1) The study demonstrates that TLR-4 is preferentially expressed by macrophages in murine and human lipid-rich atherosclerotic lesions.
2) In both mouse and human plaques, TLR-4 expression colocalized with macrophage staining.
3) In vitro, oxidized LDL upregulated TLR-4 mRNA expression in human macrophages in a dose-dependent manner, whereas native LDL had no effect. This suggests TLR-4 may provide a link between lipids and inflammation in atherosclerosis.
The Influence of Various Drags on Mortality of Mice and the Concentration of ...CrimsonPublishersAICS
The Influence of Various Drags on Mortality of Mice and the Concentration of Proinflammatory Cytokines in Blood at Sepsis Caused by E. Coli by Zabrodskii PF* in Advancements in Case Studies
The Influence of Various Drags on Mortality of Mice and the Concentration of ...CrimsonPublishersAICS
The Influence of Various Drags on Mortality of Mice and the Concentration of Proinflammatory Cytokines in Blood at Sepsis Caused by E. Coli by Zabrodskii PF* in Advancements in Case Studies
“A Chimeric Human-Mouse Model of Sjögren's Syndrome” Nicholas Young
My talk from The Federation of Clinical Immunology Societies (FOCIS) annual meeting June 24-28, 2014 in Chicago, IL: “A Chimeric Human-Mouse Model of Sjögren's Syndrome”. Recipient of a travel award given to selected abstracts to attend this conference.
Monocytosis and Angiotensin II-Induced HypertensionKimberly Williams
A Christian’s relationship with God is vital to a Christian walk but other relationships and friendships require a reflection on the Christians personal walk with the superior being.
Summary on biblical friendships
Relationships gain more strength through proximity. The physical presence of friends is a source of joy and strength to a Christian. Distance makes friendship more difficult. For example, according to II John 12, John knew that he would be happier if he went to his own people and speak to them face to face (Dietrich B. 19).
“A Chimeric Human-Mouse Model of Sjögren's Syndrome” Nicholas Young
My talk from The Federation of Clinical Immunology Societies (FOCIS) annual meeting June 24-28, 2014 in Chicago, IL: “A Chimeric Human-Mouse Model of Sjögren's Syndrome”. Recipient of a travel award given to selected abstracts to attend this conference.
Monocytosis and Angiotensin II-Induced HypertensionKimberly Williams
A Christian’s relationship with God is vital to a Christian walk but other relationships and friendships require a reflection on the Christians personal walk with the superior being.
Summary on biblical friendships
Relationships gain more strength through proximity. The physical presence of friends is a source of joy and strength to a Christian. Distance makes friendship more difficult. For example, according to II John 12, John knew that he would be happier if he went to his own people and speak to them face to face (Dietrich B. 19).
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The role of surface charge of ISCOMATRIX nanoparticles on the type of immune ...Nanomedicine Journal (NMJ)
Abstract
Objective(s):
ISCOMATRIX vaccines have now been shown to induce strong antigen-specific cellular or humoral immune responses to a broad range of antigens of viral, bacterial, parasite or tumor. In the present study, we investigated the role of ISCOMATRIX charge in induction of a Th1 type of immune response and protection against Leishmania major infection in BALB/c mice.
Materials and Methods:
Positively and negatively charged ISCOMATRIX were prepared. BALB/C mice were immunized subcutaneously, three times with 2-week intervals, with different ISCOMATRIX formulations. Soluble Leishmania antigens (SLA) were mixed with ISCOMATRIX right before injection. The extent of protection and type of immune response were studied in different groups of mice.
Results:
The group of mice immunized with negatively charged ISCOMATRIX showed smaller footpad swelling upon challenge with L. major and the highest IgG2a production compared with positively charged one. The mice immunized with positively charged ISCOMATRIX showed the lowest splenic parasite burden compared to the other groups. Cytokine assay results indicated that the highest level of IFN- γ and IL-4 secretion was observed in the splenocytes of mice immunized with negatively charged ISCOMATRIX as compared to other groups.
