The I Factor - Inflammation, Immunity,

Illness

Presented by Dr William Barnes, BSc, MB ChB

• Healing is a Personal Journey

• Science and Healing

• Science and Theory

• Science and Observation

Outline• Background • Immunity and Inflammation • Inflammation and Macrophages• Macrophage Pathology• Foam Cell, Viruses, TAMs • The Brune Theory & Nitric Oxide • Macrophages in Disease States

– Treatment Strategies • Colds and Influenza

– Treatment Strategies

Immunity and Inflammation

Immunity and Inflammation

• Inflammational dysregulation is at the root or all chronic illnesses.– Cancer, heart disease, arthritis, asthma,

bronchitis, Alzheimer’s disease, schizophrenia, multiple sclerosis, etc.

Abnormal Immunity

• Innate immune system becomes dysfunctional and perpetuates the disease process.

The Innate Immune System in Action

Components of the Immune System

Omega-6 and Omega-3 Conversion Pathways

Macrophages

Macrophages• Makros (large) + phagein (eat) = big eaters.• Monocytes enter damaged tissue through the

endothelium of a blood vessel and undergoes transformation to a macrophage.

• Monocytes are attracted to the site by chemotaxis, triggered by a range of stimuli including damaged cells, pathogens and cytokines released by macrophages already at the site. They can survive up to several months.

Fixed Macrophages• Alveolar

macrophages• Histiocytes• Kupffer cells• Microglia• Epithelioid• Osteoclasts• Sinusoidal lining

cells• Mesangial cells

• Lungs• Connective tissue• Liver• Neural tissue• Granulomas• Bone• Spleen• Kidney

Phagocytosis

Macrophage ActivationTNF-α

LPS

NO

Nitric Oxide SynthesisCitrulline

Ornithine

NO

UreaArginase

Nitric Oxide Synthase (NOS)Arginine

Nitric Oxide

• Two main types:- iNOS (inducible nitric oxide synthase),

produced by macrophages- eNOS (endothelial nitric oxide synthase),

responsible for endothelial dilatation.

Macrophages and Disease• Macrophages called foam cells may be

predominant in the pathogenesis of atherosclerosis.

• Macrophages play a role in HIV, they become infected and then become incubators for the human immunodeficiency virus.

• Macrophages called TAMs (tumour associated macrophages) are associated with cancer proliferation and spread partially by activation of NFkB activation and the inhibition of apoptosis, and also through production of TNF-α.

Macrophages

• Environmental oestrogens promote IL-1β activity in macrophages.

• Genistein and certain xeno-oestrogens synergised with LPS to activate IL-1β promoter activity. Environmental oestrogens have agonist activity both on reproductive tissue along with haemopoietic tissue. – Endocrine 1998 Oct;9(2):207-11.

Foam Cells• In the arterial intima, monocytes are transformed

into macrophages which then proceed to ingest oxidised LDL.

• This foam cell transformation alters the phagocytic and antimicrobial function of the macrophage and may well allow the documented associated infection with microbes, such as chlamydia pneumonia, within the atherosclerotic plaques.

• Cholesterol accumulation by macrophages impairs phagosome maturation.

– J Biol Chem. 2008 Dec 19;283(51):35745-55.

Flavonoids and Endothelial Health

• Dietary flavonoids reduce oxidised LDL in endothelial cells.

• Endothelial apoptosis is a driving force in the development of atherosclerosis.

• EGCG, hesperetin and quercetin decrease the influence of LDL signalling on the induction of apoptosis.– J Nutr. 2008 Jun;138(6):983-90.

TAMs

• Tumour associated macrophages are obligate partners for malignant cell migration, invasion and metastasis.

• The more TAMs the worse the prognosis.– J Exp Med. 2001 Mar 19;193(6):727-40.

TAMs Effectors of Angiogenesis and Tumour Progression

• TAMs are a prominent inflammatory cell population in many tumour types residing in both perivascular and avascular, hypoxic regions of these tissues.

