A novel cellular RNA helicase, RH116, differentially regulates cell growth, programmed cell death and human immunodeficiency virus type 1 replication.

In an effort to define novel cellular factors regulating human immunodeficiency virus type 1 (HIV-1) replication, a differential display analysis has been performed on endogenously infected cells stimulated with the HIV-suppressive immunomodulator Murabutide. In this study, the cloning and identification of a Murabutide-downregulated gene, named RH116, bearing classical motifs that are characteristic of the DExH family of RNA helicases, are reported. The 116 kDa encoded protein shares 99.9 % similarity with MDA-5, an inducible RNA helicase described recently. Ectopic expression of RH116 in HeLa-CD4 cells inhibited cell growth and cell proliferation but had no measurable effect on programmed cell death. RH116 presented steady state cytoplasmic localization and could translocate to the nucleus following HIV-1 infection. Moreover, the endogenous expression of RH116, at both the transcript and protein levels, was found to be considerably upregulated after infection. Overexpression of RH116 in HIV-1-infected HeLa-CD4 cells also resulted in a dramatic increase in the level of secreted viral p24 protein. This enhancement in virus replication did not stem from upregulated proviral DNA levels but correlated with increased unspliced and singly spliced viral mRNA transcripts. These findings implicate RH116 in the regulation of HIV-1 replication and point to an apoptosis-independent role for this novel helicase in inducing cell growth arrest.

SS-56, a novel cellular target of autoantibody responses in Sjögren syndrome and systemic lupus erythematosus.

Certain autoimmune disorders, including Sjögren syndrome (SS) and systemic lupus erythematosus (SLE), are characterized by autoantibodies against the Ro/SSA and La/SSB cellular antigens. Although the implication of these autoantibodies in disease pathogenesis is still unclear, it is believed that the aberrant responses against autoantigens may extend to other proteins that are not yet well defined. In an attempt to analyze the regulated gene expression in lymphocytes by an HIV-suppressive immunomodulator, we have identified and cloned a novel gene encoding a 56-kDa protein, named SS-56, which is structurally related to the 52-kDa Ro/SSA antigen. The new protein showed primarily perinuclear cytoplasmic localization, and recombinant SS-56 was found to react in ELISA with sera from most patients with SS or SLE. Western blot analysis confirmed the autoantigenic nature of native SS-56 in extracts from HeLa cells. Interestingly, the incidence of antibodies to SS-56 was associated with visceral complications in SLE, and roughly half of the 17 SS or SLE patients with no detectable antibodies to SSA and SSB antigens presented measurable antibodies against recombinant SS-56. Thus, SS-56 represents a new member of the SS family of autoantigens and could become an additional and important diagnostic marker for SS and SLE.

Selective regulation of human immunodeficiency virus-infected CD4(+) lymphocytes by a synthetic immunomodulator leads to potent virus suppression in vitro and in hu-PBL-SCID mice.

We have previously observed that the synthetic immunomodulator Murabutide inhibits human immunodeficiency virus type 1 (HIV-1) replication at multiple levels in macrophages and dendritic cells. The present study was designed to profile the activity of Murabutide on CD8-depleted phytohemagglutinin-activated lymphocytes from HIV-1-infected subjects and on the outcome of HIV-1 infection in severe combined immunodeficiency mice reconstituted with human peripheral blood leukocytes (hu-PBL-SCID mice). Maintaining cultures of CD8-depleted blasts from 36 patients in the presence of Murabutide produced dramatically reduced levels of viral p24 protein in the supernatants. This activity correlated with reduced viral transcripts and proviral DNA, was evident in cultures harboring R5, X4-R5, or X4 HIV-1 isolates, was not linked to inhibition of cellular DNA synthesis, and did not correlate with beta-chemokine release. Moreover, c-myc mRNA expression was down-regulated in Murabutide-treated cells, suggesting potential interference of the immunomodulator with the nuclear transport of viral preintegration complexes. On the other hand, daily treatment of HIV-1-infected hu-PBL-SCID mice with Murabutide significantly reduced the viral loads in plasma and the proviral DNA content in human peritoneal cells. These results are the first to demonstrate that a clinically acceptable synthetic immunomodulator with an ability to enhance the host's nonspecific immune defense mechanisms against infections can directly regulate cellular factors in infected lymphocytes, leading to controlled HIV-1 replication.

Identification of a developmentally regulated Schistosoma mansoni serine protease homologous to mouse plasma kallikrein and human factor I.

The isolation of 2 genomic clones has allowed us to further characterize a Schistosoma mansoni serine protease designated SmSP1. The deduced amino acid sequence (248aa) considered as a 'light chain' encoding the active site, presents significant homologies with mouse plasma kallikrein and human factor I light chain. The secondary structure of SmSP1 'light chain' is correctly predicted and may be sufficient by itself to constitute an active enzyme. The biological function of SmSP1 is unknown, however, the homology with 2 serine proteases suggests that SmSP1 may play a role in the evasion of the host immune response. This is supported by the presence of the native protein corresponding to SmSP1 particularly in schistosomula released products (SRP) and in male dorsal spines. The expression of this enzyme is differentially regulated throughout the parasite life-cycle. However, infected animals with S. mansoni did not produce specific antibodies to recombinant SmSP1. The lack of such response could be advantageous to the parasite by protecting itself from host effector mechanisms.

Profiles of Th1 and Th2 cytokines after primary and secondary infection by Schistosoma mansoni in the semipermissive rat host.

