A Novel Sandwich ELISA for Tartrate-Resistant Acid Phosphatase 5a and 5b Protein Reveals that Both Isoforms are Secreted by Differentiating Osteoclasts and Correlate to the Type I Collagen Degradation Marker CTX-I In Vivo and In Vitro.

Tartrate-resistant acid phosphatase type 5 (TRAP) exists as two isoforms, 5a and 5b. 5b is a marker of osteoclast number and 5a of chronic inflammation; however, its association with bone resorption is unknown. In this study, a double-TRAP 5a/5b sandwich ELISA measuring 5a and 5b protein in the same sample was developed. TRAP 5a and 5b protein levels were evaluated as osteoclast differentiation/activity markers in serum and in culture, and their correlation to the resorption marker CTX-I was examined. Serum TRAP 5a and 5b concentrations in healthy men were 4.4 ± 0.6 ng/ml and 1.3 ± 0.2 ng/ml, respectively, and they correlated moderately to each other suggesting that their secretion is coupled under healthy conditions. A correlation was also observed between serum TRAP 5a and 5b with CTX-I, suggesting that both TRAP isoforms associate with osteoclast number. During osteoclast differentiation on plastic/bone, predominantly 5b increased in media/lysate from M-CSF/RANKL-stimulated CD14+ PBMCs. However, substantial levels of 5a were detected at later stages suggesting that both isoforms are secreted from differentiating OCs. More TRAP 5b was released on bone indicating a connection to osteoclast resorptive activity, and a peak in TRAP 5b/5a-ratio coincided with rapid CTX-I release. At the end of the culture period of M-CSF + RANKL-stimulated CD14+ PBMCs, there was a correlation between the secretion of TRAP 5a and 5b proteins with CTX-I. The correlation of not only 5b but also 5a with collagen degradation, both in serum and osteoclast cultures indicates that a considerable proportion of the TRAP 5a originates from osteoclasts and may reflect a hitherto undisclosed regulatory mechanism during bone resorption and bone remodeling.

Intensive weight gain therapy in patients with anorexia nervosa results in improved serum tartrate-resistant acid phosphatase (TRAP) 5a and 5b isoform protein levels.

AIM: Tartrate-resistant acid phosphatase (TRAP) exists as isoforms 5a and 5b. TRAP 5a is a biomarker of chronic inflammation and influences adipose tissue and 5b associates with bone metabolism/pathologies. The aim was to investigate the association of serum TRAP 5a/5b isoforms with fat and bone markers and anthropometric parameters in patients with anorexia nervosa (AN) during weight gain therapy. METHODS: Twenty-five Swedish female AN patients, age 16-24 years, were treated for 12 weeks with a high-energy diet with six meals daily. Serum TRAP 5a/5b, markers of fat/glucose metabolism, markers of bone resorption and formation were measured. Parameters of bone and body composition were assessed by dual-energy X-ray absorptiometry and peripheral quantitative computed tomography. RESULTS: BMI increased from median 15.4 kg/m2 to 19.0 kg/m2, p < 0.0001. TRAP 5a and 5a/5b ratio increased but TRAP 5b decreased during the study. TRAP Δ5a and Δ5b correlated with Δinsulin and Δadiponectin, respectively. TRAP 5b correlated with trabecular density at start but not at week 12. At 12 weeks, TRAP 5b correlated with CTX, and Δ decrease in TRAP 5b correlated to Δ increase in bone-specific alkaline phosphatase. CONCLUSIONS: This clinical interventional study resulted in increased BMI in patients with AN. The decreased TRAP 5b protein levels confirm a role for TRAP 5b as a marker of bone resorption, whereas increased TRAP 5a seemed to derive from systemic changes in bone as well as metabolic changes. The combined detection of TRAP 5a and TRAP 5b in serum could be an indicator of improved bone metabolism. LEVEL OF EVIDENCE: Level III, prospective interventional cohort study.

The adipokine tartrate-resistant acid phosphatase 5a in serum correlates to adipose tissue expansion in obesity.

