Dede...Immunotoxicology...is this part of the article?

[Guest guest]

Yes, Patty, it is, and I found the one that I copied

completely......thank you ! ! !

I wonder what the FDA did to that article.......I tried

to look up immunotoxicology on their revised site and

it is not there......and the old web page says it is no longer

available...

Makes ya wonder !

Again, thanks Patty ! !

Hugs

Dede

When the power of love overcomes the love of power

the world will know peace. ~ Jimi Hendrix

Love the life you live, live the life you love ~ Bob Marley **************A Good Credit Score is 700 or Above. See yours in just 2 easy steps! (http://pr.atwola.com/promoclk/100126575x1222585106x1201462830/aol?redir=http://www.freecreditreport.com/pm/default.aspx?sc=668072 & hmpgID=115 & bcd=JulystepsfooterNO115)

[Guest guest]

http://www.implants.clic.net/tony/Ant1/G/27.htmParfumGigi@... ParfumGigi@...

13 juin, 2006 23:30

IMMUNOTOXICOLOGY/TOXICOLOGY

.... Immunological Responses to Silicone Breast Implants. Key words: autoantibodies, silicone breast implants, immunopathology. The breast ...

www.fda.gov/cdrh/ost/reports/fy98/IMMUNOTOX.HTM - 06-20-2001 - Cached

Back to Table of Contents]

IMMUNOTOXICOLOGY/TOXICOLOGY

Latex-Associated Allergies: Latex Protein Assay Development and Allergen Identification

Key words: latex, allergy, protein measurement, Lowry method, round robin studies, ASTM D5712, ELISA development

Natural latex in medical devices may induce a Type 1

allergy which may be life threatening in individuals highly sensitized

to latex proteins. Although awareness of allergy to latex proteins has

increased in the last 5 years, the prevalence of latex sensitization is

increasing in the general population, as well as in health care

providers who use latex gloves in their occupations.

OST scientists are undertaking collaborative

research projects that are focused on reducing protein levels on

finished latex products and identifying allergenic proteins. One major

thrust involves OST participation in the ASTM effort to revise the

present ASTM D5712 Modified Lowry method for measurement of soluble

latex proteins. Variables such as pH, buffer composition, extraction

conditions, and sample clean-up methods in the testing lab were

evaluated in order to improve the assay for quantitation of soluble

latex proteins. The ongoing round robin studies are addressing these

results as it concerns reproducibility, sensitivity, and effects of

interfering substances. These questions are answered jointly by the FDA

and industry in order to establish a reliable and reproducible protein

assay for finished latex products, especially those with a "low

protein" claim.

Present laboratory and clinical studies include several in vitro and in vivo

approaches for evaluation of allergenic potential of latex products and

identification of latex-sensitized individuals. However, the clinical

relevance of various in vitro methods has not been established,

and the identity of all latex allergens has not been determined yet.

The purpose of this project was to evaluate the specificity of the

anti-latex IgE antibodies in human sera reacting with latex proteins

from various sources. These findings have been correlated with the

medical history of test subjects and specific exposure profiles. This

study revealed (a) the existence of a number of major allergenic

proteins that are present in various latex products, and ( that the

specificity of allergenic response depends on the type of product and

the pattern of exposure. While the profile of allergenic proteins

varies from product to product, it was assumed that all proteins that

may be allergenic are present in nonammoniated raw latex. Therefore,

the studies were extended to evaluate the relationship between total

protein content vs. allergen content, comparing raw latex extracts with

extracts of finished latex products.

Latex protein extracts from three major sources

(nonammoniated and ammoniated raw latex and latex products) were

evaluated for total protein content by Lowry assay. The level of

allergenic proteins was determined in vitro, using a pool of

human sera and compared to the intensity of skin reactions in

sensitized individuals. In clinical studies performed at the s

Hopkins University, skin testing was performed with these three

extracts. The intensity of skin reactions in each patient was the same

for all three extracts. These findings indicated that the total protein

level may be a reliable measure of the potential allergenicity of latex

products. To confirm this finding, OST scientists extended the study to

make similar comparisons on the wide range of extracts from finished

latex products by three in vitro tests. Protein extracts from

surgical and examination gloves and other latex products were evaluated

for: a) total protein levels by the Lowry assay, total antigen level

using an ELISA with rabbit anti-latex antiserum, and c) allergenic

protein levels using an ELISA with immune human sera.

The comparison of these three methods was intended

to reveal which one would be the most suitable predictor of potential

allergenicity of latex products. The initial results indicated that the

total protein values in the Lowry assay are 10- to 20-fold higher than

the total antigen values in ELISA test. A better correlation was

observed in the samples with high or very low protein levels.

