Gender influences the incidence and severity of some bacterial and viral infections and autoimmune diseases in animal models and humans. To determine a gender-based difference, comparisons were made between male and female mice inoculated with herpes simplex virus type 1 (HSV-1) by the corneal route. Mortality was higher in the male mice of the three strains tested: 129/Sv//Ev wild type, gamma interferon (IFN-␥) knockout (GKO), and IFN-␥ receptor knockout (RGKO). Similarly, in vivo HSV-1 reactivation occurred more commonly in male mice, but the male-female difference in reactivation was restricted to the two knockout strains and was not seen in the 129/Sv//Ev control. Comparison among male mice of the three strains showed a higher mortality of the RGKO mice and a higher reactivation rate of the GKO and RGKO mice than of the 129/Sv//Ev males. In contrast, female RGKO and GKO mice did not differ from female 129/Sv//Ev controls in either mortality or reactivation. HSV-1 periocular and eyelid disease was also more severe in male and dihydrotestosterone (DHT)-treated female mice than in control female mice. These results show a consistent gender difference in HSV-1 infection, with a worse outcome in male mice. In addition, the results comparing GKO and RGKO mice to controls show differences only in male mice, suggesting that some effects of IFN-␥, a key immunoregulatory molecule, are gender specific.

Recently, prokaryotic DNAs containing unmethylated CpG motifs have been shown to be intrinsically immunostimulatory both in vitro and in vivo, tending to promote Th1-like responses. In contrast, CpG dinucleotides in mammalian DNAs are extensively methylated on cytosines and hence immunologically inert. Since the herpes simplex virus (HSV) genome is unmethylated and G؉C rich, we predicted that CpG motifs would be highly prevalent in the HSV genome; hence, we examined the immunostimulatory potential of purified HSV DNA in vitro and in vivo. Historically, DNA has been viewed as immunologically inert. However, numerous recent studies have established that bacterial, but not mammalian, DNAs can activate both innate and adaptive immune responses. This indicates that the vertebrate immune system has evolved to discriminate basic structural differences between invertebrate and mammalian DNAs (27, 62). The motifs that mediate immunostimulation and discrimination of bacterial DNAs have been identified in Escherichia coli DNA as nonmethylated CpG dinucleotides flanked by specific bases (32). CpG dinucleotides are present at 25% of the expected frequency in mammalian DNA, and when they occur, they are invariably methylated on cytosines and usually flanked by bases that constitute immune-neutralizing rather immunostimulatory motifs (6, 31) Nonmethylated CpG DNA induces direct activation of professional antigen-presenting cells (APCs), including dendritic cells, macrophages, and B cells, but not T cells. CpG DNA upregulates expression of major histocompatibility complex (MHC) class II and costimulatory molecules (e.g., B7-1 and B7-2), induces cytokine production by macrophages and DCs, and additionally promotes polyclonal activation of B cells (26,30,32), but it does not directly activate T cells (27). Synthetic oligodeoxyribonucleotides (ODNs) containing unmethylated consensus CpG motifs can mimic immunostimulatory bacterial DNAs, and, remarkably, a single nucleotide change as in the case of GpC or methylation of the cytosine within the CpG motif is sufficient to abolish immunostimulatory activity (36,37). It is now established that innate defense mechanisms are triggered by host reactions to pathogen-associated molecular patterns that distinguish infectious entities from the host itself and additionally discriminate among different invading pathogens (3, 41). Several studies have implicated members of the Toll-like receptor (TLR) family (originally identified in Drosophila) as the receptors for pathogen-associated molecular patterns. Thus, TLR4 and TLR2 react to lipopolysaccharides (LPS) in gram-negative bacteria and peptidoglycans and lipopeptides in gram-positive bacteria, respectively. Hemmi et al. (18) recently demonstrated that cells from TLR9-deficient mice fail to respond to CpG DNA but remain fully responsive to bacterial LPS. In contrast, inactivation of TLR4 blocked responses to LPS but not CpG DNA (22). These results show that unmethylated CpG motifs in bacterial DNAs are yet another molecular pattern for which ...

