Interpretation of time-to-event outcomes in randomized trials: an online randomized experiment.

Background: Multiple features in the presentation of randomized controlled trial (RCT) results are known to influence comprehension and interpretation. We aimed to compare interpretation of cancer RCTs with time-to-event outcomes when the reported treatment effect measure is the hazard ratio (HR), difference in restricted mean survival times (RMSTD), or both (HR+RMSTD). We also assessed the prevalence of misinterpretation of the HR. Methods: We carried out a randomized experiment. We selected 15 cancer RCTs with statistically significant treatment effects for the primary outcome. We masked each abstract and created three versions reporting either the HR, RMSTD, or HR+RMSTD. We randomized corresponding authors of RCTs and medical residents and fellows to one of 15 abstracts and one of 3 versions. We asked how beneficial the experimental treatment was (0-10 Likert scale). All participants answered a multiple-choice question about interpretation of the HR. Participants were unaware of the study purpose. Results: We randomly allocated 160 participants to evaluate an abstract reporting the HR, 154 to the RMSTD, and 155 to both HR+RMSTD. The mean Likert score was statistically significantly lower in the RMSTD group when compared with the HR group (mean difference -0.8, 95% confidence interval, -1.3 to -0.4, P < 0.01) and when compared with the HR+RMSTD group (difference -0.6, -1.1 to -0.1, P = 0.05). In all, 47.2% (42.7%-51.8%) of participants misinterpreted the HR, with 40% equating it with a reduction in absolute risk. Conclusion: Misinterpretation of the HR is common. Participants judged experimental treatments to be less beneficial when presented with RMSTD when compared with HR. We recommend that authors present RMST-based measures alongside the HR in reports of RCT results.

Whole mount immunofluorescence analysis of placentas from normotensive versus preeclamptic pregnancies.

INTRODUCTION: Defects in placental angiogenesis and spiral artery remodeling have been proposed to play essential roles in the development of preeclampsia. However, the specific molecular mechanism(s) responsible for aberrant placental angiogenesis in preeclampsia are incompletely understood. The vascular endothelial growth factor receptors (VEGFR1, R2, R3) and STAT3 have critical functions in normal blood vessel development, but their potential roles in preeclampsia are currently unclear. In this study, we utilized a novel whole mount immunofluorescence (WMIF) method to compare expression of VEGFR1, R2, R3 and activated, phosphorylated STAT3 (pSTAT3) in placentas of preeclamptic (PE) versus normotensive (NT) pregnancies. METHODS: Placental biopsies collected from NT and PE pregnant women were fixed and stained with fluorochrome-conjugated antibodies to identify specific cell populations as follows: CD31 for blood vessel endothelial cells, cytokeratin-7 for trophoblast cells, and CD45 for immune cells. Expression of the VEGFRs and pSTAT3 were subsequently characterized by WMIF in conjunction with confocal microscopy. RESULTS: A total of 18 PE and 18 NT placentas were evaluated. No significant differences in the cell type-specific expression patterns or expression levels of VEGFR1, VEGFR2 or VEGFR3 were detected between NT and PE placentas. In contrast, statistically significant increases in pSTAT3 staining were detected in endothelial cells of PE placentas versus NT controls. DISCUSSION: Our study demonstrates that increased pSTAT3 expression in placental endothelial cells is associated with PE. We speculate that elevated pSTAT3 expression in the blood vessels of PE placentas may be due to aberrant angiogenesis, increased pro-inflammatory cytokine expression, and/or placental stress.

Mechanism of IL-12 mediated alterations in tumour blood vessel morphology: analysis using whole-tissue mounts.

New blood vessel formation within tumours is a critical feature for tumour growth. A major limitation in understanding this complex process has been the inability to visualise and analyse vessel formation. Here, we report on the development of a whole-tissue mount technique that allows visualisation of vessel structure. Mice expressing green fluorescent protein (GFP) made it possible to easily see GFP(+) vessels within non-GFP-expressing B16 melanoma tumours. The small fragments of tumour used in this technique were also effectively stained with fluorescent probe-conjugated antibodies, allowing characterisation of the vessels based on surface marker phenotype. The vessels within tumour tissue were much more irregular and tortuous compared to those within surrounding normal muscle. B16 tumours stably transfected with the genes for IL-12 were used to assess the effects of this cytokine on tumour growth and vessel formation. The IL-12-expressing tumours grew more slowly and had much smaller blood vessels than the large, webbed vessels characteristic of the parental tumours, effects that were dependent on interferon gamma (IFN-gamma). Vessels in the parental tumours were found to express VEGFR-3, the receptor for VEGF-C and VEGF-D. Expression of this receptor by the endothelial cells of the blood vessels was lost in the cytokine expressing tumours, thus suggesting a mechanism for the antiangiogenic effects of IL-12. The combination of the whole mount technique and the GFP transgenic mice provides a powerful method for visualising tumour vasculature and characterising the effects of agents such as cytokines.