Complement Activation

Key words: complement activation, cellulose acetate, dialysis, sepharose, protein-A, perfusion, research, standards.

The term complement describes a series of serum proteins involved in mediating immune reactions. Complement activation

is a tightly regulated process which, in addition to direct cell

cytolysis, can have profound affects on the immune, vascular, and

coagulation systems. Though complement activation is an important

defense mechanism of the host, particularly against microbial

infections, inappropriate activation (such as by implanted or external

medical devices which encounter human blood) may result in serious

acute or chronic reactions.

Examples of devices whose materials might activate

complement include perfusion devices, columns for treating blood

externally, indwelling artificial vascular grafts, encapsulated drugs

or cells, and vascular shunts. At the request of ODE, OST is conducting

research to acquire baseline information needed, in particular, for

industry standards concerning testing of materials to be used in

blood-contacting devices.

A microassay was developed for assaying

whole-complement depletion by solid materials used in the construction

of blood-contacting medical devices. This method has been submitted as

a draft Standard Practice to the ASTM Committee F-4 on Medical and

Surgical Materials and Devices via Subcommittee F04.16 on

Biocompatibility. Cellulose acetate fibers and powders used in the

manufacture of dialysis membranes were tested using this standard

practice. Although cellulose powder potently activated complement (57%

reduction from control levels when exposed to an equal volume of serum

for 1 hour at 37oC), there was no difference between

molecular weight 50,000 versus 30,000. Cellulose acetate fibers were

less potent than the precursor powders (only 19% reduction from control

levels), though storage time (from dates of manufacture) did not

produce significant differences between the fibers (9/96 versus 2/89.)

These results suggest that adverse patient reactions to dialysis by

materials could be related to complement activation, which might not be

influenced by molecular weight or age of degradation particles.

Sepharose is used to immobilize protein-A in

perfusion columns for removing antibodies from patient blood. Both raw

sepharose and sepharose-conjugated protein-A were tested for whole

complement depletion. Both resulted in a 75% or greater reduction in

whole complement activity versus controls. No significant difference

was seen between raw sepharose and sepharose-conjugated protein-A,

indicating that the majority of the complement activation was due to

the matrix, rather than to the attached protein-A. The activation of

complement by sepharose was by the alternative pathway (as documented

by Bb generation), rather than by the classical pathway (documented by

lack of C4d generation). Washing the sepharose with citrate buffers

(used in preparing columns for patient blood) resulted in a temporary

blockage of complement activation that was rapidly lost upon sequential

exposure to additional serum aliquots. These results indicate the

potential for complement activation to be a hazard and/or confounding

systemic modifier in therapeutic use of antibody-depleting columns.

Endocrine Disruption by Medical Device Materials

Key words: estrogen disruption, uterotrophic assay, heat shock proteins, bisphenol A

CDRH is concerned with the potential for certain

medical device materials to mimic or interfere with endogenous hormone

actions. Because the hormone estrogen is a potent molecule having

profound effects at remarkably low doses, assays are needed to assess

the potential for harm from materials that may induce unwanted effects

due to interference with normal estrogen homeostasis. OST scientists

are collaborating with researchers at the Department of Biology,

Washington University, on projects focused on improving the use of a

key biomarker of exposure to estrogenic compounds and determining the

characteristics of the estrogenic activity of bisphenol A, a

plasticizer found in some medical devices.

OST scientists are modifying and enhancing the

traditional assay for estrogenic effects. The traditional assay

utilizes the ability of estrogen to cause a hypertrophy of the uterus

in ovariectomized or immature female mice. The purpose of these

modifications is to provide mechanistic information that will reduce

the number of uncertainties in assessing risk from exposure to

estrogenic materials. OST scientists are developing an assay that

promises to do just that: obtain mechanistic information while

examining estrogenic responses in the whole animal. This is

accomplished by a side-by-side comparison of the traditional

uterotrophic assay and a specific cellular event, the induction of

stress proteins, that occurs when estrogen or estrogen-mimicking

materials bind to its specific cellular receptor.

Stress, or heat shock, proteins are synthesized

rapidly by most cells in response to various chemical and physical

stressors, especially heat. This response is thought to serve a

cellular protective function. Other functions have been found, and heat

shock proteins are called "chaperones" because they appear to associate

with other proteins and hold them in proper conformations. This appears

to be their function in relation to steroid hormone receptors: they

appear to hold the receptor molecule in an open position, so that the

receptor can bind to ligand easily. When the ligand binds, the heat

shock protein dissociates; the receptor is free to assume a new shape,

one that retains the bound ligand but which also enables the receptor

to bind the correct sequence on the genomic DNA, called the hormone

responsive element. Thus, OST has tried to identify changes in heat

shock proteins as a marker of estrogen receptor binding in tissues from

whole animals treated with estrogens.