Conclusion:
The results indicated that ISCOMATRIX formulations generate an immune response with mixed Th1/Th2 response that was not protective against challenge against L. major.
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Lung Cancer: Artificial Intelligence, Synergetics, Complex System Analysis, S...Oleg Kshivets
RESULTS: Overall life span (LS) was 2252.1±1742.5 days and cumulative 5-year survival (5YS) reached 73.2%, 10 years – 64.8%, 20 years – 42.5%. 513 LCP lived more than 5 years (LS=3124.6±1525.6 days), 148 LCP – more than 10 years (LS=5054.4±1504.1 days).199 LCP died because of LC (LS=562.7±374.5 days). 5YS of LCP after bi/lobectomies was significantly superior in comparison with LCP after pneumonectomies (78.1% vs.63.7%, P=0.00001 by log-rank test). AT significantly improved 5YS (66.3% vs. 34.8%) (P=0.00000 by log-rank test) only for LCP with N1-2. Cox modeling displayed that 5YS of LCP significantly depended on: phase transition (PT) early-invasive LC in terms of synergetics, PT N0—N12, cell ratio factors (ratio between cancer cells- CC and blood cells subpopulations), G1-3, histology, glucose, AT, blood cell circuit, prothrombin index, heparin tolerance, recalcification time (P=0.000-0.038). Neural networks, genetic algorithm selection and bootstrap simulation revealed relationships between 5YS and PT early-invasive LC (rank=1), PT N0—N12 (rank=2), thrombocytes/CC (3), erythrocytes/CC (4), eosinophils/CC (5), healthy cells/CC (6), lymphocytes/CC (7), segmented neutrophils/CC (8), stick neutrophils/CC (9), monocytes/CC (10); leucocytes/CC (11). Correct prediction of 5YS was 100% by neural networks computing (area under ROC curve=1.0; error=0.0).
CONCLUSIONS: 5YS of LCP after radical procedures significantly depended on: 1) PT early-invasive cancer; 2) PT N0--N12; 3) cell ratio factors; 4) blood cell circuit; 5) biochemical factors; 6) hemostasis system; 7) AT; 8) LC characteristics; 9) LC cell dynamics; 10) surgery type: lobectomy/pneumonectomy; 11) anthropometric data. Optimal diagnosis and treatment strategies for LC are: 1) screening and early detection of LC; 2) availability of experienced thoracic surgeons because of complexity of radical procedures; 3) aggressive en block surgery and adequate lymph node dissection for completeness; 4) precise prediction; 5) adjuvant chemoimmunoradiotherapy for LCP with unfavorable prognosis.
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Title: Sense of Smell
Presenter: Dr. Faiza, Assistant Professor of Physiology
Qualifications:
MBBS (Best Graduate, AIMC Lahore)
FCPS Physiology
ICMT, CHPE, DHPE (STMU)
MPH (GC University, Faisalabad)
MBA (Virtual University of Pakistan)
Learning Objectives:
Describe the primary categories of smells and the concept of odor blindness.
Explain the structure and location of the olfactory membrane and mucosa, including the types and roles of cells involved in olfaction.
Describe the pathway and mechanisms of olfactory signal transmission from the olfactory receptors to the brain.
Illustrate the biochemical cascade triggered by odorant binding to olfactory receptors, including the role of G-proteins and second messengers in generating an action potential.
Identify different types of olfactory disorders such as anosmia, hyposmia, hyperosmia, and dysosmia, including their potential causes.
Key Topics:
Olfactory Genes:
3% of the human genome accounts for olfactory genes.
400 genes for odorant receptors.
Olfactory Membrane:
Located in the superior part of the nasal cavity.
Medially: Folds downward along the superior septum.
Laterally: Folds over the superior turbinate and upper surface of the middle turbinate.
Total surface area: 5-10 square centimeters.
Olfactory Mucosa:
Olfactory Cells: Bipolar nerve cells derived from the CNS (100 million), with 4-25 olfactory cilia per cell.
Sustentacular Cells: Produce mucus and maintain ionic and molecular environment.