PURPOSE: Tartrate-resistant acid phosphatase (TRAP) exists as two isoforms, 5a and 5b. TRAP 5a is elevated in adipose tissue of obese women, interacts with pre-adipocytes and is linked to insulin-sensitive hyperplastic obesity when overexpressed in mice. The aim of this study was to investigate the relation between serum TRAP 5a, adiposity indices and metabolic syndrome risk markers in lean and obese women, using a newly developed TRAP 5a-specific ELISA. MATERIALS AND METHODS: A TRAP 5a sandwich ELISA was optimized using TRAP 5a-specific monoclonal antibodies and tested in sera of healthy males. TRAP 5a levels were quantitated in sera from healthy males and lean and obese women. RESULTS: Serum TRAP 5a protein levels were lower in obese women in comparison with lean. In obese, but not in lean women, serum TRAP 5a correlated positively to % fat mass, BMI, waist- and hip circumference, waist-to-hip ratio and PAI, while no correlations to serum leptin, HOMA, glucose, insulin, FFA, HDL, TG, APO-A1 and APO-B were observed. CONCLUSIONS: TRAP 5a serum levels correlated positively to anthropometric obesity parameters but not to metabolic syndrome risk factors, indicating that serum TRAP 5a is associated with pathological adipose tissue expansion.

Caveolae-mediated endocytosis of the glucosaminoglycan-interacting adipokine tartrate resistant acid phosphatase 5a in adipocyte progenitor lineage cells.

Adipogenesis depends on growth factors controlling proliferation/differentiation of mesenchymal stem cells (MSCs). Membrane binding and endocytosis of growth factors are often coupled to receptor activation and downstream signaling leading to specific cellular responses. The novel adipokine tartrate-resistant acid phosphatase (TRAP) 5a exhibits a growth factor-like effect on MSCs and pre-adipocytes and induces hyperplastic obesity in vivo. However its molecular interaction with pre-adipocytes remains unknown. Therefore, this study aimed to investigate membrane interaction of TRAP and its endocytosis routes in pre-adipocytes. Confocal and/or electron microscopy were used to detect TRAP in untreated or TRAP 5a/b treated pre-adipocytes under conditions that allow or inhibit endocytosis in combination with co-staining of endocytotic vesicles. TRAP interaction with heparin/heparan sulfate was verified by gel filtration. It could be shown that TRAP 5a, but not 5b, binds to the membrane of pre-adipocytes where it co-localizes with heparin-sulfate proteoglycan glypican-4. Also in vitro, TRAP 5a exhibited affinity for both heparin and heparan sulfate with heparin inhibiting its enzyme activity. Upon caveolae-mediated endocytosis of saturating levels of TRAP 5a, TRAP 5a co-localized intracellularly with glypican-4 and late endosomal marker Rab-7 positive vesicles. The protein was also located in multivesicular bodies (MVBs) but did not co-localize with lysosomal marker LAMP-1. TRAP 5a endocytosis was also detectable in pre-osteoblasts, but not fibroblasts, embryonic MSCs or mature adipocytes. These results indicate that TRAP 5a exhibits binding to cell surface, endocytosis and affinity to glucosaminoglycans (GAGs) in pre-adipocyte and pre-osteoblast lineage cells in a manner similar to other heparin-binding growth factors.

Th2-skewed T cells correlate with B cell response to α-Gal and tick antigens in α-Gal syndrome.

Tick bites have been shown to transmit a novel form of severe food allergy, the galactose-α-1,3-galactose (α-Gal) syndrome (AGS). Cellular responses to α-Gal in patients with AGS have, to date, not been thoroughly scrutinized. Therefore, we investigated T and B cell proliferation, activation, and cytokine profiles in response to tick protein extract (TE) and α-Gal-free TE in patients with AGS and in healthy controls. T and B cells from both patients and controls proliferated in response to TE, but significantly more in patients with AGS. B cell proliferation, but not T cell proliferation, in patients with AGS was reduced by removing α-Gal from the TE. In addition, TE induced a clear Th2 cytokine profile in patients with AGS. Expression of CD23 by B cells correlated only to T cell proliferation. However, both B cell proliferation and CD23 expression were reduced when CD40L and IL-4 were blocked. A large portion of the IgG1 and IgE antibodies binding TE in patients with AGS were directed against the α-Gal epitope. We have, for what we believe to be the first time, investigated T and B cell responses to α-Gal carrying tick proteins in patients with AGS, which will be essential for the understanding of the immune response against an allergenic carbohydrate transmitted by ticks.

