Anti-TNF Treatment and Mortality Assessment

1

Lethal irradiation of recipient mice

Recipient mice older than 3 months are exposed to lethal irradiation from a Cesium source

2-3 minutesnot specified

Note: Dose: 800 rad

View evidence from paper

“Recipient mice >3 mo old, were irradiated by a Cesium source (800 rad, delivered during 2-3 min)”

2

Intravenous injection of bone marrow cells and T lymphocytes

Lethally irradiated mice are injected intravenously with T-depleted bone marrow cells, either supplemented with T lymphocytes from lymph nodes or alone as control

not specifiednot specified

Note: T lymphocytes: 2 × 10^6 B10 LN cells; control group receives BMC alone

View evidence from paper

“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”

3

Observation of GVHD development

Monitor mice for clinical signs of GVHD including weight loss and hair ruffling

approximately 2 weeks until clinical symptoms appearnot specified

Note: GVHD becomes clinically apparent around day 14; mortality reaches ~60% within 40 days and >90% at 80 days

View evidence from paper

“Injection of this number of parental T cells leads to a GVHD that becomes clinically apparent (weight loss, hair ruffling) in about 2 wk”

4

First antibody injection

Beginning on day 8 (before clinical symptoms), inject mice intravenously with either rabbit anti-TNF IgG or normal rabbit IgG control

not specifiednot specified

Note: Day 8 is selected to treat before clinical symptoms appear; dose: 2 mg per injection

View evidence from paper

“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”

5

Weekly antibody injections

Continue weekly injections of the same antibody (anti-TNF or control IgG) at 2 mg per injection

from day 8 until day 35not specified

Note: Anti-TNF activity remains detectable in serum until day 35 with titers ranging 1:30-1:200

View evidence from paper

“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”

6

Weight measurement

Measure body weight of mice on day 18 to assess weight loss as indicator of GVHD severity

not specifiednot specified

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

View evidence from paper

“On day 18, normal rabbit IgG-injected GVHD mice showed a weight loss of 11% (4%, mean SD) compared with control mice”

7

Tissue collection for histology

On days 16-18, euthanize mice and collect skin and intestinal tissue specimens for histological examination

not specifiednot specified

Note: Days 16-18 selected for histologic study based on disease course; tissues examined for acute phase GVHD lesions

View evidence from paper

“On the 16-18th d, mice were killed to examine the skin and intestinal lesions of the acute phase of GVHD”

8

Tissue fixation for light microscopy

Fix tissue specimens in 2% formaldehyde/80% ethanol solution

not specifiednot specified

Note: Specimens prepared for paraffin or methyl methacrylate embedding

View evidence from paper

“Specimens were fixed in 2% formaldehyde/80% ethanol, embedded with paraffin or methyl metacrylate”

9

Tissue sectioning and staining for light microscopy

Prepare 5-μm and 1-μm tissue sections and stain with hematoxylin and eosin

not specifiednot specified

Note: 1-μm sections used for detailed examination of crypt cell mitoses at 1,000× magnification

View evidence from paper

“5- and 1-μm sections were stained with hematoxylin and eosin”

10

Tissue fixation for electron microscopy

Fix tissue specimens in 2.5% glutaraldehyde in 0.1 M cacodylate buffer at pH 7.4

not specifiednot specified

Note: Preparation for ultrastructural examination of epithelial cell necrosis

View evidence from paper

“For electron microscopy, specimens were fixed in 2.5% glutaraldehyde in cacodylate buffer, 0.1 M pH 7.4”

11

Electron microscopy sample preparation and examination

Prepare ultrathin sections, stain with uranyl acetate and lead citrate, and examine with Philips 400 electron microscope

not specifiednot specified

Note: Examination reveals apoptotic changes in epithelial cells including nuclear chromatin condensation and cell compaction

View evidence from paper

“ultrathin sections were stained with uranyl acetate and lead citrate, and examined with an electron microscope (Philips 400; Zurich, Switzerland)”

12

Spleen collection and cell recovery

On day 18, collect spleens from GVHD mice and recover total spleen cells

not specifiednot specified

Note: Anti-TNF-treated mice: 3.6 × 10^7 cells; normal IgG-treated: 2.3 × 10^7 cells; controls: 8.6 × 10^7 cells

View evidence from paper

“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”

13

T cell proliferation assay

Culture 5 × 10^6 spleen cells for 4 hours in presence of [3H]TdR to measure donor T lymphocyte proliferation

4 hoursnot specified

Note: Performed on day 5 after lethal irradiation and cell transfer; measures proliferative response without bone marrow reconstitution

View evidence from paper

“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”

14

Radioactivity measurement

Process cultured cells for scintillation counting to quantify [3H]TdR incorporation

not specifiednot specified

Note: Measures DNA synthesis as indicator of T cell proliferation

View evidence from paper

“the culture was then processed for scintillation counting”

15

Mortality monitoring

Monitor and record survival of mice throughout 80-day observation period

80 daysnot specified

Note: Anti-TNF treatment results in ~70% survival at days 40 and 80; normal IgG treatment shows >90% mortality at 80 days

View evidence from paper

“Anti-TNF treatment resulted in a decrease in mortality, with a survival rate of ~70% at days 40 and 80”

16

Histological quantification of skin lesions

Count and measure skin lesion components including epidermal cell necrosis (ECN), lichenoid reactions (LR), hypodermic fat presence, and lymphoid infiltration

not specifiednot specified

Note: Measurements performed on days 16-18; anti-TNF treatment markedly decreases all lesion parameters

View evidence from paper

“Quantification of the various components of these lesions in the three groups of mice is shown in Table 1”

17

Histological quantification of intestinal lesions

Measure duodenal circumference, villus length, crypt length, and count crypt cell mitoses in intestinal tissue sections

not specifiednot specified

Note: Measurements on 1-μm sections at 1,000× magnification; anti-TNF treatment prevents intestinal lesions

View evidence from paper

“the quantification of these changes is shown in Table I”

Tumor necrosis factor/cachectin is an effector of skin and gut lesions of the acute phase of graft-vs.-host disease.

Equipment6

Materials17

Software1

Protocol Steps

Lethal irradiation of recipient mice

Intravenous injection of bone marrow cells and T lymphocytes

Observation of GVHD development

First antibody injection

Weekly antibody injections

Weight measurement

Tissue collection for histology

Tissue fixation for light microscopy

Tissue sectioning and staining for light microscopy

Tissue fixation for electron microscopy

Electron microscopy sample preparation and examination

Spleen collection and cell recovery

T cell proliferation assay

Radioactivity measurement

Mortality monitoring

Histological quantification of skin lesions

Histological quantification of intestinal lesions