Lisa R. Banner

Leukaemia inhibitory factor is required for normal inflammatory responses to injury in the peripheral and central nervous systems in vivo and is chemotactic for macrophages in vitro

The cytokine leukaemia inhibitory factor (LIF) is up‐regulated in glial cells after injury to the peripheral and central nervous systems. In addition, LIF is required for the changes in neuropeptide expression that normally occur when the axons of sympathetic and sensory neurons are transected. We investigated whether LIF is also necessary for the initial inflammatory response that follows mechanical injury to the sciatic nerve and cerebral cortex of adult mice. We find that inflammatory cell infiltration into crushed sciatic nerve is significantly slower in LIF knock‐out (KO) mice compared with wild‐type (WT) mice. Similarly, the microglial and astroglial responses to surgical injury of the cortex are significantly slower in LIF KO mice compared with WT mice. Consistent with these in vivo results, LIF is chemotactic for peritoneal macrophages in a microchamber culture assay. Thus, LIF is a key regulator of neural injury in vivo, where it is produced by glia and can act directly on neurons, glia and inflammatory cells. We also find that the initial inflammatory response to cortical injury is diminished in interleukin (IL)‐6 KO mice. Surprisingly, however, the inflammatory response in LIF‐IL‐6 double KO mice is very similar to that of the single KO mice, suggesting that these cytokines may act in series rather than in parallel in this response.

Leukemia Inhibitory Factor Is Expressed in Astrocytes Following Cortical Brain Injury

The neuropoietic cytokine leukemia inhibitory factor (LIF) can act as a trophic factor, enhancing neuronal survival, and as a differentiation factor altering neuronal and glial gene expression. LIF also plays a role in the response to injury of the peripheral nervous system, as indicated by an increase in the amount of its mRNA within nonneuronal injury response in LIF knock-out mice. To determine if LIF is regulated after injury to the central nervous system, we surgically lesioned the cortex in adult rat brain. Using a quantitative RNAse protection assay, we find that LIF mRNA increases 30-fold following injury. The amount of this transcript goes up within 6 h after injury, reaches a peak at 24 h and returns to baseline by 7 days postlesion. In situ hybridization analysis reveals LIF transcript-containing cells scattered throughout the ipsilateral cortex close, but not immediately adjacent to the lesion site. Double-labeling with a variety of antibodies reveals that LIF mRNA is induced in GFAP-positive astrocytes as well as in a small number of microglial cells. The striking induction of LIF transcripts in glia suggests that this cytokine may play a key injury-response role in the CNS as it does in the PNS, where LIF has been demonstrated to regulate neuropeptide expression both in vivo and in vitro.

Random nature of coronavirus RNA recombination in the absence of selection pressure

Abstract RNA-RNA recombination is thought to occur preferentially at certain selected sites and in only a few RNA viruses; the mechanism for these restrictions is unknown. In this paper we report the development of a recombination assay for coronavirus, using polymerase chain reaction, in the absence of selection pressure. Our results showed that within a 1-kb region of the peplomer gene, RNA recombination occurred at almost every potential crossover site. Thus, coronavirus RNA recombination appears to be more random than previously realized. However, after serial passages of the recombinant viruses in tissue culture, the recombination sites among the progeny viruses became clustered in the region which contains the previously reported “hot spot” for coronavirus recombination. These results suggest that RNA recombination is common and random in nature, but only certain recombinants can be selected. Thus, the presence of recombinational “hot spots” for coronavirus or other RNA viruses most likely resulted from selection of certain recombinant viruses and not restriction on the occurrence of RNA recombination. The failure to detect recombinants in other RNA viruses may therefore be due to unfavorable properties of recombinant viruses. This approach can be used to detect recombinants in these viruses.

Heterogeneity of gene expression of the hemagglutinin-esterase (HE) protein of murine coronaviruses☆

Abstract The hemagglutinin-esterase (HE) membrane glycoprotein is present only in some members of the coronavirus family, including some strains of mouse hepatitis virus (MHV). In the JHM strain of MHV, expression of the HE gene is variable and corresponds to the number of copies of a UCUAA pentanucleotide sequence present at the 3′-end of the leader RNA. This copy number varies among MHV strains, depending on their passage history. The JHM isolates with two copies of UCUAA in their leader RNA showed a high level of HE expression, whereas the JHM isolate with three copies had a low-level expression. In this study, the analysis of HE gene expression was extended to other MHV strains. The synthesis of HE mRNA in these viruses also correlates with the copy number of UCUAA in the leader RNA and the particular intergenic sequence preceding the HE gene. In one MHV strain, MHV-1, no detectable HE mRNA was synthesized, despite the presence of a proper transcription initiation signal. This lack of HE mRNA expression was consistent with a leader RNA containing three UCUAA copies. However, mutations and deletions within the coding region of the MHV-1 HE gene have generated a stretch of sequence which resembled the transcriptional initiation motif, and was shown to initiate the synthesis of a novel smaller mRNA. These findings strengthened the theory that interactions between leader RNA and transcriptional initiation sequences regulate MHV subgenomic mRNA transcription. Sequence analysis revealed that most MHV strains, through extensive mutations, deletions, or insertions, have lost the complete HE open reading frame, thus turning HE into a pseudogene. This high degree of variation is unusual as the other three structural proteins (spike, membrane, and nucleocapsid) are well-maintained. In contrast to bovine coronavirus, which apparently requires HE for viral replication, the HE protein in MHV may be only an accessory protein which is not necessary for viral replication. JHM and MHV-S, however, have preserved the expression of HE protein.

