Carol H. Pontzer

Identification of a Transcytosis Epitope on Staphylococcal Enterotoxins

ABSTRACT Staphylococcal enterotoxins (SE) are exoproteins produced by Staphylococcus aureus that act as superantigens and have been implicated as a leading cause of food-borne disease and toxic shock. Little is known about how these molecules penetrate the gut lining and gain access to both local and systemic immune tissues. To model movement in vitro of staphylococcal enterotoxins, we have employed a monolayer system composed of crypt-like human colonic T-84 cells. SEB and SEA showed comparable dose-dependent transcytosis in vitro, while toxic shock syndrome toxin (TSST-1) exhibited increased movement at lower doses. Synthetic peptides corresponding to specific regions of the SEB molecule were tested in vitro to identify the domain of the protein involved in the transcytosis of SE. A toxin peptide of particular interest contains the amino acid sequence KKKVTAQELD, which is highly conserved across all SE. At a toxin-to-peptide ratio of 1:10, movement of SEB across the monolayers was reduced by 85%. Antisera made against the SEB peptide recognized native SEB and also inhibited SEB transcytosis. Finally, the conserved 10-amino-acid peptide inhibited transcytosis of multiple staphylococcal enterotoxins, SEA, SEE, and TSST-1. These data demonstrate that this region of the staphylococcal enterotoxins plays a distinct role in toxin movement across epithelial cells. It has implications for the prevention of staphylococcal enterotoxin-mediated disease by design of a peptide vaccine that could reduce systemic exposure to oral or inhaled superantigens. Since the sequence identified is highly conserved, it allows for a single epitope blocking the transcytosis of multiple SE.

Interferon-tau inhibits the development of diabetes in NOD mice

Interferon-alpha (IFN-α) inhibits the development of diabetes in animal models of autoimmune diabetes. However, the mechanism of the action is not fully understood and drug toxicity could limit its potential clinical utility. Interferon-tau (IFN-τ) is another type 1 interferon, which has less toxicity but may have different biologic activity than IFN-α. This study explores the effect of IFN-τ on the diabetic process in non-obese diabetic (NOD) mice. IFN-τ by intraperitoneal, subcutaneous, or oral routes of administration decreased the development of spontaneous diabetes in NOD mice. Islet inflammation was decreased 50%. IFN-τ administration to recipient mice prevented the development of passively transferred and cyclophosphamide accelerated diabetes. IFN-τ treatment also decreased anti-islet effector activity of NOD splenic cells. Immunoregulatory activity of splenic cells was augmented by IFN-τ administration as was the number of splenic CD25+CD4+ cells. Concanavalin A (Con A)-induced release of IFN-γ was decreased in spleen cells from IFN-τ treated mice. In conclusion, IFN-τ inhibits spontaneous autoimmune diabetes and passively transferred diabetes in the NOD mouse. This diabetes sparing activity may be due to an induction of regulatory cells, possibly CD25+CD4+ T cells, which in turn inhibit anti-islet effector cell activity and the development of insulitis and diabetes. Due to the lower drug toxicity, IFN-τ could be a better drug candidate than IFN-α for experimental clinical trials.

Staphylococcal enterotoxin B causes differential expression of Rnd3 and RhoA in renal proximal tubule epithelial cells while inducing actin stress fiber assembly and apoptosis

Staphylococcal enterotoxin B (SEB) is a toxic shock-inducing agent produced by Staphylococcus aureus. The hallmark of SEB-induced lethal shock is acute vasodilation leading to severe hypotension. Animal studies reveal that approximately 70% of intravenously administered toxin localizes to renal proximal tubule epithelial cells (RPTEC). This evidence, together with the well-documented role of the kidney in regulation of vascular tone, suggests that molecular events induced in RPTEC by SEB may contribute to the blood pressure dysregulation seen in enterotoxic shock. In an attempt to elucidate these molecular mechanisms, differential display was performed on SEB-treated and untreated RPTEC, and 32 differentially expressed transcripts (DETs) were identified. One of the down-regulated DETs matched the sequence for Rnd3, which normally inhibits Rho protein function. Consistent with Rnd3 down-regulation, message for RhoA was shown to increase upon SEB exposure, and actin stress fiber formation was dramatically increased. Further, SEB-exposed cells showed both increased enzymatic activity of caspase-3 and an increase in the percentage of apoptotic cells. Taken together, these results support the hypothesis that RPTEC undergo apoptosis upon exposure to SEB. Furthermore, these data implicate the involvement of the Rho family proteins in the molecular signaling pathway induced by SEB in RPTEC.

