Induction of endothelial barrier dysfunction by serum factors in rats subjected to traumatic brain injury and hemorrhagic shock.

Traumatic brain injury (TBI) has been associated with the development of indirect acute respiratory distress syndrome (ARDS). However, the causative relationship between TBI and lung injury remains unclear. To explore potential mechanisms linking TBI with the development of ARDS, we characterized the effects of serum factors released following TBI and hemorrhagic shock (HS) in a rat model on the pulmonary endothelial cell (EC) barrier dysfunction, a key feature of ARDS. We found that serum samples from animals exposed to both controlled cortical impact (CCI) and HS, but not from sham-operated rats induced significant barrier dysfunction in human pulmonary artery EC monolayers at 2 days post injury. Thrombin inhibitor and thrombin receptor antagonist attenuated the acute phase of the serum-induced trans-endothelial resistance (TER) decline caused by CCI-HS serum, but not in later time points. However, both the early and late phases of CCI-HS-induced EC permeability were inhibited by heparin. The barrier disruptive effects of CCI-HS serum were also prevented by serum preincubation with heparin-sepharose. Pulmonary EC treated for 3 h with serum from CCI-HS rats demonstrated a significant decline in expression of EC junctional protein, VE-Cadherin, and disassembly of peripheral EC adherens junction complexes monitored by immunostaining with VE-cadherin antibody. These results suggest that exposure to CCI-HS causes early and late-phase barrier disruptive effects in vascular endothelium. While thrombin-PAR1 signaling has been identified as a mechanism of acute EC permeability increase by CCI-HS serum, the factor(s) defining long-term EC barrier disruption in CCI-HS model remains to be determined.

Continuous Vital Sign Analysis to Predict Secondary Neurological Decline After Traumatic Brain Injury.

Background: In the acute resuscitation period after traumatic brain injury (TBI), one of the goals is to identify those at risk for secondary neurological decline (ND), represented by a constellation of clinical signs that can be identified as objective events related to secondary brain injury and independently impact outcome. We investigated whether continuous vital sign variability and waveform analysis of the electrocardiogram (ECG) or photoplethysmogram (PPG) within the first hour of resuscitation may enhance the ability to predict ND in the initial 48 hours after traumatic brain injury (TBI). Methods: Retrospective analysis of ND in TBI patients enrolled in the prospective Oximetry and Noninvasive Predictors Of Intervention Need after Trauma (ONPOINT) study. ND was defined as any of the following occurring in the first 48 h: new asymmetric pupillary dilatation (>2 mm), 2 point GCS decline, interval worsening of CT scan as assessed by the Marshall score, or intervention for cerebral edema. Beat-to-beat variation of ECG or PPG, as well as waveform features during the first 15 and 60 min after arrival in the TRU were analyzed to determine physiologic parameters associated with future ND. Physiologic and admission clinical variables were combined in multivariable logistic regression models predicting ND and inpatient mortality. Results: There were 33 (17%) patients with ND among 191 patients (mean age 43 years old, GCS 13, ISS 12, 69% men) who met study criteria. ND was associated with ICU admission (P < 0.001) and inpatient mortality (P < 0.001). Both ECG (AUROC: 0.84, 95% CI: 0.76,0.93) and PPG (AUROC: 0.87, 95% CI: 0.80, 0.93) analyses during the first 15 min of resuscitation demonstrated a greater ability to predict ND then clinical characteristics alone (AUROC: 0.69, 95% CI: 0.59, 0.8). Age (P = 0.02), Marshall score (P = 0.001), penetrating injury (P = 0.02), and predictive probability for ND by PPG analysis at 15 min (P = 0.03) were independently associated with inpatient mortality. Conclusions: Analysis of variability and ECG or PPG waveform in the first minutes of resuscitation may represent a non-invasive early marker of future ND.

Intraosseous infusion rates under high pressure: a cadaveric comparison of anatomic sites.