Adjuvant properties of mesoporous silica particles tune the development of effector T cells.

Alum is the most frequently used adjuvant today, primarily inducing Th2 responses. However, Th1-type responses are often desirable within immune therapy, and therefore the development of new adjuvants is greatly needed. Mesoporous silica particles with a highly ordered pore structure have properties that make them very interesting for future controlled drug delivery systems, such as controllable particle and pore size; they also have the ability to induce minor immune modulatory effects, as previously demonstrated on human-monocyte-derived dendritic cells (MDDCs). In this study, mesoporous silica particles are shown to be efficiently engulfed by MDDCs within 2 h, probably by phagocytic uptake, as seen by confocal microscopy and transmission electron microscopy. A co-culture protocol is developed to evaluate the capability of MDDCs to stimulate the development of naïve CD4(+) T cells in different directions. The method, involving ELISpot as a readout system, demonstrates that MDDCs, after exposure to mesoporous silica particles (AMS-6 and SBA-15), are capable of tuning autologous naïve T cells into different effector cells. Depending on the size and functionalization of the particles added to the cells, different cytokine patterns are detected. This suggests that mesoporous silica particles can be used as delivery vehicles with tunable adjuvant properties, which may be of importance for several medical applications, such as immune therapy and vaccination.

Selective expansion of HIV-1 envelope glycoprotein-specific B cell subsets recognizing distinct structural elements following immunization.

The HIV-1 envelope glycoprotein (Env) functional spike has evolved multiple immune evasion strategies, and only a few broadly neutralizing determinants on the assembled spike are accessible to Abs. Serological studies, based upon Ab binding and neutralization activity in vitro, suggest that vaccination with current Env-based immunogens predominantly elicits Abs that bind nonneutralizing or strain-restricted neutralizing epitopes. However, the fractional specificities of the polyclonal mixture of Abs present in serum, especially those directed to conformational Env epitopes, are often difficult to determine. Furthermore, serological analyses do not provide information regarding how repeated Ag inoculation impacts the expansion and maintenance of Env-specific B cell subpopulations. Therefore, we developed a highly sensitive Env-specific B cell ELISPOT system, which allows the enumeration of Ab-secreting cells (ASC) from diverse anatomical compartments directed against different structural determinants of Env. In this study, we use this system to examine the evolution of B cell responses in mice immunized with engineered Env trimers in adjuvant. We demonstrate that the relative proportion of ASC specific for defined structural elements of Env is altered significantly by homologous booster immunizations. This results in the selective expansion of ASC directed against the variable regions of Env. We suggest that the B cell specificity and compartment analysis described in this study are important complements to serological mapping studies for the examination of B cell responses against subspecificities of a variety of immunogens.

Heavy-chain complementarity-determining regions determine conformation selectivity of anti-aß antibodies.

BACKGROUND/AIMS: Amyloid-ß (Aß) protofibrils are neurotoxic soluble intermediates in the Aß aggregation process eventually forming senile plaques in Alzheimer's disease. This Aß species is a potential biomarker for Alzheimer's disease and also a promising target for immunotherapy. In this study, we investigated the characteristics of conformation-dependent Aß antibodies specific for Aß protofibrils. METHODS: Mice were immunized with Aß protofibrils to generate hybridomas producing Aß-specific monoclonal antibodies. Binding of antibodies to different Aß conformations was investigated with inhibition ELISA. The antibodies' complementarity-determining region (CDR) sequences were determined and compared. RESULTS: A majority of the antibodies were of the IgM class, all selectively binding to aggregated Aß. Two IgG antibodies were generated: one with selective affinity for Aß protofibrils and the other bound Aß in all conformations. A high degree of similarity between the heavy-chain CDRs of the conformation-dependent antibodies was found, and all high-affinity Aß antibodies displayed a high degree of sequence similarity in the light-chain CDRs. CONCLUSION: Sequence similarity in the heavy-chain CDRs is associated with conformation selectivity of the antibodies, while sequence similarity in the light-chain CDRs correlates with the affinity for Aß.

Efficient internalization of mesoporous silica particles of different sizes by primary human macrophages without impairment of macrophage clearance of apoptotic or antibody-opsonized target cells.