• Analysis of TAMs in human tumour biopsies has shown they express a variety of tumour promoting factors and the evidence from transgenic murine tumour models has provided unequivocal evidence for the importance of these cells in driving angiogenesis, lymphangiogenesis, immunosuppression and metastasis.– Biochim Biophys Acta. 2009 Mar 6.

The Brune Theory• Macrophages phagocytose apoptotic cells (AC). • Phagocytosis of AC regulates the immune response by

shifting the phenotype of the macrophage to an anti-inflammatory type (M2).

• AC in the M2 macrophage enhances the expression of arginase II; this halts the induction of NO formation in IFN-gamma stimulated macrophages.

• AC triggers IL-10, TGF-β, PGE2 production; reduced capacity to produce TNF-α.

• TAMs support tumour growth survival and contibute to angiogenesis and immune evasion.

– Mol Biol Cell. 2007 Oct;18(10):3810-9.

Brune Theory - M2 Macrophage

Citrulline

Ornithine

NO

UreaArginase

Nitric Oxide Synthase (NOS)Arginine

Carnitine• Carnitine decreases arginase activity.• High levels of arginase and ornithine in different

carcinomas may indicate their relation to cancer.• Carnitine is a cofactor required for the

transformation of free long-chain fatty acids into acetylcarnitines. Carnitine may have an effect on the ornithine arginase tissue levels.

• Conclusion: NO levels were significantly higher in the carnitine treated group whilst the tissue level of arginase activity and ornithine was significantly decreased.– Cell Biol Int. 2007 Nov;31(11):1414-9.

Effect of Carnitine on Nitric Oxide Synthesis

Citrulline

Ornithine

NO

UreaArginase

Nitric Oxide Synthase (NOS)Arginine

Carnitine

Macrophages and Helicobacter pylori

• Macrophages and Helicobacter pylori infection lead to gastric cancer.

• Gastric infection by H. pylori extract (HPE) activates the production of cytokines and chemokines by mononuclear cells.

• IL-8 is one of the major HPE triggered cytokines. • This is activated by HPE mediated LPS stimulation

of macrophages; also amplifies the inflammatory response.

Characteristics of Inflammatory Infiltrate of Gastric Mucosa in Patients

with Dysplasia and Cancer• 85 patients. • Composition of the inflammatory infiltrate from gastric

mucosa (GM) was determined. • Patients with adenocarcinoma showed an increase in

lymphocytes, neutrophils and macrophages.– Klin Med (Mosk). 2008;86(11):48-53.

• Helicobacter pylori infection results in neutrophil activation and chronic gastritis, and this has a role in the development of gastric cancer.

• The greater the intensity of HPE infection, the greater the degree of neutrophil activation and, in turn, the gastritis and metaplasia. – Niger J Clin Pract. 2008 Sep;11(3):270-4.

Characteristics of Inflammatory Infiltrate of Gastric Mucosa in Patients with

Dysplasia and Cancer

Idiopathic Pulmonary Fibrosis (IPF)

• IPF is a chronic, progressive, and often fatal lung disease of unknown aetiology.

• Characterised by onset of infiltrate in the parenchyma of the lung tissue.

• This infiltrate has been identified as lymphatic endothelial cells (LECs). – Proc Natl Acad Sci U S A. 2009 Mar 10;106(10):3958-63.

• Alveolar regions of the lung are usually devoid of LECs.– Alveolar macrophages either promote the transformation of

lymphocytes into LECs, OR– Macrophages themselves become LECs and form the basis for

this granulation tissue.

MMPs• MMPs (Matrix metalloproteinases) are a family of

enzymes involved in the breakdown of extracellular matrix in normal physiological processes such as embryonic development, reproduction (endothelial menstrual breakdown) and tissue remodelling.

• They are activated in diseases such as arthritis, cancer metastasis and pulmonary fibrosis.

• LPS activated macrophages produce TNF and NO which in turn activate MMPs.

• MMP-2 and MMP-9 are two of particular interest in diseases.

