Microneedle patch-based enzyme-linked immunosorbent assay to quantify protein biomarkers of tuberculosis.

There is a clinical need for differential diagnosis of the latent versus active stages of tuberculosis (TB) disease by a simple-to-administer test. Alpha-crystallin (Acr) and early secretory antigenic target-6 (ESAT-6) are protein biomarkers associated with the latent and active stages of TB, respectively, and could be used for differential diagnosis. We therefore developed a microneedle patch (MNP) designed for application to the skin to quantify Acr and ESAT-6 in dermal interstitial fluid by enzyme-linked immunosorbent assay (ELISA). We fabricated mechanically strong microneedles made of polystyrene and coated them with capture antibodies against Acr and ESAT-6. We then optimized assay sensitivity to achieve a limit of detection of 750 pg/ml and 3,020 pg/ml for Acr and ESAT-6, respectively. This study demonstrates the feasibility of an MNP-based ELISA for differential diagnosis of latent TB disease.

Immunogenicity and protective efficacy of a pan-fungal vaccine in preclinical models of aspergillosis, candidiasis, and pneumocystosis.

Invasive fungal infections cause over 1.5 million deaths worldwide. Despite increases in fungal infections as well as the numbers of individuals at risk, there are no clinically approved fungal vaccines. We produced a "pan-fungal" peptide, NXT-2, based on a previously identified vaccine candidate and homologous sequences from Pneumocystis, Aspergillus,Candida, and Cryptococcus. We evaluated the immunogenicity and protective capacity of NXT-2 in murine and nonhuman primate models of invasive aspergillosis, systemic candidiasis, and pneumocystosis. NXT-2 was highly immunogenic and immunized animals had decreased mortality and morbidity compared to nonvaccinated animals following induction of immunosuppression and challenge with Aspergillus, Candida, or Pneumocystis. Data in multiple animal models support the concept that immunization with a pan-fungal vaccine prior to immunosuppression induces broad, cross-protective antifungal immunity in at-risk individuals.

Contextualizing social class and leadership in fraternity and sorority communities.

This chapter examines leadership and social class in the context of fraternities and sororities. With no extensive research in this area, recommendations provided may help educators create a plan to address the intersection of social class, leadership education, and membership in a fraternity or sorority.

Immunomodulatory in vitro effects of oclacitinib on canine T-cell proliferation and cytokine production.

BACKGROUND: Oclacitinib is a Janus kinase inhibitor used to control pruritus and skin lesions in canine allergic skin disease; its effect on canine T cells is not well-characterized. HYPOTHESIS/OBJECTIVES: To evaluate the impact of oclacitinib on cultured T cells using peripheral blood mononuclear cells from dogs. ANIMALS: Six bluetick coonhounds. METHODS AND MATERIALS: Lymphocyte-enriched cells were incubated with or without the T-cell mitogen concanavalin A (Con A), oclacitinib (0.5, 1 or 10 µM), ciclosporin (200 ng/mL), Con A + oclacitinib 1 µM and Con A + ciclosporin. We assessed both T-cell proliferation and the secretion of cytokines. RESULTS: Ciclosporin and oclacitinib both inhibited the spontaneous proliferation of T cells; this effect was significant only after incubation with oclacitinib at 10 µM. At this concentration, oclacitinib significantly reduced the spontaneous secretion of clonal activator cytokines [interleukin (IL)-2, IL-15], pro-inflammatory cytokines (interferon-gamma (IFN-γ), IL-18) and the regulatory cytokine IL-10; tumour necrosis factor alpha (TNF-α) and IL-6 cytokine production was mildly inhibited. After Con A stimulation, only T cells co-treated with ciclosporin achieved a significant proliferation inhibition and reduction of IL-2, IL-10, IL-15, IL-18, IFN-γ and TNF-α. Surprisingly, oclacitinib at 1 µM (337 ng/mL, corresponding to the oral dosage of 0.4-0.6 mg/kg) did not significantly affect Con A-stimulated T-cell proliferation nor cytokine production (IL-2, IL-10, IL-15, IL-18, IFN-γ and TNF-α). CONCLUSIONS: Although a limited number of dogs were investigated, these preliminary results suggest that oclacitinib appears to have immunosuppressive properties, but only at dosages above those used to treat allergic pruritus in dogs.

Avian influenza viruses infect primary human bronchial epithelial cells unconstrained by sialic acid α2,3 residues.

