Visualizing the kinetics of tumor-cell clearance in living animals

Evaluation of potential antineoplastic therapies would be enhanced by noninvasive detection of tumor cells in living animals. Because light is transmitted through mammalian tissues, it was possible to use bioluminescence to monitor (both externally and quantitatively) growth and regression of labeled human cervical carcinoma (HeLa) cells engrafted into immunodeficient mice. The efficacy of both chemotherapy and immunotherapeutic treatment with ex vivo expanded human T cell-derived effector cells was evaluated. In the absence of therapy, animals showed progressive increases in signal intensity over time. Animals treated with cisplatin had marked reductions in tumor signal; 5'-fluorouracil was less effective, and cyclophosphamide was ineffective. Immunotherapy dramatically reduced signals at high effector-to-target cell ratios, and significant decreases were observed with lower ratios. This model system allowed sensitive, quantitative, real-time spatiotemporal analyses of the dynamics of neoplastic cell growth and facilitated rapid optimization of effective treatment regimens.     

Monitoring for undertransfusion

BACKGROUND: Most published reviews and audits of blood and blood component transfusion have focused on the issue of overtransfusion and on the inappropriate use of red cell components. There is growing concern that efforts to curb unnecessary transfusions may result in a trend toward undertransfusion of patients. There is little published information that addresses this issue or the magnitude of this practice. STUDY DESIGN AND METHODS: Undertransfusion was evaluated by examining the transfusion records from a 3-month period for 55 patients who met the study criteria of having either a hemoglobin level < 7 g per dL or a platelet count of < 10 × 10(9) per L. If the identified patient did not receive a transfusion within 24 hours of the reported hemoglobin level or platelet count, the medical record was reviewed by a resident physician. RESULTS: A total of 213 individual hemoglobin levels and platelet counts, representing the 55 patients, met our transfusion criteria. All except 8 of the identified patients received red cells and/or platelet transfusions. Reasons for not transfusing red cells included the patient's response to nutritional support and iron supplementation, refusal of blood, and noncompliance. Reasons for not transfusing platelets included falsely low platelet count because of platelet clumping in vitro, contraindication based on clinical diagnosis (e.g., immune thrombocytopenic purpura), and the patient's death before transfusion. CONCLUSION: Red cell and platelet transfusions were appropriately ordered for all patients who met the transfusion criteria. Undertransfusion is not a problem at this institution according to the criteria established. It is recommended that other institutions expand their blood utilization audits to include investigation for evidence of undertransfusion. Further research regarding the issue of undertransfusion is warranted and could be expanded to include other components.     

Oral sumatriptan in the acute treatment of migraine and migraine recurrence in general practice

We investigated the efficacy, safety and tolerability compared with placebo of a second dose of oral sumatriptan 100 mg in 1349 general practice patients who had already treated a moderate or severe migraine headache with 100 mg sumatriptan 4 h earlier. Headache was relieved by the first sumatriptan dose in about 70% of patients, but the second dose did not produce significantly more relief than placebo, either in nonresponders or in the group as a whole, nor did it reduce other symptoms (photophobia, nausea, vomiting, etc,) at 8 h, or influence the incidence of headache recurrence. The drug was well-tolerated, and a further single dose was effective in treating recurrence after initial relief. A single 100 mg dose of sumatriptan is an effective acute treatment for migraine. A second dose should be reserved for treating headache recurrence.      

Antigen-specific T cell-mediated gene therapy in collagen-induced arthritis

Autoantigen-specific T cells have tissue-specific homing properties, suggesting that these cells may be ideal vehicles for the local delivery of immunoregulatory molecules. We tested this hypothesis by using type II collagen-specific (CII-specific) CD4(+) T hybridomas or primary CD4(+) T cells after gene transfer, as vehicles to deliver an immunoregulatory protein for the treatment of collagen-induced arthritis (CIA), a mouse model of rheumatoid arthritis (RA). CII-specific T cells or hybridomas were transduced using retroviral vectors to constitutively express the IL-12 antagonist, IL-12 p40. Transfer of engineered CD4(+) T cells after immunization significantly inhibited the development of CIA, while cells transduced with vector control had no effect. The beneficial effect on CIA of IL-12 p40-transduced T cells required TCR specificity against CII, since transfer of T cells specific for another antigen producing equivalent amounts of IL-12 p40 had no effect. In vivo cell detection using bioluminescent labels and RT-PCR showed that transferred CII-reactive T-cell hybridomas accumulated in inflamed joints in mice with CIA. These results indicate that the local delivery of IL-12 p40 by T cells inhibited CIA by suppressing autoimmune responses at the site of inflammation. Modifying antigen-specific T cells by retroviral transduction for local expression of immunoregulatory proteins thus offers a promising strategy for treating RA.     

Selective intratumoral amplification of an antiangiogenic vector by an oncolytic virus produces enhanced antivascular and anti-tumor efficacy.

The development of effective cancer therapy will require the simultaneous targeting of multiple steps in tumor development. We have previously described an antiangiogenic gene therapy vector, Ad Flk1-Fc, which expresses a soluble VEGF receptor capable of inhibiting tumor angiogenesis and growth. We have also described an oncolytic virus, dl922/947, whose replication and subsequent cytotoxicity are restricted to cancer cells with a loss of the G1-S cell cycle checkpoint. Here we have optimized methods for combining these therapies, yielding significantly greater anti-tumor effects than the respective monotherapies. In cultured tumor lines, co-infection with both Ad Flk1-Fc and dl922/947 allowed replication and repackaging of the replication-deficient Ad Flk1-Fc and enhanced soluble VEGF receptor expression. Similar repackaging and increased gene expression were demonstrated in vivo using bioluminescence imaging studies. Finally, coadministration of these therapeutic viral therapies in vivo produced significantly enhanced anti-tumor effects in colon HCT 116 and prostate PC-3 xenografts in mice. This increased therapeutic benefit correlated with replication of Ad Flk1-Fc viral genomes, increased intratumoral levels of Flk1-Fc protein, and decreased microvessel density, consistent with enhanced antiangiogenic activity.     

