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BackgroundStaphylococcal enterotoxins (SEs), SE-like (SEl) toxins, and toxic shock syndrome toxin-1 (TSST-1), produced by Staphylococcus aureus, belong to the subgroup of microbial superantigens (SAgs). SAgs induce clonal proliferation of T cells bearing specific variable regions of the T cell receptor β chain (Vβ). Quantitative real time PCR (qRT-PCR) has become widely accepted for rapid and reproducible mRNA quantification. Although the quantification of Vβ subgroups using qRT-PCR has been reported, qRT-PCR using both primers annealing to selected Vβ nucleotide sequences and SYBR Green I reporter has not been applied to assess Vβ-dependent expansion of T cells by SAgs.MethodsHuman peripheral blood mononuclear cells were stimulated with various SAgs or a monoclonal antibody specific to human CD3. Highly specific expansion of Vβ subgroups was assessed by qRT-PCR using SYBR Green I reporter and primers corresponding to selected Vβ nucleotide sequences.ResultsqRT-PCR specificities were confirmed by sequencing amplified PCR products and melting curve analysis. To assess qRT-PCR efficiencies, standard curves were generated for each primer set. The average slope and R2 of standard curves were -3.3764 ± 0.0245 and 0.99856 ± 0.000478, respectively, demonstrating that the qRT-PCR established in this study is highly efficient. With some exceptions, SAg Vβ specificities observed in this study were similar to those reported in previous studies.ConclusionsThe qRT-PCR method established in this study produced an accurate and reproducible assessment of Vβ-dependent expansion of human T cells by staphylococcal SAgs. This method could be a useful tool in the characterization T cell proliferation by newly discovered SAg and in the investigation of biological effects of SAgs linked to pathogenesis.

Resistance of hypoxic solid tumor niches to chemotherapy and radiotherapy remains a major scientific challenge that calls for conceptually new approaches. Here we exploit a hitherto unrecognized ability of sickled erythrocytes (SSRBCs) but not normal RBCs (NLRBCs) to selectively target hypoxic tumor vascular microenviroment and induce diffuse vaso-occlusion. Within minutes after injection SSRBCs, but not NLRBCs, home and adhere to hypoxic 4T1 tumor vasculature with hemoglobin saturation levels at or below 10% that are distributed over 70% of the tumor space. The bound SSRBCs thereupon form microaggregates that obstruct/occlude up to 88% of tumor microvessels. Importantly, SSRBCs, but not normal RBCs, combined with exogenous prooxidant zinc protoporphyrin (ZnPP) induce a potent tumoricidal response via a mutual potentiating mechanism. In a clonogenic tumor cell survival assay, SSRBC surrogate hemin, along with H2O2 and ZnPP demonstrate a similar mutual potentiation and tumoricidal effect. In contrast to existing treatments directed only to the hypoxic tumor cell, the present approach targets the hypoxic tumor vascular environment and induces injury to both tumor microvessels and tumor cells using intrinsic SSRBC-derived oxidants and locally generated ROS. Thus, the SSRBC appears to be a potent new tool for treatment of hypoxic solid tumors, which are notable for their resistance to existing cancer treatments.

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Plasma of a patient with metastatic colon carcinoma was perfused over Formalin and heat-killed S. aureus, in an extracorporeal filtration apparatus, in order to nonspecifically remove IgG and its complexes. Twenty ex vivo absorption procedures were done, over a five-month period, with a minimum of discomfort to the patient. Extracorporeal perfusion of plasma on S. aureus effectively reduced the levels of IgG and immune complexes in the perfused plasma. The nonspecific removal of IgG resulted in 1) slight biochemical alterations in the serum, 2) a transient reduction in the serum blocking activity and appearance of complement-dependent serum cytotoxicity, 3) an increase in the serum IgM levels, 4) a transient increase in the Ig surface-bearing lymphocytes and a decrease in "E" rosetting lymphocytes in the first 24-48 hours postperfusion, particularly during the early treatments, 5) an improvement in general condition of the patient and decrease in tumor size, and 6) histological changes in the tumor consistent with tumor destruction. The potential problems and clinical applications of procedures involving ex vivo specific or nonspecific immunoabsorbents are discussed.

Selective drug delivery to hypoxic tumor niches remains a significant therapeutic challenge that calls for new conceptual approaches. Sickle red blood cells (SSRBCs) have shown an ability to target such hypoxic niches and induce tumoricidal properties when used together with exogenous pro-oxidants. Here we determine whether the delivery of a model therapeutic encapsulated in murine SSRBCs can be enhanced by ex vivo photosensitization under conditions that delay autohemolysis to a time that coincides with maximal localization of SSRBCs in a hypoxic tumor. Hyperspectral imaging of 4T1 carcinomas shows oxygen saturation levels <10% in a large fraction (commonly 50% or more) of the tumor. Using video microscopy of dorsal skin window chambers implanted with 4T1 tumors, we demonstrate that allogeneic SSRBCs, but not normal RBCs (nRBCs), selectively accumulate in hypoxic 4T1 tumors between 12-24 hours after systemic administration. We further show that ex vivo photo-oxidation can program SSRBCs to postpone hemolysis/release of a model therapeutic to a point that coincides with their maximum sequestration in hypoxic tumor microvessels. Under these conditions, drug-loaded photosensitized SSRBCs show a 3-4 fold greater drug delivery to tumors compared to non-photosensitized SSRBCs, drug-loaded photosensitized nRBCs, and free drug. These results demonstrate that photo-oxidized SSRBCs, but not photo-oxidized nRBCs, sequester and hemolyze in hypoxic tumors and release substantially more drug than photo-oxidized nRBCs and non-photo-oxidized SSRBCs. Photo-oxidation of drug-loaded SSRBCs thus appears to exploit the unique tumor targeting and carrier properties of SSRBCs to optimize drug delivery to hypoxic tumors. Such programmed and drug-loaded SSRBCs therefore represent a novel and useful tool for augmenting drug delivery to hypoxic solid tumors.

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