Arthritis

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Arthritis and the Immune System

Natural Products - A Gold Mine for Arthritis Treatment

• Study conclusions: – Although various treatments for arthritis exist they

suffer from various drawbacks such as lack of efficacy, excessive side effects and high cost.

– Arthritis requires a lifetime of treatment.– Plant-derived products offer promise but require

investigation, however owing to the lack of intellectual property rights, the pharmaceutical industry has little motivation to pursue the studies.

• Potential treatment strategies for arthritis:– Suppress the expression of:

• TNF-α, • IL-1β, • COX-2,• LOX, • MMPs.

– Suppress NFkB.• Curcumin, resveratrol, boswellic acids, quercetin, tea

polyphenols, etc.– Curr Opin Pharmacol 2007 Jun;7(3):344-51.

Natural Products - A Gold Mine for Arthritis Treatment

Curr Opin Pharmacol. 2007 Jun;7(3):344-51.

Synergy • Different polyphenol herbs have slightly different

actions on different survival pathways. • Quercetin on its own only starts its action at

10mM. – Quercetin action is mediated through reduction of

iNOS, TNF-α, or LOX. – Synergy between green tea and curcumin or

resveratrol will bring stronger influence on other pathways such as Akt inhibition where these other herbs have shown greater effect. Curcumin for instance is a stronger inhibitor of the COX pathway.

• This synergy will also be of effect in other inflammatory conditions and with pain in general.

Flavonoids

Flavonoids

• High intake of dietary isoflavones is linked to the inhibition of COX activity in human macrophages.– Nutr Cancer. 2004:49(1):89-93.

Flavonoids• Inhibition of iNOS and COX-2 expression by flavonoids

in macrophages.• Flavonoids quercetin, galangin, apigenin and naringenin

markedly decreased PGE2 and COX-2 expression in a concentration dependent manner.

• The same flavonoids modulated the expression of iNOS between 5-50μM.– Achievable with 1500mg quercetin orally.

– Life Sci. 2001 Jan 12;68(8):921-31.

Flavonoids

• Flavonoids affect oral anti-inflammatory activity and inhibit systemic TNF-α activity.

• Study in mice showed that oral adminstration of flavonoids significantly inhibited the TNF-α production.– Biosci Biotechnol Biochem. 2004 Jan;68(1):119-25.

Quercetin

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Quercetin

• Belongs to a polyphenolic class of flavonoids.• Aglycone; basically rutin without the sugar.• Major bioflavonoid in the human diet.• Average daily intake 25mg. • Foods with highest concentrations include

onions, apples, grapes; however, it is ubiquitous.• Absorption from diet can be up to 20%. • 64mg in diet can give up to 0.6μM three hours

after ingestion, with the half-life up to 17-25h.

Quercetin

• Plasma concentration required to be effective in cancer is above 10μM. The oral dose to achieve this may be in the vicinity of 1500mg. With a relatively long half-life (17-25h), there will be an accumulative effect over time.

• Anti-inflammatory effects in conditions such as prostatitis have been shown at 100mg/d.

• In our clinic, we have used doses up to 1000mg as a single IV and 500mg 3 times a week for months without side effects except K lowering.

Quercetin and Immunity

• Quercetin blocks inflammation, cell migration and angiogenesis through reduction in iNOS, TNF-α, NFkB, MMP inactivation, etc.

• Its actions are relevant to all chronic inflammatory diseases.

• As its anti-inflammatory actions are primary, the question remains - Where should it be used in acute infections?

Quercetin Attenuates NFkB Activation and Nitric Oxide Production

• Cell culture studies with hepatocytes.• Cells stimulated with IL-1β.• IL-1β is a multifunctional cytokine that plays a critical role in

inflammation, immunity, antiviral responses, and a variety of diseases.• IL-1β stimulates T cells. It is very important in the liver and is implicated

in inflammatory liver diseases.• IL-1β stimulates iNOS production in hepatocytes during inflammation.

NO binds with SO2 to produce peroxynitrites.• Peroxynitrites are cytotoxic and capable of injuring invading pathogens

and eliminating altered cells. However indiscriminate destruction of cells and tissues is implicated in the pathology of liver disease such as hepatitis.