Basal Cells: Replace worn-out olfactory cells with an average lifespan of 1-2 months.
Bowman’s Gland: Secretes mucus.
Stimulation of Olfactory Cells:
Odorant dissolves in mucus and attaches to receptors on olfactory cilia.
Involves a cascade effect through G-proteins and second messengers, leading to depolarization and action potential generation in the olfactory nerve.
Quality of a Good Odorant:
Small (3-20 Carbon atoms), volatile, water-soluble, and lipid-soluble.
Facilitated by odorant-binding proteins in mucus.
Membrane Potential and Action Potential:
Resting membrane potential: -55mV.
Action potential frequency in the olfactory nerve increases with odorant strength.
Adaptation Towards the Sense of Smell:
Rapid adaptation within the first second, with further slow adaptation.
Psychological adaptation greater than receptor adaptation, involving feedback inhibition from the central nervous system.
Primary Sensations of Smell:
Camphoraceous, Musky, Floral, Pepperminty, Ethereal, Pungent, Putrid.
Odor Detection Threshold:
Examples: Hydrogen sulfide (0.0005 ppm), Methyl-mercaptan (0.002 ppm).
Some toxic substances are odorless at lethal concentrations.
Characteristics of Smell:
Odor blindness for single substances due to lack of appropriate receptor protein.
Behavioral and emotional influences of smell.
Transmission of Olfactory Signals:
From olfactory cells to glomeruli in the olfactory bulb, involving lateral inhibition.
Primitive, less old, and new olfactory systems with different path
- Video recording of this lecture in English language: https://youtu.be/lK81BzxMqdo
- Video recording of this lecture in Arabic language: https://youtu.be/Ve4P0COk9OI
- Link to download the book free: https://nephrotube.blogspot.com/p/nephrotube-nephrology-books.html
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Title: Sense of Taste
Presenter: Dr. Faiza, Assistant Professor of Physiology
Qualifications:
MBBS (Best Graduate, AIMC Lahore)
FCPS Physiology
ICMT, CHPE, DHPE (STMU)
MPH (GC University, Faisalabad)
MBA (Virtual University of Pakistan)
Learning Objectives:
Describe the structure and function of taste buds.
Describe the relationship between the taste threshold and taste index of common substances.
Explain the chemical basis and signal transduction of taste perception for each type of primary taste sensation.
Recognize different abnormalities of taste perception and their causes.
Key Topics:
Significance of Taste Sensation:
Differentiation between pleasant and harmful food
Influence on behavior
Selection of food based on metabolic needs
Receptors of Taste:
Taste buds on the tongue
Influence of sense of smell, texture of food, and pain stimulation (e.g., by pepper)
Primary and Secondary Taste Sensations:
Primary taste sensations: Sweet, Sour, Salty, Bitter, Umami
Chemical basis and signal transduction mechanisms for each taste
Taste Threshold and Index:
Taste threshold values for Sweet (sucrose), Salty (NaCl), Sour (HCl), and Bitter (Quinine)
Taste index relationship: Inversely proportional to taste threshold
Taste Blindness:
Inability to taste certain substances, particularly thiourea compounds
Example: Phenylthiocarbamide
Structure and Function of Taste Buds:
Composition: Epithelial cells, Sustentacular/Supporting cells, Taste cells, Basal cells
Features: Taste pores, Taste hairs/microvilli, and Taste nerve fibers
Location of Taste Buds:
Found in papillae of the tongue (Fungiform, Circumvallate, Foliate)
Also present on the palate, tonsillar pillars, epiglottis, and proximal esophagus
Mechanism of Taste Stimulation:
Interaction of taste substances with receptors on microvilli
Signal transduction pathways for Umami, Sweet, Bitter, Sour, and Salty tastes
Taste Sensitivity and Adaptation:
Decrease in sensitivity with age
Rapid adaptation of taste sensation
Role of Saliva in Taste:
Dissolution of tastants to reach receptors
Washing away the stimulus
Taste Preferences and Aversions:
Mechanisms behind taste preference and aversion
Influence of receptors and neural pathways
Impact of Sensory Nerve Damage:
Degeneration of taste buds if the sensory nerve fiber is cut
Abnormalities of Taste Detection:
Conditions: Ageusia, Hypogeusia, Dysgeusia (parageusia)
Causes: Nerve damage, neurological disorders, infections, poor oral hygiene, adverse drug effects, deficiencies, aging, tobacco use, altered neurotransmitter levels
Neurotransmitters and Taste Threshold:
Effects of serotonin (5-HT) and norepinephrine (NE) on taste sensitivity
Supertasters:
25% of the population with heightened sensitivity to taste, especially bitterness
Increased number of fungiform papillae
micro teaching on communication m.sc nursing.pdfAnurag Sharma
Microteaching is a unique model of practice teaching. It is a viable instrument for the. desired change in the teaching behavior or the behavior potential which, in specified types of real. classroom situations, tends to facilitate the achievement of specified types of objectives.