Adhesion and guidance in compatible pollination.

The mechanisms of compatible pollination are less studied than those of incompatible pollination and yet most of the angiosperms show self-compatibility. From the release of pollen from anthers to the penetration of the micropyle by the pollen tube tip, there are numerous steps where the interaction between pollen and the pistil can be regulated. Recent studies have documented some diverse ways in which pollen tubes carrying sperm cells are guided to the ovules through the pistil extracellular matrices of the transmitting tract. What is still missing is an understanding of pollen tube cell biology in vivo. A recent finding supports the role of the synergids in the crucial guidance cue for the pollen tube tip at the micropyle, but experimental evidence for other 'guidepost' cells in the pistil is still lacking. The fact that the pollen tube must first travel through the matrices of the stigma and style before it can respond to the cue from the ovule makes it likely that there is a hierarchy of signalling events in pollen-pistil interactions starting at the stigma and ending at the micropyle. On the pistil side, several model systems have been used in the discovery of molecules implicated in either physical or chemical guidance. In lily, which has a hollow style, adhesion molecules (pectin and SCA) are implicated in guidance. SCA alone is also capable of inducing pollen chemotropism in an in vitro assay, suggesting that this peptide plays a dual role in lily pollination: chemotactic in the stigma and haptotactic (adhesion mediated) in the style.

Dendritic cells transduced with HSV-1 amplicons expressing prostate-specific antigen generate antitumor immunity in mice.

There is currently much interest in generating cytotoxic T lymphocyte (CTL) responses against tumor antigens as a therapy for cancer. This work describes a novel gene transfer technique utilizing dendritic cells (DCs), an extremely potent form of antigen-presenting cell (APC), and herpes simplex virus-1 (HSV-1) amplicons. HSV-1 amplicons are plasmid-based viral vectors that are packaged into HSV-1 capsids, but lack viral coding sequences. Amplicon vectors have been constructed that encode the model tumor antigen ovalbumin (HSV-OVA) and human prostate-specific antigen (HSV-PSA), a protein that is expressed specifically in prostate epithelium and prostate carcinoma cells. These amplicons were packaged using a helper virus-free system that produces vector stocks that are devoid of contaminating cytotoxic helper virus. Transduction of DCs with HSV-OVA or HSV-PSA and co-culture with CTL hybridomas results in specific activation, indicating that transduced DCs express these transgenes and process the tumor antigens for class I MHC presentation to CTL. Mice immunized with HSV-PSA-transduced DCs generate a specific CTL response that can be detected in vitro by a (51)Cr-release assay and are protected from challenge with tumors that express PSA. These results indicate that DCs transduced with HSV-1 amplicon vectors may provide a tool for investigation of the biology of CTL activation by DCs and a new modality for immunotherapy of cancer.

T-cell antigen discovery (T-CAD) assay: a novel technique for identifying T cell epitopes.

The identification of T cell epitopes is a critical step in evaluating and monitoring T cell mediated immune responses. Here, we describe a novel technique for simultaneously identifying class I and class II MHC restricted epitopes using a one-step protein purification system. This method uses Ni/chelate coated magnetic beads and magnetic separation to isolate poly-histidine tagged recombinant antigen from bacterial lysates. These beads, once coated with antigen, are also used to deliver antigen to APC where it is processed and presented to T cells. A colorimetric assay and ovalbumin specific, lacZ inducible, T cell hybridomas were used to validate the system. Further, using PSA specific hybrids, generated from T cells isolated from PSA secreting tumors, both class I and class II MHC restricted epitopes of PSA were identified. Additional characterization has shown that these peptides contribute significantly to the overall PSA specific response in vivo, and may represent the dominant epitopes of PSA.

