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websearch for: Key words: autoantibodies, silicone

breast implants, immunopathology

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 potentialallergenicity 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. t

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 withestrogens.

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 areassociated 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

diamondparticle 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 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.

[Guest guest]

Yes it is interesting. Thank you Rogene....

>

> websearch for: Key words: autoantibodies, silicone

> breast implants, immunopathology

>

>

> 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 potentialallergenicity 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. t

> 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 withestrogens.

>

> 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 areassociated 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

> diamondparticle 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 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.

>

[Guest guest]

Maybe Sherry will find the section " Immunological

Responses to Silicone Breast Implants " meets her

standard of proof.

Rogene

--- sedwards41255 <sedwards41255@...> wrote:

> Yes it is interesting. Thank you Rogene....

>