Characterization of N-Terminal Interferon τ Mutants: P26L Affords Enhanced Activity and Lack of Toxicity

Interferon (IFN)-τ is a type I IFN that is responsible for the maternal recognition of pregnancy in ruminants. This protein also has classic IFN-like properties, including antiviral, antiproliferative, and immunomodulatory functions. Using IFN-τ as a model, we examined the structural basis for the activity of type I IFNs, focusing on amino acids within helix A and the first section of the AB loop, which have been proposed as a site for receptor interaction. Six amino-acid substitutions were made that replaced a residue in ovine IFN-τ1 mod with the corresponding residue in human IFN-αA. Receptor binding was enhanced by a P26L mutation and was reduced by a conservative lysine-to-histidine substitution at residue 34. Alterations in the antiviral and antiproliferative activities of the IFN- τ mutants were not always correlated, but both functions were maintained or enhanced relative to the wild-type IFN-τ by the proline-to-leucine mutation at residue 26. In contrast, this mutation did not affect the low in vitro cytotoxicity that is characteristic of ovine IFN-τ1 mod. Thus, the IFN- τ P26L mutant may have potential as an improved IFN-based therapeutic.

[1] Measurement of interferons

Publisher Summary There are three major groups of interferons called alpha (α), beta (β), and gamma (γ) based on protein structure and antigenic properties. The interferons (IFN) are a family of proteins that were initially identified by their ability to make cells resistant to infection by virus. Subsequently, other important IFN functions have been identified. For example, the interferons regulate cell growth and differentiation and are essential for proper functioning of the immune system. One type of interferon is necessary for establishment of pregnancy in a number of animal species, primarily ruminants. The IFN proteins are primarily α-helical in structure with molecular weights which vary from 16,000 to 24,000.

Increased T cell cytotoxicity by Betathine™-induced upregulation of TNFα

Betathine (BT) is a low molecular weight disulfide that has previously been shown to exhibit in vivo antitumor activity in murine myeloma and melanoma models. We have shown that BT treatment of both human T cells and monocytes is associated with an increase in surface tumor necrosis alpha (TNFalpha) expression. Further, in T cells and monocytes that have been stimulated with PMA and ionomycin, the addition of BT results in a dose and time dependent increase in the percentage of high TNFalpha-expressing cells. Unlike TNFalpha upregulation produced by the commonly used thiol antioxidant N-acetyl-L-cysteine (NAC), the BT-induced increase in TNFalpha is observed consistently in different donors. This increase in surface TNFalpha is associated with elevated levels of TNFalpha mRNA. In addition, expression of TNFalpha receptor I is also significantly enhanced by BT treatment. The upregulation of surface TNFalpha by BT has functional consequences, in that, BT-treated T cells exhibit enhanced cytotoxic activity. Thus, increased TNFalpha expression may be one mechanism responsible for the antineoplastic activity of BT.

The Role of Type I Interferon Subtypes and Interferon-Gamma in Type I Interferon Diabetes Inhibitory Activity in the NOD Mouse

As in bacterial infections and endotoxin shock, type I interferons (IFNs) also have complex and often opposing effects in various models of autoimmune disease. We have shown that type I IFN paradoxically inhibits autoimmune diabetes in the nonobese diabetic mouse (NOD) and biobreeding (BB) rat. We hypothesize that type I IFN activity differs by IFN subtype and interaction with IFN-gamma. We examined the structure-function relationship of the type I IFN molecule and the mechanism of its diabetes-sparing activity in the NOD mouse. While both recombinant human IFN-alpha A/D (bgl 11) (rHuIFN-alphaA/D) and ovine IFN-tauImod (ovIFN-tau) potently inhibited the development of diabetes (P < 0.01), neither recombinant human IFN-alpha B/D (rHuIFN-alphaB/D) nor recombinant human IFN-alpha consensus (CIFN) were efficacious. The activity of IFN subtypes correlate with their NH3-terminal amino acid sequences. All type I IFN save CIFN, which has no diabetes-sparing activity, inhibited the accessory cell function. IFN-tau administration decreased the expression of Fas and ICAM on total cells, class II MHC expression on B cells, and CD40L expression on T cells by 39%, 45%, 45%, and 60%, respectively. In addition, IFN-tau inhibited the development of diabetes in the NOD.IL4(null) but not the NOD.IFN-gamma(null) mice, suggesting a coordinated interaction between type I and type II IFNs to suppress diabetes development. Thus, the amino terminal portion of the type I IFN molecule influences its ability to inhibit the development of autoimmune diabetes in NOD mice. These data also support the contention that IFN-gamma may have a role in mediating the diabetes-sparing effect of high-dose type I IFNs by the inhibition of the IFN-gamma-inducible immune modulators, class II MHC, Fas, ICAM, and CD40L.