Source Paper
Tumor necrosis factor/cachectin is an effector of skin and gut lesions of the acute phase of graft-vs.-host disease.
P F Piguet, G E Grau, B Allet, P Vassalli
The Journal of Experimental Medicine • 1987
Source Paper
P F Piguet, G E Grau, B Allet, P Vassalli
The Journal of Experimental Medicine • 1987
Lethally irradiated mice were injected with semiallogeneic, T-depleted bone marrow cells and an amount of peripheral T lymphocytes sufficient to induce graft-vs.-host disease (GVHD) becoming apparent on the second week after the graft and leading to an increasing mortality rate within the following weeks (greater than 90% mortality within 80 d). Mice receiving bone marrow cells alone had no GVHD and were used as controls. Beginning on day 8, mice with GVHD were injected weekly with 2 mg of either rabbit anti-mouse recombinant tumor necrosis factor/cachectin (TNF-alpha) IgG, or normal rabbit IgG. On the 16-18th d, mice were killed to examine the skin and intestinal lesions of the acute phase of GVHD. The anti-TNF treatment resulted in an almost complete prevention of the severe lesions seen in the mice treated with normal rabbit IgG, i.e., the skin epidermal cell necrosis, foci of lichenoid hyperplastic reactions, and loss of the hypodermic fat; in the gut dilatation with marked flattening of the villi and elevation of the crypts, with increased numbers of mitoses and isolated crypt cell necrosis. In addition to preventing these acute lesions, anti-TNF treatment resulted in a significantly decreased mortality (approximately 70% survival at 80 d). These results suggest that during acute GVHD, the activation of grafted lymphocytes leads to a local release of TNF in the cutaneous and intestinal mucosae, which induces epithelial cell alterations and increases the inflammatory reaction.
Objective: To assess the effect of anti-TNF antibody treatment on mortality rates and tissue lesions in mice with graft-versus-host disease (GVHD) over an 80-day period
This is a Anti-TNF Treatment and Mortality Assessment protocol using mouse as the model organism. The procedure involves 17 procedural steps, 6 equipment items, 17 materials. Extracted from a 1987 paper published in The Journal of Experimental Medicine.
Model and subjects
mouse • C57BL/10 (B10), CBA/ca, C57BL/6 (B6) H-2k, and (B10 × CBA)F1 hybrid • unknown • greater than 3 months old • not specified
Study window
~11.4 week study window | ~8.1 hours hands-on
Core workflow
Lethal irradiation of recipient mice • Intravenous injection of bone marrow cells and T lymphocytes • Observation of GVHD development
Primary readouts
Key equipment and reagents
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Recipient mice older than 3 months are exposed to lethal irradiation from a Cesium source
Note: Dose: 800 rad
“Recipient mice >3 mo old, were irradiated by a Cesium source (800 rad, delivered during 2-3 min)”
Lethally irradiated mice are injected intravenously with T-depleted bone marrow cells, either supplemented with T lymphocytes from lymph nodes or alone as control
Note: T lymphocytes: 2 × 10^6 B10 LN cells; control group receives BMC alone
“Lethally irradiated mice were injected with semiallogeneic, T-depleted bone marrow cells and an amount of peripheral T lymphocytes sufficient to induce graft-vs.-host disease”
Monitor mice for clinical signs of GVHD including weight loss and hair ruffling
Note: GVHD becomes clinically apparent around day 14; mortality reaches ~60% within 40 days and >90% at 80 days
“Injection of this number of parental T cells leads to a GVHD that becomes clinically apparent (weight loss, hair ruffling) in about 2 wk”
Beginning on day 8 (before clinical symptoms), inject mice intravenously with either rabbit anti-TNF IgG or normal rabbit IgG control
Note: Day 8 is selected to treat before clinical symptoms appear; dose: 2 mg per injection
“Beginning on day 8, mice with GVHD were injected weekly with 2 mg of either rabbit anti-mouse recombinant tumor necrosis factor/cachectin (TNF-alpha) IgG, or normal rabbit IgG”
Continue weekly injections of the same antibody (anti-TNF or control IgG) at 2 mg per injection