• Quercetin inhibited the accumulation of nitrite, and decreased the production of NO.

• Quercetin inhibits the production of NO from LPS stimulated Kupffer cells.

• In macrophages quercetin and resveratrol decrease LPS stimulated iNOS.

– J Nutr. 2005 Jun;135(6):1359-65.

Quercetin and Cancer• To date there has been one phase 1 trial of

quercetin IV in cancer.• Two patients had a positive response with

reductions in tumour markers and tumour size over the 30-day trial.

• One case was stage IV ovarian, one hepatocellular cancer.

• In vitro studies show quercetin to be effective in a wide range of cancers ranging from leukaemias to lung cancer, melanoma, breast, colon, etc.

• My clinical practice: long-term stabilisation of lymphoma, breast, myeloma, colon, lung, mesothelioma and prostate cancers, with both oral and IV regimes.

• Blockade of the epidermal growth factor (EGF) receptor tyrosine kinase activity by quercetin leads to growth inhibition in pancreatic tumour cells.

• EGF increased the levels of MMP-2 and MMP-9, whilst quercetin appeared to decrease these levels.

• MMP-2 and MMP-9 are implicated in the spread of metastasis.

• EGF stimulation is associated with increased cell proliferation.– Anticancer Res. 2002 May-Jun;22(3):1615-27.

Quercetin and Cancer

Quercetin in Myocardial Infarction

• Intravenous quercetin significantly reduces mortality in myocardial infarction. – Ukrainian study.– 57 patients randomised to receive placebo or be

treated with IV quercetin over five days.– Results:

• reduced size of the heart muscle damage• reduced size of final infarct • improved injection fraction • reduced the risk of subsequent heart failure.

• “This is a safe inhibitor; it is non-toxic, we have very good long-term survival rate improvement.”

• Results presented at heart failure congress, Milan 2008.

Green Tea

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Green Tea

• MMPs and green tea.• Biochemical research catches up with folk

medicine. • EGCG from green tea is an effective blocker of

MMP-2 and MMP-9.

Green Tea Inhibits Helicobacter pylori Growth in vitro and in vitro• Components of green tea have been

shown to:– be bactericidal – be bacteriostatic– inhibit growth of HPE – prevent the inflammatory effect of HPE on the

gastric mucosa.– Int J Antimicrob Agents. 2009 May;33(5):473-8.

Harpagophytum procumbens (Devil’s Claw)

• Anti-inflammatory action related to inhibition of LPS stimulated iNOS, NFkB, COX-2 in macrophages and mesangial cells.

• Anti-inflammatory action reported as effective pain control in osteoathritis.

• 50mg of harpagoside gives equivalent analgesia as synthetic COX inhibitors, but with lower toxicity.

• Potential use in kidney inflammation.

Uncaria tomentosa (Cat’s Claw)

• In vitro studies suggest U. tomentosa inhibits LPS stimulated TNF-α production 5.5-fold.

• Uses in gastritis and osteoarthritis have been reported.

• May have a role in Alzheimer’s disease as it has been shown to bind to beta-amyloid.

• May have some antibacterial activity in both strept and staph isolates but not fungal nor pseudomonas.

Macrophage Phagocytosis• Immunomodulatory effects of herbs on

macrophage function.• Conclusion:

– Standardised extracts of echinacea, cat’s claw and saw palmetto; main action on macrophages was in stimulation of phagocytosis.

– J Med Food. 2007 Mar;10(1):73-9.

Obesity and Inflammation • “These findings provide significant evidence that obesity is

linked to tumour promotion in the pancreas ... suggests alterations in inflammatory responses and bioenergetic pathways as the potential underlying cause.”– Proc Natl Acad Sci U S A. 2009 Mar 3;106(9):3354-9.

• “Chronic infections, obesity, alcohol, tobacco, radiation, environmental pollutants, and high-calorie diet have been recognised as major risk factors for the most common types of cancer. All these risk factors are linked to cancer through inflammation.”– Clin Cancer Res. 2009 Jan 15;15(2):425-30.