Acute scrotum is a general term referring to an emergency condition affecting the contents or the wall of the scrotum.
There are a number of conditions that present acutely, predominantly with pain and/or swelling
A careful and detailed history and examination, and in some cases, investigations allow differentiation between these diagnoses. A prompt diagnosis is essential as the patient may require urgent surgical intervention
Testicular torsion refers to twisting of the spermatic cord, causing ischaemia of the testicle.
Testicular torsion results from inadequate fixation of the testis to the tunica vaginalis producing ischemia from reduced arterial inflow and venous outflow obstruction.
The prevalence of testicular torsion in adult patients hospitalized with acute scrotal pain is approximately 25 to 50 percent
These simplified slides by Dr. Sidra Arshad present an overview of the non-respiratory functions of the respiratory tract.
Learning objectives:
1. Enlist the non-respiratory functions of the respiratory tract
2. Briefly explain how these functions are carried out
3. Discuss the significance of dead space
4. Differentiate between minute ventilation and alveolar ventilation
5. Describe the cough and sneeze reflexes
Study Resources:
1. Chapter 39, Guyton and Hall Textbook of Medical Physiology, 14th edition
2. Chapter 34, Ganong’s Review of Medical Physiology, 26th edition
3. Chapter 17, Human Physiology by Lauralee Sherwood, 9th edition
4. Non-respiratory functions of the lungs https://academic.oup.com/bjaed/article/13/3/98/278874
Prix Galien International 2024 Forum ProgramLevi Shapiro
June 20, 2024, Prix Galien International and Jerusalem Ethics Forum in ROME. Detailed agenda including panels:
- ADVANCES IN CARDIOLOGY: A NEW PARADIGM IS COMING
- WOMEN’S HEALTH: FERTILITY PRESERVATION
- WHAT’S NEW IN THE TREATMENT OF INFECTIOUS,
ONCOLOGICAL AND INFLAMMATORY SKIN DISEASES?
- ARTIFICIAL INTELLIGENCE AND ETHICS
- GENE THERAPY
- BEYOND BORDERS: GLOBAL INITIATIVES FOR DEMOCRATIZING LIFE SCIENCE TECHNOLOGIES AND PROMOTING ACCESS TO HEALTHCARE
- ETHICAL CHALLENGES IN LIFE SCIENCES
- Prix Galien International Awards Ceremony
Pulmonary Thromboembolism - etilogy, types, medical- Surgical and nursing man...VarunMahajani
Disruption of blood supply to lung alveoli due to blockage of one or more pulmonary blood vessels is called as Pulmonary thromboembolism. In this presentation we will discuss its causes, types and its management in depth.