Note: Anti-TNF activity remains detectable in serum until day 35 with titers ranging 1:30-1:200
“After this first injection, mice were reinjected weekly with the same amounts of IgG until day 35; anti-TNF activity was detectable in the serum of treated mice until day 35 with titers ranging between 1:30-1:200”
Measure body weight of mice on day 18 to assess weight loss as indicator of GVHD severity
Note: Normal IgG-treated GVHD mice show 11% weight loss (±4% SD) compared to controls; anti-TNF-treated mice show no significant weight difference from controls
“On day 18, normal rabbit IgG-injected GVHD mice showed a weight loss of 11% (4%, mean SD) compared with control mice”
On days 16-18, euthanize mice and collect skin and intestinal tissue specimens for histological examination
Note: Days 16-18 selected for histologic study based on disease course; tissues examined for acute phase GVHD lesions
“On the 16-18th d, mice were killed to examine the skin and intestinal lesions of the acute phase of GVHD”
Fix tissue specimens in 2% formaldehyde/80% ethanol solution
Note: Specimens prepared for paraffin or methyl methacrylate embedding
“Specimens were fixed in 2% formaldehyde/80% ethanol, embedded with paraffin or methyl metacrylate”
Prepare 5-µm and 1-µm tissue sections and stain with hematoxylin and eosin
Note: 1-µm sections used for detailed examination of crypt cell mitoses at 1,000 × magnification
“5- and 1-µm sections were stained with hematoxylin and eosin”
Fix tissue specimens in 2.5% glutaraldehyde in 0.1 M cacodylate buffer at pH 7.4
Note: Preparation for ultrastructural examination of epithelial cell necrosis
“For electron microscopy, specimens were fixed in 2.5% glutaraldehyde in cacodylate buffer, 0.1 M pH 7.4”
Prepare ultrathin sections, stain with uranyl acetate and lead citrate, and examine with Philips 400 electron microscope
Note: Examination reveals apoptotic changes in epithelial cells including nuclear chromatin condensation and cell compaction
“ultrathin sections were stained with uranyl acetate and lead citrate, and examined with an electron microscope (Philips 400; Zurich, Switzerland)”
On day 18, collect spleens from GVHD mice and recover total spleen cells
Note: Anti-TNF-treated mice: 3.6 × 10^7 cells; normal IgG-treated: 2.3 × 10^7 cells; controls: 8.6 × 10^7 cells
“On day 18, the spleens of GVHD mice were smaller than those of controls, with a recovery of 2.3 and 3.6 X 10^7 cells”
Culture 5 × 10^6 spleen cells for 4 hours in presence of [3H]TdR to measure donor T lymphocyte proliferation
Note: Performed on day 5 after lethal irradiation and cell transfer; measures proliferative response without bone marrow reconstitution
“5 × 10^6 spleen cells were cultured for 4 h in the presence of [3H]TdR, and the culture was then processed for scintillation counting”
Process cultured cells for scintillation counting to quantify [3H]TdR incorporation
Note: Measures DNA synthesis as indicator of T cell proliferation
“the culture was then processed for scintillation counting”
Monitor and record survival of mice throughout 80-day observation period
Note: Anti-TNF treatment results in ~70% survival at days 40 and 80; normal IgG treatment shows >90% mortality at 80 days
“Anti-TNF treatment resulted in a decrease in mortality, with a survival rate of ~70% at days 40 and 80”
Count and measure skin lesion components including epidermal cell necrosis (ECN), lichenoid reactions (LR), hypodermic fat presence, and lymphoid infiltration
Note: Measurements performed on days 16-18; anti-TNF treatment markedly decreases all lesion parameters
“Quantification of the various components of these lesions in the three groups of mice is shown in Table 1”
Measure duodenal circumference, villus length, crypt length, and count crypt cell mitoses in intestinal tissue sections
Note: Measurements on 1-µm sections at 1,000 × magnification; anti-TNF treatment prevents intestinal lesions
“the quantification of these changes is shown in Table I”
This section explains what the experiment is doing, which readouts matter, what the data artifacts usually look like, and how the analysis should flow from raw capture to reported result.