BACKGROUND: When traditional vascular access methods fail, emergency access through the intraosseous (IO) route can be lifesaving. Fluids, medications, and blood components have all been delivered through these devices. We sought to compare the performance of IO devices placed in the sternum, humeral head, and proximal tibia using a fresh human cadaver model. METHODS: Commercially available IO infusion devices were placed into fresh human cadavers: sternum (FAST-1), humeral head (EZ-IO), and proximal tibia (EZ-IO). Sequentially, the volume of 0.9% saline infused into each site under 300 mm Hg pressure over 5 minutes was measured. Rates of successful initial IO device placement and subjective observations related to the devices were also recorded. RESULTS: For 16 cadavers over a 5-minute bolus infusion, the total volume of fluid infused at the three IO access sites was 469 (190) mL for the sternum, 286 (218) mL for the humerus, and 154 (94) mL for the tibia. Thus, the mean (SD) flow rate infused at each site was as follows: (1) sternum, 93.7 (37.9) mL/min; (2) humerus, 57.1 (43.5) mL/min; and (3) tibia, 30.7 (18.7) mL/min. The tibial site had the greatest number of insertion difficulties. CONCLUSION: This is the first study comparing the rate of flow at the three most clinically used adult IO infusion sites in an adult human cadaver model. Our results showed that the sternal site for IO access provided the most consistent and highest flow rate compared with the humeral and tibial insertion sites. The average flow rate in the sternum was 1.6 times greater than in the humerus and 3.1 times greater than in the tibia.

Phenotype, functions and fate of adoptively transferred tumor draining lymphocytes activated ex vivo in mice with an aggressive weakly immunogenic mammary carcinoma.

BACKGROUND: Regression of established tumors can be induced by adoptive immunotherapy with tumor draining lymph node lymphocytes activated with bryostatin and ionomycin. We hypothesized that tumor regression is mediated by a subset of the transferred T lymphocytes, which selectively infiltrate the tumor draining lymph nodes and proliferate in vivo. RESULTS: Adoptive transfer of B/I activated tumor draining lymphocytes induces regression of advanced 4T1 tumors, and depletion of CD8, but not CD4 T cells, abrogated tumor regression in mice. The predominant mediators of tumor regression are CD8+ and derived from CD62L- T cells. Transferred lymphocytes reached their peak concentration (10.5%) in the spleen 3 days after adoptive transfer and then rapidly declined. Adoptively transferred cells preferentially migrated to and/or proliferated in the tumor draining lymph nodes, peaking at day 5 (10.3%) and remained up to day 28. CFSE-stained cells were seen in tumors, also peaking at day 5 (2.1%). Bryostatin and ionomycin-activated cells proliferated vigorously in vivo, with 10 generations evident in the tumor draining lymph nodes on day 3. CFSE-stained cells found in the tumor draining lymph nodes on day 3 were 30% CD8+, 72% CD4+, 95% CD44+, and 39% CD69+. Pre-treatment of recipient mice with cyclophosphamide dramatically increased the number of interferon-gamma producing cells. CONCLUSIONS: Adoptively transferred CD8+ CD62L(low) T cells are the principal mediators of tumor regression, and host T cells are not required. These cells infiltrate 4T1 tumors, track preferentially to tumor draining lymph nodes, have an activated phenotype, and proliferate in vivo. Cyclophosphamide pre-treatment augments the anti-tumor effect by increasing the proliferation of interferon-gamma producing cells in the adoptive host.

Incubation of antigen-sensitized T lymphocytes activated with bryostatin 1 + ionomycin in IL-7 + IL-15 increases yield of cells capable of inducing regression of melanoma metastases compared to culture in IL-2.

Regression of established tumors can be induced by adoptive immunotherapy (AIT) with tumor draining lymph node (DLN) lymphocytes activated with bryostatin and ionomycin (B/I). We hypothesized that B/I-activated T cells cultured in IL-7 + IL-15 might proliferate and survive in culture better than cells cultured in IL-2, and that these cells would have equal or greater anti-tumor activity in vivo. Tumor antigen-sensitized DLN lymphocytes from either wild-type or T cell receptor transgenic mice were harvested, activated with B/I, and expanded in culture with either IL-2, IL-7 + IL-15 or a regimen of alternating cytokines. Cell yields, proliferation, apoptosis, phenotypes, and in vitro responses to tumor antigen were compared for cells grown in different cytokines. These T cells were also tested for anti-tumor activity against melanoma lung metastases established by prior i.v. injection of B16 melanoma cells. IL-7 + IL-15 or alternating cytokines resulted in much faster and prolonged proliferation and much less apoptosis of B/I-activated T cells than culturing the same cells in IL-2. This resulted in approximately tenfold greater yields of viable cells. Culture in IL-7 + IL-15 yielded higher proportions of CD8+ T cells and a higher proportion of cells with a central memory phenotype. Despite this, T cells grown in IL-7 + IL-15 had higher IFN-gamma release responses to tumor antigen than cells grown in IL-2. Adoptive transfer of B/I-activated T cells grown in IL-7 + IL-15 or the alternating regimen had equal or greater efficacy on a "per-cell" basis against melanoma metastases. Activation of tumor antigen-sensitized T cells with B/I and culture in IL-7 + IL-15 is a promising modification of standard regimens for production of T cells for use in adoptive immunotherapy of cancer.