Porcine epidemic diarrhea virus (PEDV) is an enteric coronavirus causing acute intestinal infection in pigs, with high mortality often seen in neonatal pigs. The newborns rely on innate immune responses against invading pathogens because of lacking adaptive immunity. However, how PEDV disables the innate immunity of newborns towards severe infection remains unknown. We found that PEDV infection led to reduced expression of histone deacetylases (HDACs), especially HDAC1 in porcine IPEC-J2 cells. HDACs are considered as important regulators of innate immunity. We hypothesized that PEDV might interact with certain host factors to regulate HDAC1 expression in favor of its replication. We show that HDAC1 acted as a negative regulator of PEDV replication in IPEC-J2 cells, as shown by chemical inhibition, gene knockout and overexpression. A GC-box ( GCCCCACCCCC ) within the HDAC1 promoter region was identified for Sp1 binding in IPEC-J2 cells. Treatment of the cells with Sp1 inhibitor, mithramycin A, inhibited HDAC1 expression, indicating direct regulation of HDAC1 expression by Sp1. Of the viral proteins that were overexpressed in IPEC-J2 cells, the N protein was found to be present in the nuclei and more inhibitory to HDAC1 transcription. The putative NLS 261 PKKNKSR 267 contributed to its nuclear localization. The N protein interacted with Sp1 and interfered with its binding to the promoter region, thereby inhibiting its transcriptional activity for HDAC1 expression. Our findings reveal a novel mechanism of PEDV evasion of the host responses, offering implications for studying the infection processes of other coronaviruses. Importance The enteric coronavirus porcine epidemic diarrhea virus (PEDV) causes fatal acute intestinal infection in neonatal pigs that rely on innate immune responses. Histone deacetylases (HDACs) play important roles in innate immune regulation. Our study found PEDV suppresses HDAC1 expression via the interaction of its N protein and porcine Sp1, which identified a novel mechanism of PEDV evasion of the host responses to benefit its replication. This study suggests that other coronaviruses, including SARS-CoV and SARS-CoV-2, may also make use of their N proteins to intercept the host immune responses in favor of their infection.

A novel mass spectrometry-based assay system for determining protein kinase activity employing mass-tagged substrate peptide probes was used for the diagnosis of tumors. Two peptide probes (H-type and D-type) were synthesized containing the same substrate peptide sequence for protein kinase C (PKC). The molecular weights of the two probes differ because of the incorporation of deuterium into the acetyl groups of the D-type probe. The lysates of the normal and tumor tissue were prepared and reacted with the H-and D-type peptide probes, respectively. The PKC activities of the normal and tumor tissues can be compared simply and directly by calculating the phosphorylated ratio to each peptide probe, obtained from the peak intensity of the mass spectrum after mixing of the two reaction solutions. The phosphorylation ratio for the reaction of the H-type peptide probe with the tumor tissue lysate (B16 melanoma) was more than three times higher than that of the D type peptide probe with the normal skin tissue lysate. These results show that the novel assay system for detecting protein kinase activity using mass-tag technology can be a simple and useful means to profile protein kinase activity for cell or tissue lysate samples, and can be applied to the diagnosis of tumors. (J Am Soc Mass Spectrom 2007, 18, 106 -112)

Despite tremendous advances in cancer treatment and survival rates, pancreatic cancer remains one of the most deadly afflictions and the fourth leading cause of cancer deaths in the world. Matrix Metalloproteinases (MMPs) are thought to be involved in cancer progression. Matrix metalloproteinase (MMP)-2 is known to play a pivotal role in tumor invasion, metastasis and angiogenesis, and validated to be the anticancer target. Inhibition of MMP-2 activity is able to reduce the cancer cell invasion and suppress tumor growth in vivo. Two novel peptides, M204C4 and M205C4, which could specially inhibit MMP-2 activity, were identified by a phage display library screening. We showed that M204C4 and M205C4 inhibited the activity of MMP-2 in a dose dependent manner in vitro. Two peptides reduced MMP-2 mediated invasion of the pancreatic cancer cell lines PANC-1 and CFPAC-1, but not affected the expression and release of MMP-2. Furthermore, these two peptides could suppress tumor growth in vivo. Our results indicated that two peptides selected by phase display technology may be used as anticancer drugs in the future.