Intravascular HBO(2) saturations, perfusion and hypoxia in spontaneous and transplanted tumor models.

Clinical trials utilizing strategies to manipulate tumor oxygenation, blood flow and angiogenesis are under way, although limited quantitative information exists regarding basic tumor pathophysiology. The current study utilized murine KHT fibrosarcomas, spontaneous mammary carcinomas and first-generation spontaneous transplants to examine heterogeneity in vascular structure and function, to relate these changes to the distribution of tumor hypoxia and to determine whether fundamental relationships among the different pathophysiological parameters exist. Three methods were included: (i) immunohistochemical staining of anatomical and perfused blood vessels, (ii) cryospectrophotometric measurement of intravascular oxyhemoglobin saturations and (iii) fluorescent detection of the EF5 hypoxic marker. While a distinct pattern of decreasing oxygenation with increasing distance from the tumor surface was observed for KHT tumors, striking intertumor variability was found in both spontaneous and first-generation transplants, with a reduced dependence on tumor volume. EF5 hypoxic marker uptake was also much more heterogeneous among individual spontaneous and first-generation tumors compared to KHT. Although mammary carcinomas demonstrated fewer anatomical blood vessels than fibrosarcomas, the proportion of perfused vessels was substantially reduced in KHT tumors, especially at larger tumor volumes. Vascular morphology, tissue histological appearance and pathophysiological parameters differed substantially between KHT tumors and both spontaneous and first-generation tumors. Such differences in vascular structure and function are also likely to correlate with altered response to therapies targeted to the vascular system. Finally, spontaneous differentiation status, tumor morphology, vascular configuration and function were well preserved in first-generation transplanted tumors, suggesting a close relationship between vascular development and function in early-generation transplants and spontaneous tumor models.

Tumours can act as adjuvants for humoral immunity.

Tumour cells transfected with cDNAs encoding non-self proteins were used to investigate the ability of the immune system to respond to immunogenic antigens expressed by tumours. Secreted, intracellular and surface proteins were used as model antigens, as these reflect the potential forms of tumour antigens. Syngeneic BALB/c mice injected with viable line 1 lung carcinoma or EMT6 mammary tumour cells secreting ovalbumin (OVA) or prostate-specific antigen (PSA) produced very high immunoglobulin G (IgG) antibody titres, equivalent to those of mice injected with protein in Freund's complete adjuvant (FCA). Secretion of the antigens was not necessary as tumour cells expressing a cell-surface antigen (HER-2/Neu) or an intracellular antigen - green fluorescence protein (GFP) - also generated high-titre antigen-specific IgG antibodies. In interleukin-4 (IL-4)-deficient mice, both IgG1 and IgG2a were produced in response to OVA administered in FCA, whereas in response to tumour-produced antigen, the antibodies switched from predominantly IgG1 to IgG2a, indicating that the mechanisms responsible for antibody induction differed between these forms of immunization. In contrast to the line 1 and EMT6 tumours, which are of BALB/c origin, OVA- or PSA-producing B16 melanoma cells, which are of C57BL/6 origin, failed to elicit antibody production. This was not the result of strain differences, as a similar finding was observed when the tumours were grown in (BALB/c x C57BL/6)F1 mice, but appeared to be caused by intrinsic differences in the tumours. Furthermore, co-injection of both B16/OVA and line 1 tumours resulted in production of anti-OVA antibody, indicating that B16 tumours were not immunosuppressive, but instead line 1 tumours appear to exert an adjuvant effect.

Effects of radiation on tumor intravascular oxygenation, vascular configuration, development of hypoxia, and clonogenic survival.

The underlying physiological mechanisms leading to tumor reoxygenation after irradiation have elicited considerable interest, but they remain somewhat unclear. The current study was undertaken to determine the effects of a single dose of 10 Gy gamma radiation on both tumor pathophysiology and radiobiologically hypoxic fraction. Immunohistochemical staining and perfusion markers were used to quantify tumor vasculature, uptake of the hypoxia marker EF5 to assess the distribution of hypoxia, and intravascular HbO(2) measurements to determine oxygen availability. Tumor radiosensitivity was measured by a clonogenic assay. At 24 h postirradiation, oxygen availability increased, perfused vessel numbers decreased, EF5 uptake decreased, and the radiobiologically hypoxic fraction was unchanged. Together, these results demonstrate that tumor hypoxia develops at an increased distance from perfused blood vessels after irradiation, suggesting a decrease in oxygen consumption at 24 h. By 72 h postirradiation, all physiological parameters had returned to the levels in volume-matched, nonirradiated controls. These studies clearly show that single measures of either tumor oxygenation or vascular structure are inadequate for assessing the effects of radiation on tumor clonogenicity. Although such direct measurements have previously proven valuable in predicting tumor response to therapy or oxygen manipulation, a combination of parameters is required to adequately describe the mechanisms underlying these changes after irradiation.