Clinical Use of Interferon-gamma.

Interferon gamma (IFN-gamma), a pleotropic cytokine, has been shown to be important to the function of virtually all immune cells and both innate and adaptive immune responses. In 1986, early clinical trials of this cytokine began to evaluate its therapeutic potential. The initial studies focused on the tolerability and pharmacology of IFN-gamma and systematically determined its antitumor and anti-infection activities. In the 20-plus years since those first trials, IFN-gamma has been used in a wide variety of clinical indications, which are reviewed in this article.

Bryostatin 1/ionomycin (B/I) ex vivo stimulation preferentially activates L-selectinlow tumor-sensitized lymphocytes.

We have shown that tumor vaccine-sensitized draining lymph node (vDLN) cells activated ex vivo with bryostatin and ionomycin (B/I) were capable of inducing antigen-specific regression of a murine mammary tumor, 4T07. vDLN cells not activated with B/I were ineffective. We hypothesized that B/I selectively activates tumor-sensitized (CD62Llow) lymphocytes, to account for the highly potent and tumor-specific activity. We hypothesized that CD8+ CD62Llow cells may be preferentially activated by B/I treatment, infiltrate the tumors and mediate tumor regression in mice. 4T07-IL2 tumor cells were injected into one hind footpad of BALB/c mice. Ten days later, vDLN were harvested and separated based on CD62L expression. After separation, cells were activated with B/I, expanded with IL2 (40 IU/ml) for 10 days, and adoptively transferred to 4T07 tumor bearing mice. Naive mice were also treated with different subsets of T cells and later were challenged with 4T07 tumor cells. To test in vitro responses to antigen, expanded lymphocytes were cultured either alone or with irradiated 4T07 tumor cells. Supernatants were harvested after 24 h and tested by ELISA for IFN-gamma. The importance of the host immune response was tested by AIT into 4T07-bearing nude athymic mice. Host mice were depleted in vivo of CD4 or CD8 T cells after vDLN AIT to ascertain the mediators of tumor regression. In order to track B/I activated vDLN cells, they were prestained with CFSE prior to adoptive transfer into tumor-bearing hosts. At various time points, tumors, spleens and lymph nodes of host mice were harvested, dual stained for activation marker expression and analyzed by flow cytometry. CD62Llow cells expanded 12-fold more than CD62Lhigh lymphocytes during the 10 day culture period. Supernatant from CD62Llow cells + 4T07 cultures contained 33-fold more IFN-gamma than supernatant from CD62Lhigh cells + 4T07 cultures (843.9 pg/ml +/- 135.8 vs 25.89 pg/ml +/- 0.01). Adoptive transfer of CD62Llow lymphocytes induced complete tumor regressions in all mice, while tumors regressed in only 17% of mice treated with CD62Lhigh lymphocytes. Naive mice that received B/I-activated CD62Llow cells were protected from future tumor challenges, while mice given CD62Lhigh cells did not exhibit the same resistance to tumor growth. Tumors in nude host mice regressed after AIT treatment. In vivo depletion of CD4 T cells after AIT did not inhibit tumor regression, but CD8 T cell depletion abrogated tumor regression. vDLN cells tracked preferentially to tumor draining lymph nodes and proliferated in vivo, persisting for at least 21 days, and were 95% CD44+ and 39% CD69+. Bryostatin 1 and ionomycin, by increasing PKC activity and intracellular calcium, respectively, mimic intracellular signals that result in T cell activation. CD62Llow cells are preferentially activated by B/I, leading to a highly effective anti-tumor T cell population.