Avian influenza viruses (AIV) are an important emerging threat to public health. It is thought that sialic acid (sia) receptors are barriers in cross-species transmission where the binding preferences of AIV and human influenza viruses are sias α2,3 versus α2,6, respectively. In this study, we show that a normal fully differentiated, primary human bronchial epithelial cell model is readily infected by low pathogenic H5N1, H5N2 and H5N3 AIV, which primarily bind to sia α2,3 moieties, and replicate in these cells independent of specific sias on the cell surface. NHBE cells treated with neuraminidase prior to infection are infected by AIV despite removal of sia α2,3 moieties. Following AIV infection, higher levels of IP-10 and RANTES are secreted compared to human influenza virus infection, indicating differential chemokine expression patterns, a feature that may contribute to differences in disease pathogenesis between avian and human influenza virus infections in humans.

Identification and characterization of a spontaneous ovarian carcinoma in Lewis rats.

BACKGROUND: Ovarian carcinoma is the fourth most common cause of death from cancer in women. Limited progress has been made toward improving the survival rate of patients with this disease in part because of the lack of a good animal model. We present here a model of spontaneous ovarian carcinoma arising in a normal Lewis rat. METHODS: A spontaneously occurring tumor of the left ovary was found in a normal Lewis rat during necropsy, which was sectioned for histological examination and placed into single cell suspension. Tumor cells were passaged in vivo by intraperitoneal injection into immunocompetent Lewis rats, and in vitro culture resulted in generation of a cell line. Tumor cells were examined by flow cytometry for expression of estrogen receptor alpha, progesterone receptor, androgen receptor, her-2/neu, epithelial cell adhesion molecule, and CA125. beta-catenin expression and cellular localization was assessed by immunocytochemistry. RNA was harvested for gene expression profiling and studying the expression of cytokines. RESULTS: The tumor, designated FNAR, could be serially transplanted into Lewis rats and propagated as a cell line in vitro, maintaining the properties of the original tumor. The FNAR cells displayed striking morphologic similarities to human ovarian carcinoma, resembling the endometrioid carcinoma subtype of surface epithelial neoplasms. The cells expressed estrogen receptor alpha, progesterone receptor, androgen receptor, her-2/neu, epithelial cell adhesion molecule, CA125, and nuclear beta-catenin. A gene expression profile showed upregulation of a number of genes that are also upregulated in human ovarian carcinoma. CONCLUSION: This reliable model of ovarian carcinoma should be helpful in better understanding the biology of the disease as well as the development of novel treatment strategies.

Respiratory syncytial virus F and G proteins induce interleukin 1alpha, CC, and CXC chemokine responses by normal human bronchoepithelial cells.

Human respiratory syncytial virus (RSV) is a ubiquitous respiratory virus that causes serious lower respiratory tract disease in infants and young children worldwide. Studies have shown that RSV infection modulates chemokine expression patterns, suggesting that particular cytokine expression profiles may be indicators of disease severity. In this study, we show that RSV F or G protein treatment of fully differentiated primary normal human bronchial epithelial cells induces apical and basolateral secretion of interleukin 8 (IL-8), interferon-inducible protein 10 (IP-10), monocyte chemotactic protein 1 (MCP-1), and RANTES (regulated on activation, normal T cell expressed and secreted). Purified RSV G (attachment) protein was shown to stimulate the secretion of interleukin 1alpha and RANTES, whereas purified F (fusion) protein elicited the production of IL-8, IP-10, and RANTES. Studies of ultraviolet-inactivated RSV showed that treatment of normal human bronchial epithelial cells induces apical IL-8, IP-10, and MCP-1 secretion independent of infection, suggesting that RSV proteins alone modify the chemokine response pattern, which may affect the early immune response before infection.

Circulating clonotypic B cells in classic Hodgkin lymphoma.

Although Hodgkin and Reed-Sternberg (HRS) cells are B lymphoid cells, they are unlike any normal cells of that lineage. Moreover, the limited proliferative potential of HRS cells belies the clinical aggressiveness of Hodgkin lymphoma (HL). More than 20 years ago, the L428 HL cell line was reported to contain a small population of phenotypic B cells that appeared responsible for the continued generation of HRS cells. This observation, however, has never been corroborated, and such clonotypic B cells have never been documented in HL patients. We found that both the L428 and KM-H2 HL cell lines contained rare B-cell subpopulations responsible for the generation and maintenance of the predominant HRS cell population. The B cells within the HL cell lines expressed immunoglobulin light chain, the memory B-cell antigen CD27, and the stem cell marker aldehyde dehydrogenase (ALDH). Clonal CD27(+)ALDH(high) B cells, sharing immunoglobulin gene rearrangements with lymph node HRS cells, were also detected in the blood of most newly diagnosed HL patients regardless of stage. Although the clinical significance of circulating clonotypic B cells in HL remains unclear, these data suggest they may be the initiating cells for HL.

Glycosylphosphatidylinositol-anchored protein deficiency confers resistance to apoptosis in PNH.