Common Cold

• Things to consider: – Influenza or URTI? – Presentation in otherwise healthy individual.– Presentation in patient with inflammational

disease already present.– Secondary infections and complications.

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Feature Colds Flu Aetiological age nt >100 viral strains; rhinov irus

most common 3 strains of influenza virus: influenza A, B, and C

Site of infection Upper respiratory tract Entire respiratory system Symptom onset Gradual: 1-3 days Sudden: within a few hours Fever, chills Occasional, low grade

(<38.3ÞC) Characteristic, higher (>38.3ÞC), lasting 2-4 days

Headache Frequent, usually mild Characteristic, more severe General aches, pains Mild, if any Characteristic, often severe

and affecting the entire body Cough, chest congestion Mild to moderate, with

hacking cough Common, may become severe

Sore throat Common, usually mild Sometimes present Runny, stu ffy nose Very common, accompanied

by bouts of sneezing Sometimes present

Fatigue, weakness Mild, if any Usual, may be severe and last 2-3 weeks

Extreme exhaustion Never Frequent, usually in early stages of illness

Season Year round, peaks in winter months

Most cases between November and February (American winter)

Antibiotics helpfu l? No, unless secondary bacterial infection develops

No, unless secondary bacterial infection develops

Adapted from: Roxas M, Jurenka J. Colds and influenza: a review of diagnosis and conventional, botanical, and nutritional considerations. Altern Med Rev 2007 Mar;12(1):25-48.

Common Cold and Influenza Characteristics

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Advances in Diagnosis and Management of Influenza

• H5N1 avian flu (H haemagglutinin surface protein, N neuraminidase surface protein).

• H1N1 swine flu.• H1N1 virus is not new; the current flu is a new form of an

old virus responsible for the Spanish flu of 1918; re-emerged in China 1957 and Russia 1977.

• Older people may well have immunity through previous contact.

• Influenza vaccines modified yearly - usually influenza A H3N2 and H1N1 and one B virus; only effective in 3 strains in one year.

• There may be up to several hundred strains in any one year.

• Vaccines for the haemagglutinin and/or neuraminidase antigen confer up to 70% protection in the healthy population with those three viruses considered to be most virulent.

• Less effective in institutionalised elderly patients - down to 30%.

Flu Vaccines

Neuraminidase (NA) Inhibitors• Influenza viruses attach to cells via sialic acid

receptors.• NA is needed to remove sialic acids so the

budded virus can detach and disperse.• Mutations in the sialic binding site can change

the need for NA to increase dispersion.• Drugs that inhibit NA - oseltamivir (Tamiflu),

zanamir - can be effective in reducing the duration of the illness. Resistance has been observed.

Echinacea

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Echinacea purpurea

• Echinacea alkylamides suppressed IL-2 secretion by stimulated lymphocytes.

• This effect was lessened upon oxidation through the P450 enzymes to carboxylic acids.

• This shows the difference between in vitro effects of echinacea and potential in vivo effects.– Planta Med 2006 Dec;72(15):1372-7.

Echinacea Induces Macrophage Activation

• In vivo trial in mice have shown that E. purpurea, when administered orally, can up-regulate macrophage function in listeria infections.

• Echinacea stimulates the innate immune system by stimulating IL-6, TNF, IL-12 and iNOS from macrophages.

• This resulted in decreased bacterial burden during infection.– Immunopharmacol Immunotoxicol. 2008:30(3);553-74.

Echinacea purpurea Stimulates Alveolar Macrophage Function

• Increase in macrophage activity.• Dose dependent increase in NO and TNF-α on

stimulation with LPS.• Echinacea stimulates macrophage function in

lung and spleen in rats.– Int Immunopharmacol. 2002 Feb;2(2-3): 381-7.

• Caution in fibrosis and autoimmune diseases.

Echinacea purpurea• A recent review of PubMed revealed 123 papers on

echinacea and infection. • Prophylactic treatment with commercially available E.

purpurea did not significantly alter the frequency of upper respiratory tract symptoms compared with placebo.