A lily stylar pectin is necessary for pollen tube adhesion to an in vitro stylar matrix.

Pollen tube cells adhere to the wall surface of the stylar transmitting tract epidermis in lily. This adhesion has been proposed as essential for the proper delivery of the sperm cells to the ovule. An in vitro adhesion bioassay has been used to isolate two stylar molecules required for lily pollen tube adhesion. The first molecule was determined to be a small, cysteine-rich protein with some sequence similarity to lipid transfer proteins and now called stigma/stylar cysteine-rich adhesin (SCA). The second, larger, molecule has now been purified from style fragments and characterized. Chemical composition, specific enzyme degradations, and immunolabeling data support the idea that this molecule required for pollen tube adhesion is a pectic polysaccharide. In vitro binding assays revealed that this lily stylar adhesive pectin and SCA are able to bind to each other in a pH-dependent manner.

In vivo behavior of peptide-specific T cells during mucosal tolerance induction: antigen introduced through the mucosa of the conjunctiva elicits prolonged antigen-specific T cell priming followed by anergy.

The mucosa of the conjunctiva is an important site of entry for environmental Ags as well as Ags emanating from the eye itself. However, very little is known about T cell recognition of Ag introduced through this important mucosal site. We have characterized the in vivo process of CD4 T cell recognition of Ag delivered via the conjunctival mucosa. Application of soluble OVA to the conjunctiva of BALB/c mice induced potent T cell tolerance. APC-presenting OVA peptide in vivo was only found in the submandibular lymph node and not in other lymph nodes, spleen, or nasal-associated lymphoid tissue. Similarly, in TCR transgenic DO11. 10 adoptive transfer mice, OVA-specific CD4+ T cell clonal expansion was only observed in the submandibular lymph node following conjunctival application of peptide. These experiments thus define a highly specific lymphatic drainage pathway from the conjunctiva. OVA-specific T cell clonal expansion peaked at day 3 following initiation of daily OVA administration and gradually declined during the 10-day treatment period, but remained elevated compared with nontreated adoptive transfer mice. During this period, the T cells expressed activation markers, and proliferated and secreted IL-2 in vitro in response to OVA stimulation. In contrast, these cells were unable to clonally expand in vivo, or proliferate in vitro following a subsequent OVA/CFA immunization. These results suggest that Ag applied to a mucosal site can be efficiently presented in a local draining lymph node, resulting in initial T cell priming and clonal expansion, followed by T cell anergy.

Detection of hypoxia in human squamous cell carcinoma by EF5 binding.

Localization and quantitation of 2-nitroimidazole drug binding in low pO2 tumors is a technique that can allow the assessment of hypoxia as a predictive assay. EF5 [2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl) acetamide] is such a drug, and it has been shown to be predictive of radiation response in rodent tumors. Using fluorescence immunohistochemical techniques, we provide data on the presence, distribution, and levels of EF5 binding as a surrogate for hypoxia in human head and neck and uterine cervix squamous cell cancers (SCCs). Six patients with SCC were studied. Four patients had head and neck tumors, and two had uterine cervix cancers. The incubation of fresh tissue cubes in EF3 under hypoxic conditions ("reference binding") demonstrated that all tumors were capable of binding drug, and that this binding varied by a factor of 2.9-fold (174.5-516.1) on an absolute fluorescence scale. In the five patients treated at the lowest drug doses (9 mg/kg), in situ binding was quantitatable. For all six patients, the maximum rate of in situ binding varied by a factor of 6.7 between the lowest and highest binding tumor (24.8-160.3) on an absolute fluorescence scale. In tumors with high binding regions, intratumoral heterogeneity was large, extending from minimal fluorescence (<1%) up to 88.6% of reference binding. In tumors with minimal binding, there was little intratumoral heterogeneity. These studies demonstrate substantial heterogeneity of in situ binding between and within individual squamous cell tumors.