OST scientists have shown that such changes can be

detected in a number of heat shock proteins in the uterus in response

to estrogen and that the response is specific to uterine and estrogenic

compounds. The effect of exposure time on changes in heat shock

proteins was assessed in ovariectomized mice treated with estradiol,

the primary estrogen form in the body. Increased relative uterus weight

increased linearly between the 4-hour , 8-hour, and 12-hour

post-exposure time points, and remained for 1 and 2 days. The

histopathology of the uterine lining showed a similar pattern. Changes

in the pattern of heat shock protein expression were observed between 6

and 12 hours post-treatment. Thus all three endpoints examined appear

at approximately the same time after treatment. This is consistent with

the idea that the heat shock protein changes are related specifically

to the estradiol effects.

Other steroid and steroid-like compounds were used

in order to examine the specificity of the response. The compounds

included an androgen, an androgen-receptor antagonist, a progestin, and

a partial estrogen. These compounds induced changes in heat shock

protein expression, but did not induce uterine swelling. This result

demonstrates that the heat shock protein changes are not the result of

the stresses to the cells caused by uterine swelling, and the heat

shock response may be more sensitive than the uterine response.

Compound ICI 182,780, a potent anti-estrogen, had no effect on uterine

weights and did not induce changes in heat shock proteins. However, the

highest dose of the ICI compound blocked the uterine and heat shock

protein effects of co-administered estradiol. Thus heat shock protein

induction: 1) is an estrogenic effect independent of uterine swelling;

and 2) is a more sensitive indicator of estrogenic effect than is

uterine swelling. The histological changes occur in parallel with the

uterine swelling effects.

The three endpoints (uterine hypertrophy, histology,

and stress protein expression, were used to examine the estrogenic

response of the plasticizer bisphenol A. Bisphenol A was determined to

be less estrogenic that estradiol (by more than 1000-fold), but caused

similar effects. Histological changes in the uterine epithelia were

observed at doses 40-fold lower than doses that caused uterine

swelling. Although responses were variable, heat shock protein

induction was also more sensitive than the uterotrophic response.

Immunological Responses to Silicone Breast Implants

Key words: autoantibodies, silicone breast implants, immunopathology

The breast implant experiments were accomplished in

two parts: first to demonstrate immune responses to silicone gel

implants in an animal model; and second, to apply the experimental

protocols to samples from women with/without breast implants. The

objective of these studies was to determine which autoimmune-like

symptoms and other symptoms are associated with implanted materials.

OST has developed a rat model to study and

understand immune responses to silicone gel and oil used in breast

implants. This model included normal rats and a strain of autoimmune

rats, that mimicked clinical conditions found in patients with silicone

gel breast implants. Mixtures of silicone gel/oil were injected into

the mammary area of female rats. Scientists then measured the levels of

autoantibodies to collagen and to nuclear proteins developed over 2

years in response to these mixtures.

Results from the rat model indicate that specific

autoantibodies may be induced by certain biomaterials. The immune

system recognizes a biomaterial-connective tissue protein association

as altered-self or as foreign. OST scientists have demonstrated that

medical grade silicone oil can stimulate serum autoantibodies against

collagen and against DNA when this oil is injected into mammary tissues

of rats. Autoantibody production against connective tissue proteins is

an immune response that is consistent with reports from women with

silicone breast implants. The results also demonstrated pathological

changes in animals that may result from the autoimmune response and

that silicone gel can migrate to distant anatomical sites or localize

at the implantation site. This work has been presented at The FDA

Science Forum, The American Association of Immunology, and The American

Association of Biochemistry and Molecular Biology.

The implication from these findings is that leaked

oil from a breast implant via leaching or with rupture might provide

stimulus for the production of autoantibodies in clinical patients.

Therefore, OST scientists evaluated autoantibody levels (in blinded

experiments) in serum samples from 150 patients representing four

groups: women with silicone implants without connective tissue disease,

women with silicone implants with connective tissue disease (CTD),

women with connective disease but no implants, and healthy women

volunteers. Results from these experiments show in a statistically

significant manner that elevated autoantibodies to collagen type I,

collagen type II and anti-DNA were detected in serum of patients with

CTD, CTD + silicone implants, and silicone implants without CTD.

Using two different assays, autoantibodies to

connective tissue proteins (e.g., collagen) and to DNA and intranuclear

proteins have been detected in women with silicone breast implants.