2. the signaling receptor for endotoxin22 as well as human and
chlamydial HSP60.23,24
Currently, more than 10 human TLRs have been identified,
and at least 10 human homologues of drosophila Toll have
been sequenced. Whereas TLR-4 is used by enteric Gram-
negative bacteria and LPS, TLR-2 is used by Gram-positive
bacterial, mycobacterial, fungal, and spirochetal cell-wall
components.25,26 TLRs are evolutionarily conserved innate
immune receptors that recognize pathogen-associated molec-
ular patterns and contain a common intracytoplasmic domain
that conveys signals by molecules that are shared by
interleukin-1 receptor signaling to activate the NF-B path-
way and release inflammatory cytokines.21,27 Because TLR-2
and TLR-4 play an important role in the innate immune and
inflammatory responses, we investigated the expression of
these receptors in murine aortic and human coronary athero-
sclerotic plaques. Here, we report preferential expression of
TLR-4 in lipid-rich and macrophage-infiltrated murine and
human atherosclerotic plaques. In vitro studies demonstrated
basal expression of TLR-4 by macrophages, which was
upregulated by oxidized LDL (ox-LDL). These findings
suggest a potential role for TLR-4 in lipid-mediated proin-
flammatory signaling in atherosclerosis. Because TLR-4 is
the receptor that recognizes chlamydial antigens such as
chlamydia LPS and HSP60, it may provide a potential
molecular link between chronic infection, inflammation, and
atherosclerosis.
Methods
Preparation of Mouse Tissue
Apolipoprotein E (apoE)–deficient mice (C57BL/6J strain, 5 weeks
old, 18 to 20 g) obtained from Jackson Laboratory (Bar Harbor, Me)
(nϭ5) were fed a high-fat, high-cholesterol (atherogenic) diet con-
taining 42% (wt/wt) fat and 0.15% cholesterol from 6 weeks of age
through the duration of the experiment. After anesthesia with
eflurane, the mice were euthanized at 26 weeks of age, and their
hearts and proximal aortas were excised and embedded in OCT
compound (Tissue-Tek), frozen on dry ice, and then stored at Ϫ70°C
until sectioning. Serial sections 10 m thick were collected on slides
for immunohistochemistry as described earlier.28
Preparation of Human Tissue and Human
Monocyte-Derived Macrophages
Human coronary artery specimens from 9 autopsy cases were
collected within 24 hours of death, fixed with 10% formalin
overnight, and embedded in paraffin. Five of the 9 coronary artery
specimens included lipid-rich plaques containing a well-defined lipid
core covered by a fibrous cap, and the other 4 of the 9 specimens
included fibrous plaques, which contained mostly extracellular
matrix without a lipid core. Normal mammary artery specimens were
also obtained from 4 additional autopsy cases. Sections 5 m thick
were cut and applied to slides for both hematoxylin-eosin and
immunohistochemical staining. Peripheral-blood monocytes were
isolated from whole blood of normal human subjects by Ficoll-Paque
density gradient centrifugation. Monocyte-derived macrophages
were cultured in RPMI 1640 containing 10% FCS, 100 U/mL
penicillin, 100 g/mL streptomycin, and 0.25 g/mL amphotericin
B for 5 days as described earlier.29
Immunohistochemistry
Frozen sections of the apoE-deficient mouse aortic root were fixed
with acetone for 5 minutes at room temperature and then immuno-
stained with rabbit anti–hTLR-4 immune serum (1:100, obtained
from Dr Ruslan Medzhitov, Yale University) according to the
instructions on Dako’s immunostaining kit. Rat anti–mouse macro-
phage antibodies (1:500, Serotec) were used as macrophage marker.
Colors were developed with the Dako AES substrate system. Smooth
muscle cells were stained by a mouse anti-actin antibody conjugated
with alkaline phosphatase (1:50, Sigma). Colors were developed
with a Vector Red Alkaline Phosphatase Substrate Kit I. Rabbit IgG
or rabbit serum was used as a negative control.
For human atherosclerotic plaques, after deparaffinization in
graded alcohol, sections were immunostained with rabbit anti–
human TLR-4 and TLR-2 antiserum (1:100) raised against extracel-
lular peptide domains of TLR-4 and TLR-2 as previously de-
scribed.21 Preincubation of the anti–TLR-4 antiserum with TLR-4
peptide (FKEIRHKLTLRNNFDLSLNVMKT) was used to demon-
strate specificity of the stain, and rabbit IgG or rabbit serum instead
of primary antibody was used as negative control.
