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 induce acute graft-versus-host disease (GVHD) in lethally irradiated mice and evaluate the role of tumor necrosis factor (TNF) in cutaneous and intestinal lesions, with mortality and tissue damage as endpoints
This is a Graft-versus-Host Disease (GVHD) Induction Model protocol using mouse as the model organism. The procedure involves 17 procedural steps, 4 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 hybrids • 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 T-depleted bone marrow cells • Intravenous injection of peripheral T lymphocytes
Primary readouts
Key equipment and reagents
Verified items
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Recipient mice aged greater than 3 months old are exposed to whole-body irradiation from a Cesium source
Note: Dose is 800 rad, which is lethal without bone marrow reconstitution
“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 for hematopoietic reconstitution
Note: Mice receiving bone marrow cells alone serve as controls without GVHD
“injected intravenously with T depleted bone marrow cells (BMC), either supplemented with T lymphocytes prepared from lymph nodes (LN), or alone, as a control”
Lethally irradiated mice receiving bone marrow cells are also injected intravenously with peripheral T lymphocytes prepared from lymph nodes to induce GVHD
Note: 2 × 10^6 B10 LN cells are used as the standard dose; this leads to GVHD becoming clinically apparent around 2 weeks with >90% mortality by 80 days
“Lethally irradiated (B10 × CBA)F1 mice were injected with T-depleted BIO BMC and, unless mentioned otherwise, 2 × 10^6 B10 LN cells as a source of parental T cells”
Starting on day 8 post-transplantation (before clinical symptoms appear), mice are injected with either rabbit anti-TNF IgG or normal rabbit IgG as control
Note: Treatment begins before clinical symptoms (weight loss, hair ruffling) appear at approximately 2 weeks; injections continue weekly until day 35
“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”
Mice receive weekly intravenous or intraperitoneal injections of 2 mg anti-TNF IgG or normal rabbit IgG
Note: Anti-TNF activity remains detectable in serum of treated mice until day 35 with titers ranging from 1:30 to 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”
On days 16-18 post-transplantation, mice are killed and skin and intestinal tissues are collected for histological examination
Note: This timepoint was selected based on the course of disease and effects of anti-TNF treatment; represents the acute phase of GVHD
“On the 16-18th d, mice were killed to examine the skin and intestinal lesions of the acute phase of GVHD”
Skin and intestinal specimens are fixed in 2% formaldehyde/80% ethanol solution
Note: Specimens are then embedded in paraffin or methyl methacrylate for sectioning
“Specimens were fixed in 2% formaldehyde/80% ethanol, embedded with paraffin or methyl metacrylate”
Fixed and embedded tissues are sectioned at 5 µm and 1 µm thickness and stained with hematoxylin and eosin
Note: Sections are examined at various magnifications ( × 8, × 400, × 1000) using a light microscope with calibrated ocular
“5- and 1-µm sections were stained with hematoxylin and eosin”
Tissue specimens are fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer at pH 7.4
Note: This is the primary fixative for ultrastructural analysis
“For electron microscopy, specimens were fixed in 2.5% glutaraldehyde in cacodylate buffer, 0.1 M pH 7.4”
Glutaraldehyde-fixed tissues are processed into ultrathin sections, stained with uranyl acetate and lead citrate, and examined with an electron microscope
Note: Ultrastructural analysis identifies apoptotic changes in epithelial cells (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)”
Histological sections of skin are examined to quantify epidermal cell necrosis (ECN), lichenoid reactions (LR), epidermal atrophy, and loss of hypodermic fat
Note: Measurements include number of necrotic epidermal cells per microscopic field ( × 102), number of foci of atrophy and lichenoid reaction per 2-cm histologic section, and presence/absence of hypodermic fat
“Quantification of the various components of these lesions in the three groups of mice is shown in Table 1”
Histological sections of duodenum are examined to quantify duodenal circumference, villus length, crypt length, and number of crypt cell mitoses
Note: Measurements are made on thin (1 µm) sections examined at × 1000 magnification; lesions include dilatation, villus flattening, crypt elevation, and cell necrosis
“the quantification of these changes is shown in Table I”
Histological examination of skin and intestinal tissues to evaluate the degree of lymphoid cell infiltration in epidermis, lamina propria, and submucosa
Note: Anti-TNF treatment markedly decreases lymphoid cell infiltration in both tissues
“As can be seen by comparing Fig. 2, c and d, the anti-TNF treatment also markedly decreased lymphoid cell infiltration in the epidermis”
Mice are observed daily for survival and weighed at specified timepoints (day 18 and days 40, 80) to assess disease progression