Macrophage recognition and ingestion of apoptotic cell corpses, a process referred to as programmed cell clearance, is of considerable importance for the maintenance of tissue homeostasis and in the resolution of inflammation. Moreover, macrophages are the first line of defense against microorganisms and other foreign materials including particles. However, there is sparse information on the mode of uptake of engineered nanomaterials by primary macrophages. In this study, mesoporous silica particles with cubic pore geometries and covalently fluorescein-grafted particles were synthesized through a novel route, and their interactions with primary human monocyte-derived macrophages were assessed. Efficient and active internalization of mesoporous silica particles of different sizes was observed by transmission electron microscopic and flow cytometric analysis and studies using pharmacological inhibitors suggested that uptake occurred through a process of endocytosis. Moreover, uptake of silica particles was independent of serum factors. The silica particles with very high surface areas due to their porous structure did not impair cell viability or function of macrophages, including the ingestion of different classes of apoptotic or opsonized target cells. The current findings are relevant to the development of mesoporous materials for drug delivery and other biomedical applications.

A novel adjuvant G3 induces both Th1 and Th2 related immune responses in mice after immunization with a trivalent inactivated split-virion influenza vaccine.

A preferred adjuvant should promote both Th1 and Th2 responses. However, most adjuvants in common use are biased towards a Th2-driven response. Therefore, the ability of a novel saponin-based adjuvant G3 to inducing balanced Th1 and Th2 responses in BALB/c mice immunized with a split trivalent seasonal influenza vaccine was evaluated in comparison to that of the adjuvant Al(OH)3. Clear differences in the IgG profiles induced by G3, Al(OH)3 or non-adjuvanted vaccine were recorded. Both adjuvants enhanced high and similar levels of the Th2 associated IgG1 subtype compared to mice given vaccine alone. Only G3 enhanced the IgG2a subclass reflecting a Th1 response, whereas Al(OH)3 even abrogated the IgG2a production. Accordingly, G3 enhanced the production of IL-2 and IFN-γ and also of IL-2/IFN-γ double secreting cells, emphasizing the strong Th1 driving effect of G3. Only Al(OH)3 increased splenocyte production of IL-17. Taken together, the results indicate a strong propensity for G3 to induce both Th1 and Th2 driven immune responses.

Detection of macaque perforin expression and release by flow cytometry, immunohistochemistry, ELISA, and ELISpot.

Simian immunodeficiency virus (SIV)-infection in macaques provides an important animal model for human immunodeficiency virus-1 (HIV-1) infection. The involvement of perforin (PFN), released by cytotoxic cells to mediate killing of virus-infected cells, has been difficult to assess in this experimental model due to a lack of reagents. We therefore evaluated monoclonal antibodies (mAbs) Pf-80, Pf-164 and Pf-344, previously raised against human PFN, for cross-reactivity with macaque PFN. Mabs Pf-164 and Pf-344 reacted with intracellular PFN in peripheral blood mononuclear cells (PBMC) from cynomolgus and rhesus macaques by flow cytometry and stained PFN in rhesus lymphoid tissue by immunohistochemistry (IHC). Moreover, PFN capture enzyme-linked immunosorbent (ELISA) and enzyme-linked immunospot (ELISpot) assays utilizing mAbs Pf-164/Pf-80 for capture and mAb Pf-344 for detection were used to quantify PFN release by mitogen-stimulated cynomolgus and rhesus PBMC. The PFN ELISpot was further used to quantify antigen-specific CD8+ T cells by ex vivo stimulation of PBMC from cynomolgus macaques immunized against SIV/HIV-1. These macaque PFN-reactive mAbs and immunoassays will be valuable new tools for investigation of cytotoxic T lymphocyte (CTL) responses in non-human primate models of infectious diseases as well as for vaccine development.

Activation of Neutrophils via IP3 Pathway Following Exposure to Demodex-Associated Bacterial Proteins.