Alteration of tumour response to radiation by interleukin-2 gene transfer.

We have previously shown that BALB/c-derived EMT6 mammary tumours transfected with interleukin (IL)-2 have decreased hypoxia compared to parental tumours, due to increased vascularization. Since hypoxia is a critical factor in the response of tumours to radiation treatment, we compared the radiation response of IL-2-transfected tumours to that of parental EMT6 tumours. Because the IL-2 tumours have an altered host cell composition, which could affect the interpretation of radiation sensitivity as measured by clonogenic cells, we employed flow cytometric analysis to determine the proportion of tumour cells vs host cells in each tumour type. Using this approach, we were able to correct the plating efficiency based on the number of actual tumour cells derived from tumours, making the comparison of the two types of tumours possible. We also excluded the possibility that cytotoxic T-cells present in EMT6/IL-2 tumours could influence the outcome of the clonogenic cell survival assay, by demonstrating that the plating efficiency of cells derived from EMT6/IL-2 tumours remained unchanged after depletion of Thy-1+ cells. The in vivo radiation response results demonstrated that IL-2-transfected tumours were more sensitive to radiation than parental EMT6 tumours. The hypoxic fraction of the EMT6/IL-2 tumours growing in vivo was markedly decreased relative to parental EMT6 tumours thus the increased sensitivity results from the increased vascularity we have previously observed in these tumours. These results indicate the potential therapeutic benefit of combining radiation and immunotherapy in the treatment of tumours.

Noninvasive detection of tumor hypoxia using the 2-nitroimidazole [18F]EF1.

UNLABELLED: The noninvasive assessment of tumor hypoxia in vivo is under active investigation because hypoxia has been shown to be an important prognostic factor for therapy resistance. Various nuclear medicine imaging modalities are being used, including PET imaging of 18F-containing compounds. In this study, we report the development of 18F-labeled EF1 for noninvasive imaging of hypoxia. EF1 is a 3-monofluoro analog of the well-characterized hypoxia marker EF5, 2(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetami de, which has been used to detect hypoxia in tumor and nontumor systems using immunohistochemical methods. METHODS: We have studied 2 rat tumor types: the hypoxic Morris 7777 (Q7) hepatoma and the oxic 9LF glioma tumor, each grown in subcutaneous sites. PET studies were performed using a pharmacological dose of nonradioactive carrier in addition to [18F]EF1 to optimize and assess drug biodistribution. After PET imaging of the tumor-bearing rats, tissues were obtained for gamma-counting of the 18F in various tissues and immunohistochemical detection of intracellular drug adducts in tumors. In one pair of tumors, Eppendorf needle electrode studies were performed. RESULTS: [18F]EF1 was excreted dominantly through the urinary tract. The tumor-to-muscle (T/M) ratio of [18F]EF1 in the Q7 tumors was 2.7 and 2.4 based on PET studies and 2.1, 2.5, and 3.0 based on gamma-counting of the tissues (n = 3). In contrast, the T/M ratio of [18F]EF1 in the 9LF glioma tumor was 0.8 and 0.5 based on PET studies and 1.0, 1.2, and 1.4 based on gamma-counting of the tissues (n = 3). Immunohistochemical analysis of drug adducts for the two tumor types agreed with the radioactivity analysis. In the Q7 tumor, substantial heterogeneous binding was observed throughout the tumor, whereas in the 9LF tumor minimal binding was found. CONCLUSION: [18F]EF1 is an excellent radiotracer for noninvasive imaging of tumor hypoxia.

Enhancement of tumor perfusion and oxygenation by carbogen and nicotinamide during single- and multifraction irradiation.