Note: Weight loss is measured as percentage change from baseline; mortality curves are generated to compare treatment groups
“On day 18, normal rabbit IgG-injected GVHD mice showed a weight loss of 11% (4%, mean SD) compared with control mice without GVHD”
On day 18, spleens are harvested and total cell numbers are determined; structural organization is assessed histologically
Note: Cell recovery is compared between anti-TNF-treated and control IgG-treated GVHD mice; normal control mice have 8.6 × 10^7 spleen cells
“On day 18, the spleens of GVHD mice were smaller than those of controls, with a recovery of 2.3 and 3.6 × 10^7 cells for normal rabbit IgG- and anti-TNF-treated GVHD, respectively, compared with 8.6 × 10^7 for control mice”
Spleen cells from treated and control mice are cultured for 4 hours in the presence of tritiated thymidine ([³H]TdR) to measure DNA synthesis
Note: 5 × 10^6 spleen cells are used per culture; radioactivity is measured by scintillation counting to determine proliferative activity
“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”
In a separate experiment, mice receive a 7-day hypodermic perfusion of mouse TNF-α (4 µg/day) via osmotic minipump; anti-TNF IgG (2 mg) is injected intravenously or intraperitoneally to test prevention of TNF-induced cutaneous necrosis
Note: This validates that the anti-TNF antibody dose used in the main experiment is sufficient to neutralize TNF activity in vivo
“a single intravenous or intraperitoneal injection of 2 mg completely prevented the cutaneous necrosis induced by a 7-d hypodermic perfusion of a solution of mouse TNF-α delivered by an osmotic minipump (Alza, Palo Alto, CA) (4 µg/d)”
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 induce acute graft-versus-host disease (GVHD) in lethally irradiated mice and evaluate the role of tumor necrosis factor (TNF) in cutaneous and intestinal lesions, with mortality and tissue damage as endpoints
Objective
To induce acute graft-versus-host disease (GVHD) in lethally irradiated mice and evaluate the role of tumor necrosis factor (TNF) in cutaneous and intestinal lesions, with mortality and tissue damage as endpoints
Subjects
From papermouse • C57BL/10 (B10), CBA/ca, C57BL/6 (B6) H-2k, and (B10 × CBA)F1 hybrids • unknown • greater than 3 months old • not specified
Cohort notes
From paperC57BL/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 T-depleted bone marrow cells (not specified)
Intravenous injection of peripheral T lymphocytes (not specified)
Begin anti-TNF or control antibody treatment (not specified)
Mortality rate and survival curves (measured at days 40 and 80)
From paperResults are presented as means ± standard deviation (SD) of values observed in 6-9 individual mice per group.
Artifact type
Representative image panels with region or marker comparisons
Comparison focus
Compare staining intensity, structure, or cell counts across matched conditions
Body weight loss (percentage change from baseline, measured on day 18)
From paperResults are presented as means ± standard deviation (SD) of values observed in 6-9 individual mice per group.
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 (ECN) count, lichenoid reactions (LR), epidermal atrophy, loss of hypodermic fat
From paperResults are presented as means ± standard deviation (SD) of values observed in 6-9 individual mice per group.
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, crypt cell necrosis
From paperResults are presented as means ± standard deviation (SD) of values observed in 6-9 individual mice per group.
Artifact type
Representative image panels with region or marker comparisons
Comparison focus
Compare staining intensity, structure, or cell counts across matched conditions
Mortality rate and survival curves (measured 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 loss (percentage change from baseline, measured 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 (ECN) count, lichenoid reactions (LR), epidermal atrophy, loss of hypodermic fat
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, crypt 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 are presented as means ± standard deviation (SD) of values observed in 6-9 individual mice per group.
Scoring or quantification
Quantify the primary readouts for this experiment: Mortality rate and survival curves (measured at days 40 and 80); Body weight loss (percentage change from baseline, measured on day 18); Skin lesions: epidermal cell necrosis (ECN) count, lichenoid reactions (LR), epidermal atrophy, loss of hypodermic fat; Intestinal lesions: duodenal circumference, villus length, crypt length, crypt cell mitoses, crypt 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 and survival curves (measured at days 40 and 80), Body weight loss (percentage change from baseline, measured on day 18), Skin lesions: epidermal cell necrosis (ECN) count, lichenoid reactions (LR), epidermal atrophy, loss of hypodermic fat, Intestinal lesions: duodenal circumference, villus length, crypt length, crypt cell mitoses, crypt 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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