In contrast to most mouse strains, rats eliminate the primary schistosome burden around 4 weeks postinfection and subsequently develop protective immunity to reinfection. In rat schistosomiasis, we have shown predominant expression of a Th2-type cytokine response at the mRNA level after primary infection. In the present study, we showed a significant increase in interleukin-4 (IL-4) mRNA expression in inguinal lymph nodes early after a secondary infection. IL-5 mRNA expression showed a significant increase at days 2 and 4 postreinfection in the spleen and lymph nodes, respectively. We did not detect any gamma interferon (IFN-gamma) mRNA after a challenge infection. Analysis of cytokine secretion by stimulated spleen cells after a primary infection showed predominant expression of IL-4 with maximum production on day 21, accompanied by production of IL-5 from day 11 to day 67. A significant increase in IFN-gamma secretion was detected at day 21. Analysis of immunoglobulin G2b (IgG2b) and IgG2c (Th1-related isotypes) showed undetectable levels of IgG2b, but detectable levels of specific IgG2c antibodies were observed from day 42. The analysis of Th2-related isotypes showed high specific IgG1 and IgG2a antibody titers from day 29. After a secondary infection, only IL-4 and IL-5 secretion was sustained. This is supported by the increased production of Th2-related isotypes. These findings showed that S. mansoni infection can drive Th2 responses in rats in the absence of egg production which is required to induce a Th2 response in mice and are in favor of the role of Th2-type cytokines in protective immunity against reinfection.

Molecular cloning and sequencing of the rat interleukin-12 p40 gene.

The nucleotide sequences containing the rat interleukin-12 p40 gene was determined. Sequencing revealed the presence of six exons and five introns. Analysis of the 5' non-coding region showed the presence of several possible sites involved in cytokine gene regulation at the transcriptional level. Comparison of the deduced amino acid sequence of rat IL-12 p40 with that of the mouse and of human p40, showed 92% and 65% identity respectively.

In vivo expression of cytokine mRNA in rats infected with Schistosoma mansoni.

As an animal model, rat schistosomiasis mansoni has provided considerable knowledge of immune mechanisms involved in the expulsion of worms and in a subsequent development of immunity to reinfection. Although it is clear that ADCC mechanisms participate in immunity to reinfection; the nature of the cytokines involved in immunity is unknown. To analyse the pattern of cytokines involved, the mRNA levels of different cytokines were assessed by RT-PCR as they occur within tissues during the course of infection. In spleens from infected rats, a significant elevation in IL-2 and IL-5 mRNA was observed during the early phase of infection (day 7). Analysis of pulmonary cytokine responses showed a dramatic increase in IL-4 and IL-5 on day 7. This was accompanied with a low but significant increase in IL-2 (day 11) and IL-12 (day 7) in the absence of augmented IFN-gamma expression. The cytokine expression patterns of draining lymph nodes (LN) from infected rats showed a significant increase of IL-2, IL-4 and IL-5 on day 21. Analysis of IL-10 expression showed exclusively a significant increase in LN on day 11, IFN-gamma mRNA was not detected in any tissue sample. Thus, rats develop a predominately Th2-type cytokine response during a primary infection which may be involved at least in part, in the expression of immunity against Schistosoma mansoni infection.

Expression of rat interleukin-5 and generation of neutralizing antiserum: a comparative study of rat IL-5 produced in Escherichia coli and insect cells.

A cDNA coding for rat IL-5 was obtained by RT-PCR from total spleen RNA. With the exception of a single a.a. replacement at position 85 (L-P), it is identical to the published sequence obtained by retroviral gene transfer. This cDNA was used to express biologically active recombinant IL-5 in E. coli and in insect cells using a baculovirus system. Rat IL-5 is more active on B13, an IL-5 dependent cell line, when produced in insect cells (specific activity 1.47 x 10(11)UI/mg compared to 4.28 x 10(6)UI/mg). This increased activity seems to be associated with the presence of IL-5 homodimers in recombinant protein preparations. A rabbit antiserum raised against recombinant bacterial IL-5 specifically inhibited B13 proliferation induced by bacterial and baculoviral IL-5. The availability of such reagents should facilitate studying the role of IL-5 in different infectious diseases, experimental allergic encephalomyelitis and in transplantation biology where the rat represents a more suitable model than mice.

Molecular characterization of a partial sequence encoding a novel Schistosoma mansoni serine protease.

A PCR strategy using degenerate oligonucleotide primers based upon consensus sequences of the active site of serine proteases yielded a 467 bp fragment from genomic DNA from Schistosoma mansoni cercariae. The sequence presented a continuous open reading frame and the deduced amino acid sequence (156 aa) presented homologies with various serine proteases, in particular the highest percentage identity was observed with a mammalian plasma kallikrein. The expression of this serine protease was studied first at the mRNA level and it was only detected by RT-PCR in cercariae and in adult worms. At the protein level we were able to detect it by Western blotting and by using antigen extracts from metabolically radio-isotope labelled worms. The absence of any positive signal in Northern blot and the detection of the protein suggest that the mRNA has a very short half-life, however the protein may be accumulated in the parasite. The significance of identity with mammalian kallikrein was confirmed by cross-immunoreactivity with a native porcine pancreatic kallikrein. However, no cross-reactivity was observed with S. mansoni elastase, another serine protease. Thus, we suggest that the serine protease described in this paper is a kallikrein-like protease.