Historically, there is a strong correlation between anti-nuclear

antibodies and clinical symptoms of some autoimmune diseases. OST has

documented serum immune responses in these patients with the goal of

enhancing the ability to predict the likelihood of immunotoxic symptoms

occurring in the presence of implants, including breast prostheses.

This work has been presented to the Institute of Medicine.

OST scientists are continuing to investigate these

results by correlating this data to the clinical history of the

patients. Researchers plan to determine the clinical significance of

the data and to study a larger patient population well defined with

regard to implants and autoimmune disease symptoms.

Molecular Biomarkers for New Approaches to Safety Assessment: Studies with Mercury

Key words: mercury, stress proteins, kidney, preclinical test method development

An important part of FDA's mission is to facilitate

the development, refinement, and validation of more sensitive and

predictive preclinical methods in toxicology. One approach is to

develop technologies that define molecular biomarkers of exposure and

toxicity for ultimate use in preclinical safety evaluation and in risk

assessment activities. Prior to acceptance as a standard protocol in

preclinical and clinical safety assessment, these type of approaches

must be carefully validated with traditional standards and understood

mechanistically for risk assessment applications.

OST investigators are focusing on developing markers

at the molecular level because such targets are usually the first

responses induced by potentially hazardous materials. Ongoing studies

are evaluating the "stress", or heat shock, protein response as a

method that will more reliably predict potential adverse effects of

device materials.

Mercury is a major constituent of dental amalgam and

millions of teeth are filled annually with this material. Mercury,

which accumulates in kidney and brain tissues, is one of many

proteotoxicants that enhance the synthesis of heat shock proteins

(hsps) as part of a cellular defense mechanism. Recently, in a study

focused on expression of hsps in kidney in response to mercury

injections, OST investigators determined the differential expression of

hsps in rat renal cortex and medulla in response to mercuric chloride,

a readily soluble form of mercury. The five hsps evaluated were hsp90;

two members of the hsp70 family, the inducible hsp72 and the

constitutive hsp73; hsp25, and a glucose-regulated protein (grp94). In

whole kidney, mercury induced a time- and dose-related accumulation of

hsp72 and grp94. Interestingly, hsp72 accumulation was predominantly

localized in the cortex and not the medulla, while grp94 accumulated

primarily in the medulla but not the cortex. Mercury is toxic primarily

to the cells of the proximal tubules located in the cortex. These

results demonstrate that hsp expression in rat kidney exhibits regional

heterogeneity in response to mercury exposure. The study points out the

need to fully understand the expression of particular biomarkers in

various cell types and tissues if these new technologies are to be

incorporated as surrogates for, or adjuncts to, existing traditional

standard methods for safety assessment.

Particulate Effects on Immunologic Function

Key words: particles, cytokines, wear and degradation, macrophages, standards, research

Wear and corrosion of implanted medical devices,

such as dental and orthopedic prostheses, may produce particulate

debris which may lead to acute and chronic inflammatory responses in

the host. In addition, polymeric particles, such as

polytetrafluoroethylene (PTFE), may be injected directly into the

patients for clinical indications. When particulates are present, the

host monocytes/macrophages are activated and they synthesize or secrete

mediators of inflammation, and phagocytize particles. In order to

understand the mechanisms underlying the host immune response to

particulates and device-associated infections, OST scientists have

focused their studies on the impact of these particulates on macrophage

function. Macrophages play a pivotal role in the body's response to

foreign bodies and they also interact with other cellular components in

the immune system. OST developed an in vitro assay using

established murine macrophages. This assay system was incorporated into

an ASTM standard on the Biological Responses to Particles (F04.16.01).

The inflammatory potential of particles prepared from medical device

materials, such as PTFE, titanium oxide, hydroxyapatite (HA),

polymethylmethacrylate (PMMA), SiO2 and fumed silica, polystyrene (PS), CdCl2, CdO, Al2O3 , and diamond particle was studied.

In the in vitro assay, murine macrophage

cells were exposed to particles or chemicals with and without bacterial

lipopolysaccharide (LPS), which is a component of bacterial cell walls

that mimics bacterial infections. The cells were then evaluated for

cytotoxicity, production of nitric oxide (NO), tumor necrosis

factor-alpha (TNF-a ) and interleukin-6

(IL-6), both inflammatory cytokines. NO is induced by LPS and is

critically important in eradicating microorganisms associated with

infections, but it can also be harmful by causing tissue injury and

vascular collapse. OST studies showed that minute amounts of LPS, which

could be associated with a bacterial infection at the site of an

implanted device, induced a significant amount of TNF-a , IL-6 and NO production by macrophages.