Rosacea is a chronic inflammatory condition that predominantly affects the skin of the face. Sera from rosacea patients display elevated reactivity to proteins from a bacterium (Bacillus oleronius) originally isolated from a Demodex mite from a rosacea patient suggesting a possible role for bacteria in the induction and persistence of this condition. This work investigated the ability of B. oleronius proteins to activate neutrophils and demonstrated activation via the IP3 pathway. Activated neutrophils displayed increased levels of IP1 production, F-actin formation, chemotaxis, and production of the pro-inflammatory cytokines IL-1ß and IL-6 following stimulation by pure and crude B. oleronius protein preparations (2 µg/ml), respectively. In addition, neutrophils exposed to pure and crude B. oleronius proteins (2 µg/ml) demonstrated increased release of internally stored calcium (Ca(2+)), a hallmark of the IP3 pathway of neutrophil activation. Neutrophils play a significant role in the inflammation associated with rosacea, and this work demonstrates how B. oleronius proteins can induce neutrophil recruitment and activation.

Detection of human perforin by ELISpot and ELISA: ex vivo identification of virus-specific cells.

The perforin (PFN) protein is essential for the elimination of target cells by cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. The study of cells releasing PFN has been hampered by a lack of sensitive methods. We therefore produced PFN-reactive monoclonal antibodies (mAb) and developed capture enzyme-linked immunosorbent (ELISA) and enzyme-linked immunospot (ELISpot) assays. Three mAbs were generated and shown to react with unique determinants of PFN. All mAbs recognized intracellular PFN in human peripheral blood mononuclear cell (PBMC) as assessed by flow cytometry and immunohistochemistry. Functional PFN capture ELISA and ELISpot assays were developed utilizing two of the mAbs for capture and the third mAb for detection. When examining PFN release by the YT lymphoma cell line, the ELISpot displayed a greater detection sensitivity than the ELISA. Assessment of PFN release by a CTL clone using ELISpot gave results consistent with a parallel (51)Cr-release cytotoxicity assay. Moreover, PFN release by PBMC could be quantified by ELISpot and ELISA after ex vivo stimulation with defined CTL epitopes from common viruses. These novel immunoassays will be valuable for further investigations of the mechanisms underlying granule-mediated apoptosis. In addition, the capture immunoassays could provide tools for studying CTL responses in infectious and tumor diseases as well as for vaccine development.

ELISpot and ELISA analyses of human IL-21-secreting cells: Impact of blocking IL-21 interaction with cellular receptors.

Interleukin (IL)-21 is crucial for the regulation of lymphocytes and is implicated in autoimmune and other diseases. The relevance of being able to measure human IL-21 prompted us to develop ELISA and ELISpot assays for analysis of IL-21 levels and IL-21-producing cells, respectively. Monoclonal antibodies (mAbs) to IL-21 were made and ELISA and ELISpot assays were developed. The selected detection mAb also neutralized IL-21-mediated activation of human cells. Peripheral blood mononuclear cells (PBMCs) from healthy donors (n=24) were stimulated polyclonally (phytohemagglutinin; PHA) or with antigen (Candida albicans extract and tetanus toxoid). Using ELISpot, high numbers of IL-21-producing cells were detected after PHA activation; lower but positive responses to antigen were seen in approximately 50% of the donors. In contrast, the ELISA detected IL-21 in supernatants from PHA-activated cells but not from antigen-stimulated cells. When analyzing IL-17A in parallel, PHA and antigens induced detectable responses in ELISpot as well as in ELISA. Hypothesizing that the lack of detectable IL-21 levels after antigenic stimulation was due to a combination of low frequencies of IL-21-secreting cells and consumption of IL-21 by cellular receptors during cell culture, PBMCs (n=18) were stimulated in the presence of the neutralizing detection mAb. When preventing IL-21 from interacting with its receptor, increased IL-21 levels were found by ELISA after PHA activation and IL-21 could also be measured after antigen stimulation. ELISpot results were unaffected by the addition of the neutralizing mAb. In conclusion, IL-21 secreted by low frequencies of antigen-specific ex vivo-stimulated PBMC can be difficult to detect by ELISA but prevention of IL-21 interaction with its receptor leads to detectable IL-21 levels. In ELISpot, where the cytokine is captured by mAbs on a solid phase immediately upon secretion, blocking the receptor interaction does not affect the detection of IL-21-secreting cells.

Increased Number of Plasma B Cells Producing Autoantibodies Against Aß42 Protofibrils in Alzheimer's Disease.

