Protein engineering of interferon alphas.

Interferon (IFN)-alphas constitute a family of proteins exhibiting high degree of homology in primary, secondary, and tertiary structure and display a high level of species specificity in their biological properties. However, small structural differences in these proteins may be responsible for a significant variety of biological actions. Understanding the structure and function of human IFN-alpha is very important. Recombinant techniques are important tools for the production and modification of IFN proteins. The first IFN hybrid, IFN-alpha1/alpha2 was constructed using recombinant technology in 1981. Subsequently, a number of IFN hybrids and mutants have been constructed, expressed and characterized. These hybrids and mutants have resulted in novel IFNs that either combine different biological properties from the parental proteins or have significantly different biological activity. Therefore, IFN hybrids and mutants have provided a powerful tool for studying the structure and function of these molecules. Also, these engineered IFNs may have important new therapeutic applications and may provide greater sights into understanding of the clinical activities of these molecules.

Secretory leukocyte protease inhibitor binds to annexin II, a cofactor for macrophage HIV-1 infection.

The distribution of secretory leukocyte protease inhibitor (SLPI) at entry portals indicates its involvement in defending the host from pathogens, consistent with the ability of SLPI to inhibit human immunodeficiency virus (HIV)-1 infection by an unknown mechanism. We now demonstrate that SLPI binds to the membrane of human macrophages through the phospholipid-binding protein, annexin II. Based on the recent identification of human cell membrane phosphatidylserine (PS) in the outer coat of HIV-1, we define a novel role for annexin II, a PS-binding moiety, as a cellular cofactor supporting macrophage HIV-1 infection. Moreover, this HIV-1 PS interaction with annexin II can be disrupted by SLPI or other annexin II-specific inhibitors. The PS-annexin II connection may represent a new target to prevent HIV-1 infection.

Gonadotropin-regulated testicular RNA helicase (GRTH/Ddx25) is essential for spermatid development and completion of spermatogenesis.

Gonadotropin-regulated testicular RNA helicase (GRTH/Ddx25), a member of the DEAD-box protein family, is a testis-specific gonadotropin-regulated RNA helicase that is present in Leydig cells and germ cells (meiotic spermatocytes and spermatids). In this study, we observed that GRTH is present in the nucleus, cytoplasm and chromatoid body of germ cells, and is an integral component of messenger ribonuclear protein particles. Male mice with a null mutation in the GRTH gene displayed normal gonadotropin and androgen profiles. However, they were sterile, with azoospermia caused by a complete arrest of spermiogenesis at step 8 of round spermatids and failure to elongate. Round spermatids of the null mice showed marked diminution in the size of chromatoid bodies. The transcription of relevant messages was not altered, but their translation was abrogated in a selective manner. Protein expression of transition proteins 1 and 2 and angiotensin-converting enzyme was completely absent, whereas that of the transcriptional activator cAMP responsive element modulator was intact. These findings indicate that GRTH participates in translational-associated events during germ cell development. Although significant apoptosis was present at the metaphase of meiosis in the GRTH-null mice, spermatogenesis proceeded to step 8 of spermiogenesis when complete arrest occurred. This progression may relate to compensatory gene function(s) and/or the observed up-regulation of DNA repair proteins Rad51 and Dmc1. This study (i) demonstrates that GRTH is essential for completion of spermatogenesis, (ii) provides insights into intrinsic requirements for spermiogenesis, and (iii) establishes a model for studies of male infertility and contraception.

Conversion of peripheral CD4+CD25- naive T cells to CD4+CD25+ regulatory T cells by TGF-beta induction of transcription factor Foxp3.

CD4+CD25+ regulatory T cells (Treg) are instrumental in the maintenance of immunological tolerance. One critical question is whether Treg can only be generated in the thymus or can differentiate from peripheral CD4+CD25- naive T cells. In this paper, we present novel evidence that conversion of naive peripheral CD4+CD25- T cells into anergic/suppressor cells that are CD25+, CD45RB-/low and intracellular CTLA-4+ can be achieved through costimulation with T cell receptors (TCRs) and transforming growth factor beta (TGF-beta). Although transcription factor Foxp3 has been shown recently to be associated with the development of Treg, the physiological inducers for Foxp3 gene expression remain a mystery. TGF-beta induced Foxp3 gene expression in TCR-challenged CD4+CD25- naive T cells, which mediated their transition toward a regulatory T cell phenotype with potent immunosuppressive potential. These converted anergic/suppressor cells are not only unresponsive to TCR stimulation and produce neither T helper cell 1 nor T helper cell 2 cytokines but they also express TGF-beta and inhibit normal T cell proliferation in vitro. More importantly, in an ovalbumin peptide TCR transgenic adoptive transfer model, TGF-beta-converted transgenic CD4+CD25+ suppressor cells proliferated in response to immunization and inhibited antigen-specific naive CD4+ T cell expansion in vivo. Finally, in a murine asthma model, coadministration of these TGF-beta-induced suppressor T cells prevented house dust mite-induced allergic pathogenesis in lungs.

Estrogen modulates cutaneous wound healing by downregulating macrophage migration inhibitory factor.

Characteristic of both chronic wounds and acute wounds that fail to heal are excessive leukocytosis and reduced matrix deposition. Estrogen is a major regulator of wound repair that can reverse age-related impaired wound healing in human and animal models, characterized by a dampened inflammatory response and increased matrix deposited at the wound site. Macrophage migration inhibitory factor (MIF) is a candidate proinflammatory cytokine involved in the hormonal regulation of inflammation. We demonstrate that MIF is upregulated in a distinct spatial and temporal pattern during wound healing and its expression is markedly elevated in wounds of estrogen-deficient mice as compared with intact animals. Wound-healing studies in mice rendered null for the MIF gene have demonstrated that in the absence of MIF, the excessive inflammation and delayed-healing phenotype associated with reduced estrogen is reversed. Moreover, in vitro assays have shown a striking estrogen-mediated decrease in MIF production by activated murine macrophages, a process involving the estrogen receptor. We suggest that estrogen inhibits the local inflammatory response by downregulating MIF, suggesting a specific target for future therapeutic intervention in impaired wound-healing states.