The in vitro studies showed that TNF-a was induced by PTFE, PMMA, TiO2, HA, SiO2, both forms of cadmium, and Al2O3 particles

and by LPS. Polystyrene alone did not stimulate activity. PS in

combination with LPS stimulated no activity significantly above the

levels in response to LPS alone. Addition of particles to the

macrophages did not stimulate NO production. However, addition of LPS

to the particles affected the NO production. NO production increased in

a dose-response manner with LPS plus PMMA, increased but not in a

dose-dependent response with HA, and was inhibited by increasing

concentrations of TiO2. Polystyrene particles in combination with LPS produced minimal and variable differences of NO compared to LPS alone.

The in vitro system showed that IL-6 is stimulated by LPS. Neither Al2O3 nor PS alone stimulated IL-6 production. IL-6 production was stimulated by Al2O3 in combination with LPS, but PS in combination with LPS did not stimulate above the LPS alone level.

..

Thus far, the OST studies indicate the following: 1) this in vitro system measuring TNF-a

, IL-6 and NO responses can detect differences in biological responses

to particles; 2) no particle tested by OST thus far has induced NO

whereas LPS is a potent stimulator; 3) some particles stimulate TNF-a production and some do not; 4) some particles enhance, some inhibit, and some have no effect on the stimulation of TNF-a

and NO by macrophages in response to LPS; and 5) PS particles may serve

as a negative control for evaluating the induction of these three

biological responses.

Decontaminating Particles Exposed to Bacterial Lipopolysaccharde

Key words: particles, lipopolysaccharide, nitric oxide, macrophage, research

The presence of lipopolysaccharide (LPS) or

endotoxin associated with implanted medical devices can stimulate fever

in the host. Manufacturers must submit evidence that their sterilized

implanted devices are below a given endotoxin level. The most commonly

used assay to test for the presence of endotoxin is the Limulus

Amebocyte Lysate (LAL) test. OST scientists have observed, using an in vitro

murine macrophage assay, that LPS stimulates nitric oxide (NO)

production. Clean, sterilized medical device particles examined thus

far do not stimulate NO production, but certain particles that are

contaminated with minute amounts of lipopolysaccharide (LPS, endotoxin)

do stimulate NO production. Polymethylmethacrylate (PMMA) particles

deliberately contaminated with minute amounts of LPS were assayed for

the production of NO in the murine macrophage cell system. Treating

particles deliberately contaminated with LPS with 70% ethanol at room

temperature or higher for more than 24 hours followed by washing three

times with endotoxin-free phosphate buffered saline reduced the level

of detection of LPS in the in vitro cell system. LPS treated

with 70% ethanol also did not stimulate NO production. Both the LAL

test and the lack of LPS stimulation of NO production by murine

macrophages show that 70% ethanol can inactivate LPS and may be a

method to remove LPS from particles that are being tested for

inflammatory potential.

Allograft Heart Valves: The Role of Apoptosis-Mediated Cell Loss

Key words: allograft heart valve, apoptosis, histology

The objective of the study was to determine whether

apoptosis of endothelial and connective tissue cells is responsible for

the loss of cellularity observed in implanted aortic allograft valves.

The method involved retrieving fresh (n=6) and cryopreserved (n=4)

aortic allograft valves at 2 days to 20 weeks after implantation in an

ovine model. Sections of these valves were studied using histological

and electron microscopic methods, nick-end labeling and dual

immunostaining for Factor VIII-related antigen and proliferating cell

nuclear antigen (PCNA), followed by counterstaining for DNA (DAPI) and

laser scanning confocal fluorescence microscopic observation. Results

showed that the endothelial cells and cusp connective tissue

cells of implanted valvular allografts demonstrated loss of PCNA,

(indicative of cessation of mitotic activity), and evidence of

apoptosis (nick end labeling). The latter was manifested by nuclear

condensation and pyknosis, positive nick-end labeling and formation of

intra- and extracellular apoptotic bodies derived from the

fragmentation of apoptotic cells. These changes began to develop at 2

days after implantation, peaked at 10-14 days, and became complete by

20 weeks, at which time the valves had the typical acellular morphology

of allografts implanted for long periods of time. In conclusion,

apoptosis occurs in endothelial cells and cuspal connective tissue

cells of implanted allografts and appears to be a cause of their loss

of cellularity. This apoptosis may be related to various factors,

including immunologic and chemical injury, and hypoxia during valve

processing and reperfusion injury at the time of implantation.

[back to Table of Contents

Gigi-