Double Immunohistochemistry
Double immunostaining of human atherosclerotic plaques was per-
formed with Dako’s Doublestain System kit. After TLR-4 immuno-
staining, 3,3Ј-diaminobenzidine was used as the peroxidase chromo-
genic substrate. Mouse monoclonal anti–human CD68 antibody (360
g/mL, 1:20 dilution; Dako, Derma) for macrophages and mouse
monoclonal anti–human ␣-actin antibody (100 g/mL, 1:100 dilu-
tion; Dako, Derma) for smooth muscle cells were used with fast red
as the alkaline phosphatase chromogenic substrate.
Preparation and Modification of Lipoproteins
Human native LDL (Sigma) was dialyzed against isotonic PBS (pH
7.4) to remove EDTA by use of Slide-A-Lyzer cassette 10 000
MWCO (Pierce). Ox-LDL was prepared as described previously.30
In brief, oxidation of LDL was performed by incubating 0.1 mg of
LDL protein/mL with 5 mol/L CuSO4 for 24 hours at 37°C. All
reagents were endotoxin-free. LPS levels of LDL preparations were
confirmed with a chromogenic Limulus assay and contained Ͻ0.3 pg
of LPS/g LDL protein. The extent of oxidation of the lipoprotein
preparations was determined by the thiobarbituric acid–reactive
substance (TBARS) assay.31 The ox-LDL had 20 to 25 nmol/L
TBARS/mg cholesterol.
Reverse Transcription–Polymerase Chain Reaction
Total RNA was isolated from resting and native LDL–, ox-LDL–
stimulated human monocyte–derived macrophages with an RNA
Stat60 isolation reagent (Tel-test “B” Inc) according to the manu-
facturer’s instructions and treated with RNase-free DNase I. For the
reverse transcription (RT) reaction, the MMLV preamplification
system (Life Technologies, Inc) was applied. Polymerase chain
reaction (PCR) amplification was performed with Taq gold polymer-
ase (Perkin Elmer) for 32 cycles at 95°C for 45 seconds, 54°C for 45
seconds, and 72°C for 1 minute (for TLR-2 and TLR-4). The
oligonucleotide primers used for RT-PCR were TLR-2, 5Ј-
GCCAAAGTCTTGATTGATTGG and 5Ј-TTGAAGTTCTC-
CAGCTCCTG; for TLR-4, 5Ј-TGGATACGTTTCCTTATAAG and
5Ј-GAAATGGAGGCACCCCTTC-5Ј as described earlier.32
GAPDH primers were obtained from Clontech.
Results
TLR-4 Is Expressed in Atherosclerotic Lesions of
ApoE-Deficient Mice
In all 5 apoE-deficient mice, TLR-4 immunoreactivity was
observed in the aortic root atherosclerotic lesions, which
colocalized with macrophage immunoreactivity (Figure 1).
TLR-4 staining was absent in the normal vessels obtained
from control C56BL/6J mice. Mouse IgG staining was
negative, and preincubation of the tissue sections with the
specific peptide against which the anti–TLR-4 antiserum was
generated completely blocked the TLR-4 staining in the
apoE-deficient vessels, indicating the specific nature of the
3104 Circulation December 18/25, 2001
3. TLR-4 immunostaining. No TLR-2 immunoreactivity was
observed (data not shown) in normal or atherosclerotic
lesions.
