization classification categories: 53 typical carcinoid (TCs), 35 atypical carcinoid (ACs), 27 large‐cell neuroendocrine carcinomas, and 33 small‐cell lung carcinomas. A discovery screen was conducted on 46 samples by the use of whole‐exome sequencing and high‐coverage targeted sequencing of 418 genes. Eighty‐eight recurrently mutated genes from both the discovery screen and current literature were verified in the 46 cases of the discovery screen, and validated on additional 102 LNETs by targeted NGS; their prevalence was then evaluated on the whole series. Thirteen of these 88 genes were also evaluated for copy number alterations (CNAs). Carcinoids and carcinomas shared most of the altered genes but with different prevalence rates. When mutations and copy number changes were combined, MEN1 alterations were almost exclusive to carcinoids, whereas alterations of TP53 and RB1 cell cycle regulation genes and PI3K/AKT/mTOR pathway genes were significantly enriched in carcinomas. Conversely, mutations in chromatin‐remodelling genes, including those encoding histone modifiers and members of SWI–SNF complexes, were found at similar rates in carcinoids (45.5%) and carcinomas (55.0%), suggesting a major role in LNET pathogenesis. One AC and one TC showed a hypermutated profile associated with a POLQ damaging mutation. There were fewer CNAs in carcinoids than in carcinomas; however ACs showed a hybrid pattern, whereby gains of TERT, SDHA, RICTOR, PIK3CA, MYCL and SRC were found at rates similar to those in carcinomas, whereas the MEN1 loss rate mirrored that of TCs. Multivariate survival analysis revealed RB1 mutation (p = 0.0005) and TERT copy gain (p = 0.016) as independent predictors of poorer prognosis. MEN1 mutation was associated with poor prognosis in AC (p = 0.0045), whereas KMT2D mutation correlated with longer survival in SCLC (p = 0.0022). In conclusion, molecular profiling may complement histology for better diagnostic definition and prognostic stratification of LNETs. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

A role for astrocyte‐derived amyloid β peptides in the degeneration of neurons in an animal model of temporal lobe epilepsy

Kainic acid, an analogue of the excitatory neurotransmitter glutamate, can trigger seizures and neurotoxicity in the hippocampus and other limbic structures in a manner that mirrors the neuropathology of human temporal lobe epilepsy (TLE). However, the underlying mechanisms associated with the neurotoxicity remain unclear. Since amyloid‐β (Aβ) peptides, which are critical in the development of Alzheimer's disease, can mediate toxicity by activating glutamatergic NMDA receptors, it is likely that the enhanced glutamatergic transmission that renders hippocampal neurons vulnerable to kainic acid treatment may involve Aβ peptides. Thus, we seek to establish what role Aβ plays in kainic acid‐induced toxicity using in vivo and in vitro paradigms. Our results show that systemic injection of kainic acid to adult rats triggers seizures, gliosis and loss of hippocampal neurons, along with increased levels/processing of amyloid precursor protein (APP), resulting in the enhanced production of Aβ‐related peptides. The changes in APP levels/processing were evident primarily in activated astrocytes, implying a role for astrocytic Aβ in kainic acid‐induced toxicity. Accordingly, we showed that treating rat primary cultured astrocytes with kainic acid can lead to increased Aβ production/secretion without any compromise in cell viability. Additionally, we revealed that kainic acid reduces neuronal viability more in neuronal/astrocyte co‐cultures than in pure neuronal culture, and this is attenuated by precluding Aβ production. Collectively, these results indicate that increased production/secretion of Aβ‐related peptides from activated astrocytes can contribute to neurotoxicity in kainic acid‐treated rats. Since kainic acid administration can lead to neuropathological changes resembling TLE, it is likely that APP/Aβ peptides derived from astrocytes may have a role in TLE pathogenesis.     

A central role for IL-2 in fate determination of mature T cells--I: role in determining the Th1/Th2 profile in primary T cell cultures.

IL-2 signaling appears to play a significant role in enabling the synthesis of T(h)2 cytokines in an in vitro system for studying primary T cell responses. When T cells from C57BL/6J or BALB/c strains of mice were activated in vitro and re-stimulated through their TCR complex 48 h later, CD4(+) T cells producing the T(h)2 cytokines IL-4 and IL-10 were found only when IL-2 was present. IL-2 also enhanced IFN-gamma synthesis in C57BL/6J cells but not in BALB/c cells. By up-regulating production of anti-inflammatory T(h)2 cytokines during a primary response, IL-2 may play a critical role in limiting T(h)1-mediated responses.     

A new role for interleukin-7 in the induction of LFA-1 and VLA-4 adhesion molecules in Phorbol 12myristate 13acetate activated CD4+CD23+ T-cell subset

Background The low affinity receptor for IgE, CD23, has been described in several pathological conditions. However, the factors involved in the upregulation or downregulation of this receptor are still debated. Methods and Results We studied the effect of interleukin 7 (IL-7) on the expression of CD23 in normal PBT cells stimulated with PMA + Ca². The data indicate that activated PB-T cultured in the presence of IL-7 showed an increased expression of CD23. The induction of IL-7 on CD23 production appears to be independent of IL-2, IL-4, IL-9, IL-15. Indeed, the addition of specific MoAbs anti-IL-2, IL-4, IL-9, IL-15 oranti-IL2R was unable to block the effect of IL-7 on CD23. The addition of IL-7 to a specific subset CD4⁺CD23⁺ was able to augment the adhesiveness of T cells to parenchymal cell monalayers. The use of different cytokine (IL-2, IL-4, IL-9, IL-15) resulted in no increase of adhesiveness. In contrast the addition of IL-7 to a different T-cell subset (i.e. CD4⁺CD23^-) was unable to rescue the lack of adhesiveness observed in these cells. Blocking experiments with MHM6 MoAb were able to drastically reduce the adhesiveness observed in CD4+FCD23+ subsets. The presence of LFA-1 and VLA-4 adhesion molecules were responsible for the augmented adhesiveness of activated CD4⁺CD23⁺ T cells cultured in the presence of IL-7. Blocking experiments with anti-LFA-1, VLA-4. anti-LFA-1β plus VLA-4α MoAbs or anti-ICAM-1 MoAb added to the monolayers resulted in a complete inhibition of adhesion to parenchymal monolayers. In contrast, the addition of anti-IL-7 oranti-IL-7R MoAbs were able to block the augmented adhesiveness of CD4⁺CD23⁺ cells to monolayers observed in the presence of IL-7. Conclusions Taken together these findings point to the likelihood that IL-7 is responsible for the observed quantitative difference in the level of adhesion molecules and may open a new role of CD23 in the immune regulation.