OBJECTIVE: Investigate the contribution of PIG-A mutations to clonal expansion in paroxysmal nocturnal hemoglobinuria (PNH). MATERIALS AND METHODS: Primary CD34+ hematopoietic progenitors from PNH patients were assayed for annexin-V positivity by flow cytometry in a cell-mediated killing assay using autologous effectors from PNH patients or allogeneic effectors from healthy controls. To specifically assess the role of the PIG-A mutation in the development of clonal dominance and address confounders of secondary mutation and differential immune attack that can confound experiments using primary cells, we established an inducible PIG-A CD34+ myeloid cell line, TF-1. Apoptosis resistance was assessed after exposure to allogeneic effectors, NK92 cells (an interleukin-2-dependent cell line with the phenotype and function of activated natural killer [NK] cells), tumor necrosis factor (TNF)-alpha, and gamma-irradiation. Apoptosis was measured by annexin-V staining and caspase 3/7 activity. RESULTS: In PNH patients, CD34+ hematopoietic progenitors lacking glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-AP(-)) were less susceptible than GPI-AP+ CD34+ precursors to autologous (8% vs 49%; p < 0.05) and allogeneic (28% vs 58%; p < 0.05) cell-mediated killing from the same patients. In the inducible PIG-A model, GPI-AP(-) TF-1 cells exhibited less apoptosis than induced, GPI-AP+ TF-1 cells in response to allogeneic cell-mediated killing, NK92-mediated killing, TNF-alpha, and gamma-irradiation. GPI-AP(-) TF-1 cells maintained resistance to apoptosis when effectors were raised against GPI-AP(-) cells, arguing against a GPI-AP being the target of immune attack in PNH. NK92-mediated killing was partially inhibited with blockade by specific antibodies to the stress-inducible GPI-AP ULBP1 and ULBP2 that activate immune effectors. Clonal competition experiments demonstrate that the mutant clone expands over time under proapoptotic conditions with TNF-alpha. CONCLUSION: PIG-A mutations contribute to clonal expansion in PNH by conferring a survival advantage to hematopoietic progenitors under proapoptotic stresses.

Clonogenic multiple myeloma progenitors, stem cell properties, and drug resistance.

Many agents are active in multiple myeloma, but the majority of patients relapse. This clinical pattern suggests most cancer cells are eliminated, but cells with the clonogenic potential to mediate tumor regrowth are relatively chemoresistant. Our previous data suggested that CD138(+) multiple myeloma plasma cells cannot undergo long-term proliferation but rather arise from clonogenic CD138(neg) B cells. We compared the relative sensitivity of these distinct cell types to clinical antimyeloma agents and found that dexamethasone, lenadilomide, bortezomib, and 4-hydroxycyclophosphamide inhibited CD138(+) multiple myeloma plasma cells but had little effect on CD138(neg) precursors in vitro. We further characterized clonogenic multiple myeloma cells and stained cell lines using the Hoechst side population and Aldefluor assays. Each assay identified CD138(neg) cells suggesting that they possess high drug efflux capacity and intracellular drug detoxification activity. We also found that multiple myeloma cells expressing the memory B-cell markers CD20 and CD27 could give rise to clonogenic multiple myeloma growth in vitro and engraft immunodeficient nonobese diabetes/severe combined immunodeficient mice during both primary and secondary transplantation. Furthermore, both the side population and Aldefluor assays were capable of identifying circulating clonotypic memory B-cell populations within the peripheral blood of multiple myeloma patients. Our results suggest that circulating clonotypic B-cell populations represent multiple myeloma stem cells, and the relative drug resistance of these cells is mediated by processes that protect normal stem cells from toxic injury.

The role of growth factors in the activity of pharmacological differentiation agents.

Bryostatin-1 inhibits acute myeloid leukemia (AML) in vitroat doses that stimulate the growth of normal hematopoietic progenitors.Although bryostatin-1 has a number of distinct biological activities, those specifically responsible for its antileukemic activity are unclear. We found that bryostatin-1 (10(-8) M) inhibited cell cycling at G(1), induced phenotypic evidence of differentiation, and limited the clonogenic growth of both AML cell lines and patient specimens. This activity was markedly enhanced by granulocyte/macrophage-colony stimulating factor, whereas growth factor-neutralizing antibodies completely inhibited both the differentiating and antileukemic activities of bryostatin-1. Cell cycle inhibition and growth factors were also required for the differentiating activities of two unrelated agents, hydroxyurea and phenylbutyrate. These data suggest that many pharmacological differentiating agents require both cell cycle arrest and lineage-specific growth factors for full activity and may explain why these agents have demonstrated only limited clinical efficacy.