TLR-4 Is Expressed in Human Coronary Plaques
The human coronary atherosclerotic plaques were classified
into lipid-rich plaques containing a well-defined lipid core
covered by a fibrous cap (nϭ5) and fibrous plaques that
contained mostly extracellular matrix without a lipid core
(nϭ4). Strong TLR-4 expression (brown staining) was ob-
served around the lipid core and at the shoulder of lipid-rich
plaques, where it colocalized with macrophage immunoreac-
tivity (Figure 2). Incubation of the antiserum with the peptide
used to generate the primary antibody blocked TLR-4 immu-
noreactivity, confirming the specificity of the anti–TLR-4
antiserum. Double staining showed close spatial colocaliza-
tion of TLR-4 expression with macrophage immunoreactivity
(Figure 2). No TLR-4 immunoreactivity or macrophage
immunoreactivity was found in fibrous plaques that demon-
strated strong smooth muscle ␣-actin immunoreactivity (Fig-
ure 2). Normal mammary arteries showed only minimal or no
TLR-4 expression (Figure 2). TLR-2 immunoreactivity was
absent in all plaques, whereas control staining was positive in
THP-1 cells (data not shown).
TLR-4 mRNA Regulation by Ox-LDL
Cultured human monocyte-derived macrophages were stim-
ulated with native LDL or ox-LDL for 5 hours, RT-PCR for
TLR-2 and TLR-4 was performed, and relative intensity was
calculated by densitometry as described earlier.32 RT-PCR
showed basal TLR-2 and TLR-4 mRNA expression by
macrophages. The TLR-4 mRNA was upregulated by ox-
LDL in a dose-dependent manner up to 3-fold, whereas native
LDL had no effect. TLR-2 mRNA was not upregulated by
ox-LDL (Figure 3).
Discussion
Although precise triggers for inflammation in atherosclerosis
are not fully understood, hypercholesterolemia, modified
lipoproteins, and infection with organisms such as C pneu-
moniae and others have all been implicated. There is now
evidence that C pneumoniae infection can accelerate the
Figure 1. TLR-4 immunoreactivity is seen
within atherosclerotic plaque, in lipid core
of plaque of aortic sinus of apoE-deficient
mouse (A). B and C, Immunoreactivity of
macrophages and smooth muscle cells,
respectively, in serial section of aortic
sinus. Note close spatial localization of
macrophage immunoreactivity and TLR-4
immunoreactivity. D, Rabbit IgG staining
for negative control. E, There is no immu-
noreactivity of TLR-4 in nonatherosclerotic
aortic sinus of C57BL/6J mouse.
Figure 2. Photomicrographs showing
immunohistochemical evidence for TLR-4
expression in human atherosclerotic lipid-
rich plaques but not in fibrous plaques. A,
Atherosclerotic plaque stained with rabbit
anti–human TLR-4 antiserum (brown stain-
ing); B, negative control where primary
antibody was replaced by rabbit IgG; C,
TLR-4 immunoreactivity (brown); D, double
immunostain with TLR-4 brown and mac-
rophages red to demonstrate colocaliza-
tion. E, F, and G, Higher-magnification
view of macrophage immunoreactivity
(red), TLR-4 immunoreactivity (brown), and
macrophage plus TLR-4 immunoreactivity
(red and brown), respectively. Fibrous
plaques showed no immunoreactivity for
TLR-4 (H) or macrophages (J) but showed
only smooth muscle cell ␣-actin immuno-
reactivity (red) without TLR-4 immunoreac-
tivity (brown) on double staining in I. K,
Negative control with preabsorption of
antiserum with peptide; L, normal mam-
mary artery showing only minimal immuno-
reactivity to TLR-4 along endothelial
border.
Xu et al TLR-4 and Atherosclerosis 3105
4. progression and facilitate the induction of atherosclerosis in
cholesterol-fed rabbits and genetically modified atheroscle-
rosis-prone mice.33–37 The concept of C pneumoniae–induced
atherogenesis is further strengthened by the finding that
antibiotic therapy against chlamydia prevents acceleration of
atherosclerosis in the rabbit model.33 Ingalls et al38 have
suggested LPS, and Kol et al39,40 have implicated HSP60, as
the triggers for chlamydia-induced inflammatory responses.
Both chlamydia infection41 and its LPS have been shown to
induce foam-cell formation in monocytes.18 Persistence of
LPS and/or HSP-60 in the atheroma either within intact C
pneumoniae–infected cells or in the subendothelial space
after cell lysis may promote atherosclerosis by continued
macrophage activation. Indeed, circulating chlamydial LPS–
specific immune complexes have been detected in patients
with coronary heart disease.42 To date, however, the precise
molecular mechanisms by which infections such as C pneu-
moniae contribute to the progression of atherosclerosis and
the links among lipids, microbial antigens, and innate im-
mune and inflammatory responses are not well understood.