To assess the effect of anti-TNF antibody treatment on mortality rates and tissue lesions in mice with graft-versus-host disease (GVHD) over an 80-day period
Objective
To assess the effect of anti-TNF antibody treatment on mortality rates and tissue lesions in mice with graft-versus-host disease (GVHD) over an 80-day period
Subjects
From papermouse • C57BL/10 (B10), CBA/ca, C57BL/6 (B6) H-2k, and (B10 × CBA)F1 hybrid • unknown • greater than 3 months old • not specified
Cohort notes
From paperLethally irradiated recipient mice; C57BL/10 and CBA/ca mice purchased from Olac Ltd, Bicester, United Kingdom; C57BL/6 H-2k mice purchased from Memorial Sloan-Kettering Cancer Center, New York
Lethal irradiation of recipient mice (2-3 minutes)
Intravenous injection of bone marrow cells and T lymphocytes (not specified)
Observation of GVHD development (approximately 2 weeks until clinical symptoms appear)
First antibody injection (not specified)
Mortality rate (percentage survival at days 40 and 80)
From paperResults expressed as means ± SD; statistical significance determined by comparison between groups with P values reported (P < 0.05, P < 0.01, P < 0.1); quantification of lesion components performed on 6-9 individual mice per group on days 16-18
Artifact type
Representative image panels with region or marker comparisons
Comparison focus
Compare staining intensity, structure, or cell counts across matched conditions
Body weight change (percentage weight loss on day 18)
From paperResults expressed as means ± SD; statistical significance determined by comparison between groups with P values reported (P < 0.05, P < 0.01, P < 0.1); quantification of lesion components performed on 6-9 individual mice per group on days 16-18
Artifact type
Representative image panels with region or marker comparisons
Comparison focus
Compare staining intensity, structure, or cell counts across matched conditions
Skin lesions: epidermal cell necrosis count, lichenoid reaction foci, hypodermic fat presence, lymphoid infiltration
From paperResults expressed as means ± SD; statistical significance determined by comparison between groups with P values reported (P < 0.05, P < 0.01, P < 0.1); quantification of lesion components performed on 6-9 individual mice per group on days 16-18
Artifact type
Representative image panels with region or marker comparisons
Comparison focus
Compare staining intensity, structure, or cell counts across matched conditions
Intestinal lesions: duodenal circumference, villus length, crypt length, crypt cell mitoses count, epithelial cell necrosis
From paperResults expressed as means ± SD; statistical significance determined by comparison between groups with P values reported (P < 0.05, P < 0.01, P < 0.1); quantification of lesion components performed on 6-9 individual mice per group on days 16-18
Artifact type
Representative image panels with region or marker comparisons
Comparison focus
Compare staining intensity, structure, or cell counts across matched conditions
Mortality rate (percentage survival at days 40 and 80)
From paperRaw artifact
Field or section images captured from matched samples
Processed artifact
Selected representative panels with quantified intensity, counts, or area measurements
Final reported form
Per-group imaging summaries with representative figures and quantified endpoints
Body weight change (percentage weight loss on day 18)
From paperRaw artifact
Field or section images captured from matched samples
Processed artifact
Selected representative panels with quantified intensity, counts, or area measurements
Final reported form
Per-group imaging summaries with representative figures and quantified endpoints
Skin lesions: epidermal cell necrosis count, lichenoid reaction foci, hypodermic fat presence, lymphoid infiltration
From paperRaw artifact
Field or section images captured from matched samples
Processed artifact
Selected representative panels with quantified intensity, counts, or area measurements
Final reported form
Per-group imaging summaries with representative figures and quantified endpoints
Intestinal lesions: duodenal circumference, villus length, crypt length, crypt cell mitoses count, epithelial cell necrosis
From paperRaw artifact
Field or section images captured from matched samples
Processed artifact
Selected representative panels with quantified intensity, counts, or area measurements
Final reported form
Per-group imaging summaries with representative figures and quantified endpoints
Acquisition
Capture matched images from the relevant tissue region using the same acquisition settings across samples.
Preprocessing / cleaning
Results expressed as means ± SD; statistical significance determined by comparison between groups with P values reported (P < 0.05, P < 0.01, P < 0.1); quantification of lesion components performed on 6-9 individual mice per group on days 16-18
Scoring or quantification
Quantify the primary readouts for this experiment: Mortality rate (percentage survival at days 40 and 80); Body weight change (percentage weight loss on day 18); Skin lesions: epidermal cell necrosis count, lichenoid reaction foci, hypodermic fat presence, lymphoid infiltration; Intestinal lesions: duodenal circumference, villus length, crypt length, crypt cell mitoses count, epithelial cell necrosis.
Normalization
Normalize image-derived measurements against the matched acquisition or segmentation rules before comparing groups.
Statistical comparison
Statistical method not yet structured for this page.
Reporting output
Report representative outputs alongside summary comparisons for Mortality rate (percentage survival at days 40 and 80), Body weight change (percentage weight loss on day 18), Skin lesions: epidermal cell necrosis count, lichenoid reaction foci, hypodermic fat presence, lymphoid infiltration, Intestinal lesions: duodenal circumference, villus length, crypt length, crypt cell mitoses count, epithelial cell necrosis.
Source links and direct wording from the methods section for validation and deeper review.
Citation
P F Piguet et al. (1987). Tumor necrosis factor/cachectin is an effector of skin and gut lesions of the acute phase of graft-vs.-host disease.. The Journal of Experimental Medicine
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