Fluorescent‐activated cell‐sorting analysis of intracellular interferon‐γ and interleukin‐4 in fresh and frozen human peripheral blood T‐helper cells
Bibliographic record
Abstract
T-helper (Th) cells can be classified into at least three subsets based on their cytokine profiles: Th0, Th1, and Th2. The functional significance of each subset of Th cells can be determined in isolated human peripheral blood mononuclear cells (PBMC). Using two- or three-color cytometric detection of intracellular cytokines. These analyses have been limited by the requirement for fresh cells making sequential samples and longitudinal studies difficult. Cryopreservation of PBMC in liquid nitrogen for up to 1 year was evaluated to determine whether the Th1/Th2 ratio remained unchanged in cryopreserved lymphocytes. Aliquots of human PBMC from normal volunteers analyzed for activation using phorbol myristate acetate and evaluated using morphology showed that the surface marker expression was unchanged in fresh and frozen cells. Cytokine expression was measured using intracellular cytokine staining and three-color flow cytometric analysis. The percentages of cells producing interferon (IFN)-gamma or interleukin (IL)-4 were determined after 16 hours of phorbol myristate acetate and ionomycin stimulation in the presence of brefeldin A. No significant difference was found in cytokine production between fresh and frozen cells. The percentage of IFN-gamma and IL-4 producing CD3-positive fresh T cells was 19.2+/-5.8 percent and 0.9+/-0.4 percent vs. 17.6+/-0.75 percent and 0.9+/-0.3 percent, respectively, for frozen PBMC. The effects of thermal injury on the Th1/Th2 cytokine ratio and the development of hypertrophic scarring were then determined. Twelve burn patients examined 4 weeks postburn showed a significant shift in the Th1/Th2 ratio, compared with 13 normal human volunteers used as controls. IL-4 levels in the patient group were significantly higher than controls at 1 month postburn (12.7+/-2.6 percent vs. 3.9+/-0.5 percent, p<0.01) and IFN-gamma levels were significantly lower (9.3+/-1.7 percent vs. 15.3+/-2.3 percent, p<0.05). Thus, PBMC can be cryopreserved for up to 1 year, enabling investigation of chronologic changes in Th1/Th2 profiles. It is suggested that a "locked on" Th2 profile may contribute to the development of hypertrophic scarring after burn injury.
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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one teacher head, not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".