The Alzheimer's disease (AD)-related peptide amyloid-ß (Aß) has a propensity to aggregate into various assemblies including toxic soluble Aß protofibrils. Several studies have reported the existence of anti-Aß antibodies in humans. However, it is still debated whether levels of anti-Aß antibodies are altered in AD patients compared to healthy individuals. Formation of immune complexes with plasma Aß makes it difficult to reliably measure the concentration of circulating anti-Aß antibodies with certain immunoassays, potentially leading to an underestimation. Here we have investigated anti-Aß antibody production on a cellular level by measuring the amount of anti-Aß antibody producing cells instead of the plasma level of anti-Aß antibodies. To our knowledge, this is the first time the anti-Aß antibody response in plasma has been compared in AD patients and age-matched healthy individuals using the enzyme-linked immunospot (ELISpot) technique. Both AD patients and healthy individuals had low levels of B cells producing antibodies binding Aß40 monomers, whereas the number of cells producing antibodies toward Aß42 protofibrils was higher overall and significantly higher in AD compared to healthy controls. This study shows, by an alternative and reliable method, that there is a specific immune response to the toxic Aß protofibrils, which is significantly increased in AD patients.

Recombinant adenovirus vector activates and protects human monocyte-derived dendritic cells from apoptosis.

The aim of this study was to examine the effect of two of the most commonly used viral vectors, that is, retrovirus and adenovirus, on the antigen presentation of dendritic cells (DCs). DCs were generated from CD34(+) hematopoietic precursors and CD14(+) monocytes of the same prostate cancer patients. Adenoviral transduction of monocyte-derived DCs (MO-DCs) resulted in upregulation of CD80, CD86, and CD83 expression. Adenovirus-transduced MO-DCs were also more potent stimulators of allogeneic lymphocytes, produced increased amounts of the cytokines tumor necrosis factor alpha and interleukin 12 p70, and exhibited increased expression of NF-kappaB and antiapoptotic molecules Bcl-X(L) and Bcl-2. Enhanced expression of the antiapoptotic molecules correlated with increased resistance of adenovirus-transduced MO-DCs to spontaneous as well as Fas-mediated cell death. In contrast to the adenoviral construct, no significant transduction of MO-DCs with the retrovirus could be obtained. Transduction of CD34(+) cell-derived DCs with the retrovirus or the adenovirus did not significantly alter expression of the costimulatory molecules or cytokines studied. At lower stimulation ratios, CD34(+) cell-derived DCs transduced with retrovirus were less potent in their ability to stimulate allogeneic lymphocytes in comparison with nontransduced DCs. Our results indicate that adenoviral vectors may be more suitable for gene delivery to DCs for immunotherapy.

ELISpot and ELISA analysis of spontaneous, mitogen-induced and antigen-specific cytokine production in cynomolgus and rhesus macaques.

Evaluation of cytokine production in macaques has been hampered by a lack of availability of optimized and standardized immunoassays such as ELISA and enzyme-linked immune spot assay (ELISpot); only a limited number of macaque cytokines have been assessed by ELISpot. Using monoclonal antibodies (mAb) to human cytokines that cross-react with cynomolgus and rhesus macaque interferon-gamma (IFN-gamma), interleukin (IL)-2, IL-4, IL-5, IL-6, IL-12, IL-13 and granulocyte monocyte colony-stimulating factor, we measured macaque cytokine production by ELISA and ELISpot. Quantitation of spontaneous as well as phytohemagglutinin (PHA)-induced cytokine production in peripheral blood mononuclear cells (PBMC) from rhesus and cynomolgus macaques and humans were compared. The proportional distribution of the different cytokines, in terms of PBMC synthesizing different cytokines as well as the levels of the different cytokines produced, were similar in all species. Spontaneous- and PHA-induced cytokine productions thus appear to be similarly regulated in macaques and man. ELISpot and ELISA assays for macaque IFN-gamma were further used to measure antigen-specific immune responses of PBMC from cynomolgus macaques exposed to, or vaccinated against, simian immunodeficiency virus (SIV). The establishment of reliable immunoassays for detection of macaque cytokines is of importance for future progress of research utilizing macaques as experimental animals.

Enzyme-linked immunospot assay for detection of thioredoxin and thioredoxin reductase secretion from cells.