A clustering of RNA recombination sites adjacent to a hypervariable region of the peplomer gene of murine coronavirus.

Abstract Coronaviruses undergo RNA recombination at a very high frequency. To understand the mechanism of recombination in murine coronavirus, we have performed RNA sequencing of viral genomic RNA to determine the precise sites of recombination in a series of recombinants which have crossovers within the gene encoding the peplomer protein. We found that all of the recombination sites are clustered within a region of 278 nucleotides in the 5′-half of the gene. This region in which all of the crossovers occurred represents a small fraction of the distance between the two selection markers used for the isolation of these recombinant viruses. This result suggests that this region may be a preferred site for RNA recombination. The crossover sites are located within and immediately adjacent to a hypervariable area of the gene. This area has undergone deletions of varying sizes in several virus strains which have been passaged either in vivo or in vitro. These results suggest that a similar RNA structure may be involved in the occurrence of both recombination and deletion events.

Localization of extensive deletions in the structural genes of two neurotropic variants of murine coronavirus JHM.

Abstract The intracellular RNA of two neurotropic variants of the JHM strain of mouse hepatitis virus (MHV) independently isolated from the brain and spinal cord of an infected Wistar Furth rat were compared with that of the parental virus. The mRNAs corresponding to the genes encoding the peplomer (S) and the hemagglutinin-esterase (HE) proteins of the variant viruses were found to be smaller in size. The possible sequence changes were studied by oligonucleotide fingerprinting and direct RNA sequencing. Both variants have a large deletion of 246 amino acids in the carboxy-terminal end of the HE protein. However, this truncated protein was not detected in the infected cells, suggesting either a translational regulation or rapid degradation of the truncated protein in these cells. The variant virus isolated from the spinal cord has a second deletion of 147 amino acids in the amino-terminal half of the S protein. This deletion site corresponds to a hypervariable region where deletions have been frequently noted among MHV variants with different biological properties. These findings suggest that the changes in pathogenic properties of the two neural isolates are associated with drastic alterations of the viral structural glycoproteins.

Igf-i expression is decreased in Lif-deficient mice after peripheral nerve injury

We investigated the regulation of insulin-like growth factor I (IGF-I) expression after sciatic nerve crush using leukemia inhibitory factor (LIF)-deficient mice. One day post-crush, IGF-I mRNA levels were lower in the LIF-deficient mouse nerve than in the wild type nerve. IGF-I protein, analyzed by immunohistochemistry, was also decreased 1 day post-crush in LIF-deficient nerves relative to wild type nerves. By 3 days post-crush, IGF-I immunoreactivity was induced in Schwann cells to equivalent levels in both types of nerve. After crush, IGF-I expression was also found in mast cells, and these were initially decreased in the LIF-deficient mice. Thus, LIF appears to regulate IGF-I expression in the peripheral nerve basally and early in the regeneration response in vivo.

Early changes of LIFR and gp130 in sciatic nerve and muscle of diabetic mice

Peripheral neuropathy is a common complication of diabetes mediated by alterations of growth factors. Members of the neuropoietic cytokine family, which include IL-6, LIF, and CNTF among others, have been shown to be important regulators of peripheral nerves and the muscles that they innervate. To investigate their potential role in diabetic nerve and muscle, we studied the expression of the shared receptor subunits, LIFR and gp130 in a mouse model of streptozotocin (STZ)-induced diabetes. The results of Western blotting and densitometric analysis showed that both LIFR and gp130 protein expression were increased in diabetic sciatic nerve compared to control mice at early time points following STZ injection. In diabetic gastrocnemius muscle, LIFR and gp130 were increased from 3 days to 24 weeks following STZ injection. In contrast, both LIFR and gp130 protein expression were decreased in diabetic soleus muscle at 3-days post-injection. Our results suggest that hyperglycemia results in changes to nerve and muscle soon after the onset of diabetes and that cytokines may play a role in this process.

Murine hepatitis virus JHM variants isolated from Wistar Furth rats with viral-induced neurological disease.

Murine Hepatitis Virus (MHV) can produce neurological disease in murine species.(1) Intracerebral (ic) inoculation of 2-day old Wistar Furth rats with the JHM virus (JHMV) strain of MHV generally produces an acute encephalitis which kill the rats within one week of inoculation. (2,3) Grey matter lesions generally predominate in the central nervous system (CNS) of these rats. Wistar Furth rats which are inoculated at 10 days of age with JHMV generally develop a chronic demyelinating disease characterized by hind leg paralysis or paresis. (2,3,4) These symptoms generally do not develop until 2–4 weeks post inoculation.(2,4) The rats that live for 3 weeks post inoculation or longer generally have predominately white matter lesions. (2,3,4)