Numerous experimental and clinical studies have been completed regarding the effects of carbogen and nicotinamide on tumor oxygenation and radiosensitivity. The current study incorporates three physiological measurement techniques to further define spatial variations in oxygen availability and development of hypoxia after single- and multifraction irradiation in KHT murine fibrosarcomas. Distances to anatomical and perfused blood vessels were measured using immunohistochemical and fluorescent staining, intravascular oxygen levels were determined cryospectrophotometrically, and tumor hypoxia was quantified using uptake of EF5, a marker of hypoxia. Carbogen, nicotinamide, and the combination of both all increased intravascular oxygen availability compared to controls. While nicotinamide had no effect on the number of perfused blood vessels in nonirradiated tumors, carbogen produced a substantial closing of vessels. After a single dose of 4 Gy, only the combination of nicotinamide and carbogen produced significant improvements in oxygen availability, while numbers of perfused vessels were significantly increased for nicotinamide, unchanged for the combination of nicotinamide and carbogen, and significantly decreased for carbogen. After 4 x 4-Gy fractions, oxygen availability was increased substantially with the combination of nicotinamide and carbogen, somewhat with carbogen, and not at all with nicotinamide. Tumor oxygenation changes were estimated by EF5/Cy3 intensity distributions, which demonstrated that manipulative agents could produce disparate effects on tumor hypoxia when combined with either single- or multifraction irradiation.

A lipid transfer-like protein is necessary for lily pollen tube adhesion to an in vitro stylar matrix.

Flowering plants possess specialized extracellular matrices in the female organs of the flower that support pollen tube growth and sperm cell transfer along the transmitting tract of the gynoecium. Transport of the pollen tube cell and the sperm cells involves a cell adhesion and migration event in species such as lily that possess a transmitting tract epidermis in the stigma, style, and ovary. A bioassay for adhesion was used to isolate from the lily stigma/stylar exudate the components that are responsible for in vivo pollen tube adhesion. At least two stylar components are necessary for adhesion: a large molecule and a small (9 kD) protein. In combination, the two molecules induced adhesion of pollen tubes to an artificial stylar matrix in vitro. The 9-kD protein was purified, and its corresponding cDNA was cloned. This molecule shares some similarity with plant lipid transfer proteins. Immunolocalization data support its role in facilitating adhesion of pollen tubes to the stylar transmitting tract epidermis.

Hypoxia-mediated apoptosis from angiogenesis inhibition underlies tumor control by recombinant interleukin 12.

The role of angiogenesis inhibition in the antitumor activity of recombinant murine interleukin 12 (rmIL-12) was studied in K1735 murine melanomas, the growth of which is rapidly and markedly suppressed by rmIL-12 treatment. On the basis of the prediction that tumor ischemia should result from therapeutic angiogenesis inhibition, tumor cell hypoxia was evaluated as a marker of ischemia using the EF5 [2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)aceta mide] approach. This method measures intracellular binding of the nitroimidazole EF5, which covalently binds to cellular macromolecules selectively under hypoxic conditions. Whereas 1 week of rmIL-12 treatment effectively inhibited K1735 cell-induced angiogenesis in Matrigel neovascularization assays, 2 weeks of treatment were needed before severe tumor cell hypoxia was detected in K1735 tumors. The hypoxia that developed was regional and localized to tumor areas distant from blood vessels. The great majority of severely hypoxic tumor cells were apoptotic, and in vitro studies indicated that the degree of hypoxia present within treated tumors was sufficient to trigger K1735 apoptosis. Tumor cell apoptosis was also prevalent in the first week of rmIL-12 treatment when few cells were hypoxic. In vitro studies indicated that this non-hypoxia-related apoptosis was induced directly by IFN-gamma produced in response to rmIL-12 administration. These studies reveal that rmIL-12 controls K1735 tumors initially by IFN-gamma-induced apoptosis and later by hypoxia-induced apoptosis. They also establish hypoxia as an expected result of tumor angiogenesis inhibition and a mediator of its therapeutic effect.

Interleukin-3 and granulocyte-macrophage colony-stimulating factor enhance the generation and function of dendritic cells.

Dendritic cells, well-known for their potent antigen-presenting activity, are generally present at very low frequency in the spleens of naive mice. We examined the ability of mice to generate functional dendritic cells (DC) following exposure to the cytokines interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF). Tumours secreting these cytokines provided a continuous stimulus resulting in a greatly increased number and frequency of DC in the spleen. These cells were purified by conventional DC isolation techniques and were found to exhibit many of the characteristics of DC from unmanipulated mice, including high allo-stimulatory activity in mixed lymphocyte reactions and expression of many similar cell surface markers. Using ovalbumin-peptide specific class I- and class II-restricted hybridomas containing the lacZ reporter gene, we found that these cytokine-generated DC had a greatly increased efficacy in the uptake and processing of particulate antigen. These cells appear to have retained the ability to ingest antigen that is generally associated with immature DC, but also exhibit the peptide/major histocompatibility complex (MHC)-presenting capabilities of mature DC. Development of an assay to measure the activity of a single DC revealed that these dual activities were the properties of the majority of the cytokine-generated DC. These findings indicate that exposure in vivo to the cytokines IL-3 and GM-CSF can result in the generation of large numbers of DC with increased capability of stimulating T cells. Thus, these cells may be important in vivo in the process of cross-priming and the subsequent generation of tumour-reactive cytotoxic T lymphocytes (CTL).