Activation of monocytes/macrophages is an important
initial step in the cascades of events leading to many
inflammatory diseases, including atherosclerosis. The recent
findings that TLR-4 is the signaling LPS receptor and also
recognizes HSP60 provided a new impetus in elucidating the
role of TLR-4 in various inflammatory diseases. Furthermore,
a recent study showed that saturated fatty acids, but not
unsaturated fatty acids, induce NF-B activation and expres-
sion of cyclooxygenase-2 through the TLR-4 receptor as
well.43
In this study, we show for the first time that the proinflam-
matory signaling receptor TLR-4 is expressed in lipid-rich,
macrophage-infiltrated atherosclerotic lesions of mice and
humans and that TLR-4 mRNA in cultured macrophages is
upregulated by ox-LDL but not native LDL. Together, these
findings raise the possibility that enhanced TLR-4 expression
may play a role in inflammation in atherosclerosis, supporting
the emerging paradigm.1,44–46
Cells of the innate immune system, such as macrophages,
have the ability to recognize common and conserved struc-
tural components of microbial origin by pattern recognition
receptors. The human homologue of drosophila Toll, TLR-4,
is a pattern recognition receptor that activates NF-B and
upregulates a variety of inflammatory genes in response to
microbial pathogens.47 TLRs play a fundamental role in the
activation of innate immune responses and pathogen recog-
nition. Activation of NF-B is essential for the regulation of
a variety of genes involved in the inflammatory and prolif-
erative responses of cells critical to atherogenesis.48,49 Both
NF-B and genes regulated by NF-B are expressed in
atherosclerotic lesions.49 Because NF-B activation leads to
transcription of a number of proinflammatory genes involved
in atherothrombosis, it is tempting to speculate that infectious
agents and chlamydial antigens, such as LPS and/or HSP-60,
Figure 3. Cultured human monocyte-derived macrophages were stimulated with either native LDL or ox-LDL with different doses for 5
hours. Expression of TLR-2 (347 bp) and TLR-4 (548 bp) mRNA was analyzed by RT-PCR. RT-PCR analysis of GAPDH expression was
used as control. Graph shows relative intensity of each band (relative to GAPDH), which was measured by densitometry with Kodak ID
image analysis software (Kodak, EDAS 290). TLR-4 mRNA is upregulated by ox-LDL but not by native LDL, whereas neither native LDL
nor ox-LDL regulated TLR-2 mRNA.
3106 Circulation December 18/25, 2001
5. might contribute to enhanced and chronic inflammation by
signaling through the TLR-4 receptor, which is upregulated
by ox-LDL.
Our findings of increased expression of TLR-4 induced by
ox-LDL suggest a potential mechanism for the synergistic
effects of hypercholesterolemia and infection in acceleration
of atherosclerosis observed in experimental models35,36 and
human epidemiological observations.50 Thus, these findings
provide additional new insights into the link among lipids,
infection/inflammation, and atherosclerosis.
In summary, we observed that human TLR-4 but not
TLR-2 is expressed in murine and human lipid-rich athero-
sclerotic plaques, including areas infiltrated by macrophages.
Furthermore, we show that ox-LDL but not native LDL
induces upregulation of TLR-4 expression in macrophages.
Given that TLR-4 plays a critical role in inflammatory and
immune signaling, upregulated TLR-4 may participate in the
inflammatory responses linking lipids to chronic infection,
inflammation, and atherosclerosis. Improved understanding
of the molecular mechanisms driving TLR-4 overexpression
and signaling and the role of the resulting chronic inflamma-
tion during atherosclerosis may provide new targets for
antiatherogenic therapy.
Acknowledgment
This study was supported by NIH grants HL-51087 and AI-50699 to
Dr Arditi.
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