The Impact of Gabapentin Administration on Brain GABA and Glutamate Concentrations: A 7T 1H-MRS Study

Gamma-aminobutyric acid (GABA) and glutamate are implicated in numerous neuropsychiatric and substance abuse conditions, but their spectral overlap with other resonances makes them a challenge to quantify in humans. Gabapentin, marketed for the treatment of seizures and neuropathic pain, has been shown to increase in vivo GABA concentration in the brain of both rodents and humans. Gabapentin effects on glutamate are not known. We conducted a gabapentin (900 mg) challenge in healthy human subjects to confirm and explore its effects on GABA and glutamate concentrations, respectively, and to test the ability of single voxel localized proton magnetic resonance spectroscopy (¹H-MRS) to reliably measure GABA and glutamate in the visual cortex at the ultra-high magnetic field of 7 Tesla. Reproducibility of GABA and glutamate measurements was determined in a comparison group without drug twice within day and 2 weeks apart. Although GABA concentration changes were small both within day (average 5.6%) and between day (average 4.8%), gabapentin administration was associated with an average increase in GABA concentration of 55.7% (6.9–91.0%). Importantly, drug-induced change in GABA levels was inversely correlated to the individual''s baseline GABA level (R²=0.72). Mean glutamate concentrations did not change significantly with or without drug administration. In conclusion, localized ¹H-MRS at 7 Tesla can be successfully applied to the measurement of GABA concentration and is sensitive to acute drug-induced changes in cortical GABA. Whether baseline GABA concentrations predict clinical efficacy of gabapentin is an area worthy of exploration.     

Detection of endogenous biomolecules in Barrett's esophagus by Fourier transform infrared spectroscopy

Fourier transform infrared (FTIR) spectroscopy provides a unique molecular fingerprint of tissue from endogenous sources of light absorption; however, specific molecular components of the overall FTIR signature of precancer have not been characterized. In attenuated total reflectance mode, infrared light penetrates only a few microns of the tissue surface, and the influence of water on the spectra can be minimized, allowing for the analyses of the molecular composition of tissues. Here, spectra were collected from 98 excised specimens of the distal esophagus, including 38 squamous, 38 intestinal metaplasia (Barrett's), and 22 gastric, obtained endoscopically from 32 patients. We show that DNA, protein, glycogen, and glycoprotein comprise the principal sources of infrared absorption in the 950- to 1,800-cm(-1) regime. The concentrations of these biomolecules can be quantified by using a partial least-squares fit and used to classify disease states with high sensitivity, specificity, and accuracy. Moreover, use of FTIR to detect premalignant (dysplastic) mucosa results in a sensitivity, specificity, positive predictive value, and total accuracy of 92%, 80%, 92%, and 89%, respectively, and leads to a better interobserver agreement between two gastrointestinal pathologists for dysplasia (kappa = 0.72) versus histology alone (kappa = 0.52). Here, we demonstrate that the concentration of specific biomolecules can be determined from the FTIR spectra collected in attenuated total reflectance mode and can be used for predicting the underlying histopathology, which will contribute to the early detection and rapid staging of many diseases.     

In vivo dynamics of regulatory T-cell trafficking and survival predict effective strategies to control graft-versus-host disease following allogeneic transplantation

CD4(+)CD25(+) regulatory T cells (Tregs) suppress immune responses to alloantigens. The in vivo circulation and tissue localization of Tregs during an adaptive immune response remain unclear. We noninvasively tracked luciferase-expressing Tregs over time in an allogeneic bone marrow transplant model and demonstrated colocalization with effector T cells and initial expansion in secondary lymphoid organs before migration into inflamed tissues. Inflammation induced by irradiation and the allogeneic setting provided crucial stimuli for early Treg expansion and migration, leading to parallel reduction of effector T-cell proliferation in lymphoid organs and peripheral tissues. Treg transplants conferred long-term protection from systemic inflammatory challenge consistent with Treg in vivo survival. Suppression occurred during multiple phases of inflammation, but is optimal in the initial phase, providing protection from graft-versus-host disease while maintaining the graft-versus-tumor effect even at physiologic doses of Tregs due to their in vivo expansion, hence overcoming a major barrier to potential clinical applications of Tregs given their rarity.     

Early CD30 signaling is critical for adoptively transferred CD4+CD25+ regulatory T cells in prevention of acute graft-versus-host disease

Murine CD4+CD25+ regulatory T cells (Treg cells) reduce acute graft-versus-host disease (aGvHD). However, surface molecules critical for suppression are unclear. Deficiency of CD30 (CD30-/-) leads to impaired thymic negative selection and augmented T-cell autoreactivity. Therefore, we investigated the role of CD30 signaling in Treg-cell function during aGvHD. Treg cells derived from CD30-/- animals were significantly less effective in preventing aGvHD lethality. Early blockade of the CD30/CD153 pathway with a neutralizing anti-CD153 mAb reduced Treg-mediated protection from proinflammatory cytokine accumulation and donor-type T-cell apoptosis. In vivo bioluminescence imaging demonstrated intact homing but reduced expansion of luciferase-expressing Treg cells when CD153 was blocked during the early phase after adoptive transfer. CD30 surface expression on Treg cells increased with alloantigen exposure, and CD153 expression on recipient-type dendritic cells increased in the presence of a proinflammatory environment. These data demonstrate that early CD30 signaling is critical for Treg-mediated aGvHD protection after major MHC-mismatch bone marrow transplantation.