Detection of hypoxic cells with the 2-nitroimidazole, EF5, correlates with early redox changes in rat brain after perinatal hypoxia-ischemia.

The hypoxia-dependent activation of nitroheterocyclic drugs by cellular nitroreductases leads to the formation of intracellular adducts between the drugs and cellular macromolecules. Because this covalent binding is maximal in the absence of oxygen, detection of bound adducts provides an assay for estimating the degree of cellular hypoxia in vivo. Using a pentafluorintated derivative of etanidazole called EF5, we studied the distribution of EF5 adducts in seven-day-old rats subjected to different treatments which decrease the level of oxygen in the brain. EF5 solution was administered intraperitoneally 30 min prior to each treatment. The effect of acute and chronic hypoxia on EF5 adduct formation (binding) was studied in the brain of newborn rats exposed to global hypoxia (8% O2 for 30, 90 or 150 min) and in the brain of chronically hypoxic rat pups with congenital cardiac defects (Wistar Kyoto). The effect of combined hypoxia-ischemia was investigated in rat pups subjected to right carotid coagulation and concurrent exposure to 8% O2 for 30, 90 or 150 min. Brains were frozen immediately at the end of each treatment. Using a Cy3-conjugated monoclonal mouse antibody (ELK3-51) raised against EF5 adducts, hypoxic cells within brain regions were visualized by fluorescence immunocytochemistry. Brains from controls or vehicle-injected animals showed no EF5 binding. Notably, brains from animals which were chronically hypoxemic as a result of congenital cardiac defects also showed no EF5 binding. A short exposure (30 min) to hypoxia or to combined hypoxia-ischemia resulted in increased background stain and few scattered cells with low-intensity immunostaining. Acute hypoxia exposure of at least 90-150 min, which in this age animal does not result in frank cellular damage, produced patchy areas of low- to moderate-intensity fluorescence scattered throughout the brain. In contrast, 90-150 min of hypoxia-ischemia was associated with intense immunofluorescence in the hemisphere ipsilateral to the carotid occlusion, with a pattern similar to that reported previously for the histological damage seen in this model. This study provides a sensitive method for the evaluation of the level of oxygen depletion in brain tissue after neonatal hypoxia-ischemia at times much earlier than any method demonstrates apoptotic or necrotic cell death Since the level of in vivo formation of macromolecular adducts of EF5 depends on the degree of oxygen depletion in a tissue, intracellular EF5 binding may serve as a useful marker of regional cellular vulnerability and redox state after brain injury resulting from hypoxia-ischemia.

Quantification of tumour vasculature and hypoxia by immunohistochemical staining and HbO2 saturation measurements.

Despite the possibility that tumour hypoxia may limit radiotherapeutic response, the underlying mechanisms remain poorly understood. A new methodology has been developed in which information from several sophisticated techniques is combined and analysed at a microregional level. First, tumour oxygen availability is spatially defined by measuring intravascular blood oxygen saturations (HbO2) cryospectrophotometrically in frozen tumour blocks. Second, hypoxic development is quantified in adjacent sections using immunohistochemical detection of a fluorescently conjugated monoclonal antibody (ELK3-51) to a nitroheterocyclic hypoxia marker (EF5), thereby providing information relating to both the oxygen consumption rates and the effective oxygen diffusion distances. Third, a combination of fluorescent (Hoechst 33342 or DiOC7(3)) and immunohistological (PECAM-1/CD31) stains is used to define the anatomical vascular densities and the fraction of blood vessels containing flow. Using a computer-interfaced microscope stage, image analysis software and a 3-CCD colour video camera, multiple images are digitized, combined to form a photo-montage and revisited after each of the three staining protocols. By applying image registration techniques, the spatial distribution of HbO2 saturations is matched to corresponding hypoxic marker intensities in adjacent sections. This permits vascular configuration to be related to oxygen availability and allows the hypoxic marker intensities to be quantitated in situ.