Oxidative stress response was determined in this study by enzyme-linked immunospot (ELISpot) assays for thioredoxin (Trx) and Trx reductase (TrxR). On exposure to oxidative stress, cells can launch a variety of defense mechanisms, including release of antioxidant proteins. The Trx system, consisting of Trx, TrxR, and NADPH, constitutes one of these cellular defense systems for maintenance of a healthy reduction-oxidation (redox) balance. Trx and TrxR are rapidly upregulated and released from monocytes, lymphocytes, and other normal and neoplastic cells on exposure. Secreted Trx and TrxR have proved to be eminent indicators of oxidative stress. Trx is a small, 12-kDa protein released through a leaderless pathway, whereas TrxR, which is a 116-kDa selenoprotein and required for regeneration of Trx, is secreted through the Golgi pathway. In this chapter we present a detailed laboratory bench protocol for enumeration of single cells secreting redox-active Trx and TrxR after oxidative stress exposure. Physiological stimuli (such as interferon gamma, lipopolysaccharide, interleukin 1, and CD23 ligation; and phorbol 12-myristate 13-acetate and ionophore) as well as UV light and hydrogen peroxide were used to generate oxidative stress, and some are presented in detail. The protocol includes a description of cell isolation, preparation, handling, and development of ELISpot plates, troubleshooting notes, presentation of results, statistical evaluation, and comments on alternative sources of materials and manufacturer Web addresses. We concluded that the ELISpot assay is a useful method for detection of single cells secreting the redox-active proteins Trx and TrxR after oxidative stress exposure.

High level of cFLIP correlates with resistance to death receptor-induced apoptosis in bladder carcinoma cells.

BACKGROUND: The cellular form of FLICE-inhibitory protein (cFLIP) blocks death receptor-induced apoptosis and has been implicated in tumour progression. cFLIP interacts with caspase-8, thereby preventing activation of the caspase cascade. In this study we investigated the endogenous expression of cFLIP and caspase-8 in bladder carcinoma cells in relation to their sensitivity to death receptor-ligation. MATERIALS AND METHODS: Apoptosis was induced by agonistic anti-CD95 mAbs or recombinant TRAIL and quantified by the TUNEL technique. The relative mRNA expression of cFLIP and caspase-8 was quantified by real-time PCR. Stable expression of cFLIP long (cFLIPL) was obtained by retroviral transduction. RESULTS: The relative ratio of cFLIP and caspase-8 was directly correlated to resistance to anti-CD95 or TRAIL-mediated apoptosis. Overexpression of cFLIPL shifted the responsiveness towards resistant status. CONCLUSION: cFLIP is an important determinant of susceptibility to death receptor-induced apoptosis in bladder carcinomas and could function as a prognostic marker for death receptor sensitivity in future immune therapy.

cIAP-2 block apoptotic events in bladder cancer cells.

BACKGROUND: The Inhibitors of Apoptosis (IAPs) are negative regulators of apoptosis and their overexpression renders cells resistant to a variety of apoptotic stimuli. We investigated the mRNA expression levels of IAPs in a panel of bladder tumour cells, selected as being sensitive or resistant to death receptor-mediated apoptosis. MATERIALS AND METHODS: The mRNA expression of IAPs was quantified in a RNase protection assay (RPA). Apoptosis was induced by recombinant killerTRAIL, agonistic anti-CD95 mAbs or doxorubicin and quantified by TUNEL and MTT assays. Stable expression of cIAP-2 was obtained by retroviral transduction. RESULTS: The expression of cIAP-2 mRNA was highly correlated with resistance to TRAIL-mediated apoptosis. Overexpression of cIAP-2 conferred resistance to previously sensitive cell lines and made cells less susceptible to doxorubicin. Treatment with doxorubicin in combination with TRAIL or anti-CD95 resulted in a synergistic cytotoxic effect, which was not possible to reverse by overexpression of cIAP-2. CONCLUSION: cIAP-2 is an important regulator of apoptosis in bladder cancer and its overexpression may make tumours less susceptible to therapy involving apoptosis. The combination of immunotherapy and chemotoxic agents may represent a valid strategy to potentiate anti-tumour therapy, in particular treating tumours resisting conventional chemotherapy.