– Ann Allergy Asthma Immunol. 2008 Apr;100(4):384-8.

• Extracts from E. purpurea showed consistent and robust inhibition of HIV - up to 50% inhibition.

– Am J Clin Nutr. 2008 Feb;87(2):488S-92S.

• E. purpurea is considered safe, with no adverse or allergic reactions in children.

– Can J Physiol Pharmacol. 2007 Nov;85(11):1195-9.

Echinacea and the Common Cold

• A proprietary echinacea extract with a standardised ratio of alkylamides, cichoric acid and polysaccharides was found to decrease the severity and duration of symptoms of the common cold.

• Mechanisms need to be determined, but suggested echinacea stimulates the non-specific immune system.

• Study in 150: 62 got a cold, 26 treated, 31 controls.

• 8 x 5mL dose, then 3 x 5mL for the next six days.

Echinacea for Treatment of the Common Cold

• Second study on same extract: 282 subjects in good health: 59 echinacea, 69 placebo.

• Echinacea group severity scores for runny nose, sore throat, fatigue headache and chills lower by up to 44%.

• Cough was the same.• Echinacea group: 50% reduction in symptoms

by Day 4; placebo: same reduction halfway through Day 5.– J Clin Pharm Ther. 2004 Feb;29(1):75-83.

Echinacea purpurea• E. purpurea may attenuate the mucosal immune

suppression known to occur with intense exercise, and reduce the duration of URTI that athletes incur.

• Study involving 32 athletes in high exercise regime known to affect mucosal immunity such as secretion rate of mucosal sIgA.

• There was no significant difference in the number of URTIs but there was significant difference in duration.– Int J Sports Med. 2007 Sep;28(9):792-7.

echinamide• Developed in Canada by Natural Factors• German scientist Dr Rudolf Bauer the worlds

most authority on echinacea states• “Echinamide is unique in that it is has

certain standardized levels of polysaccharides, cichoric acid and alkylamides. This new data , which has produced pharmacolgical results is only achievable with this product”

Quercetin in Acute Infections• Combine with

– immunostimulatory actions: echinacea, andrographis, vitamin C

– fish oil– magnesium. – Prim Care. 2002 Jun;29(2):231-61.

Bromelains

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Bromelains: Mechanisms of Action

• Introduction of proteolytic activity at inflammatory sites.

• Activation of fibrinolysis activity via the plasminogen-plasmin system.

• Depletion of kininogen.

• Inhibition of pro-inflammatory prostaglandin biosynthesis and initiation of PGE1 accumulation (which inhibits the release of polymorphonuclear leukocyte lysosomal enzymes).

Bromelain and Quercetin in Sinusitis

• Thins nasal secretions.• Effective mucolytic agent.• Inhibits prostaglandins.• 1987 study showed 85% complete resolution of

sinus inflammation; placebo 40%.• Doses up to 2000mg/d can be used safely.• 1000mg/d is more typical.

Bromelains and Cytokines

• Proteolytic enzymes have been reported to have anti-inflammatory effects.

• Bromelain treatment decreased expression of pro-inflammatory cytokines in ulcerative colitis and Crohn’s disease.

• Accumulation and activation of inflammatory cells within the mucosa leads to the tissue damage that is characteristic of inflammatory bowel disease.

• Results showed a reduction in Th1 cytokines with a significant reduction in TNF-α, IFN-gamma, but not IL-6 nor IL-1β. – Clin Immunol. 2008 Mar;126(3):345-52.

Andrographis

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Andrographis paniculata

• Current evidence suggests A. paniculata alone or in combination with Acanthopanax senticosus extract may be more effective than placebo and may be appropriate alternative treatment for uncomplicated acute URTI.

– J Clin Pharm Ther 2004 Feb;29(1):37-45.

Andrographis paniculata• Double-blind trial using andrographis extract:

– 95 in treatment group, 90 in placebo group.– Medication taken for 5 days.– Symptoms included high temperature, headache,

myalgia, sore throat, cough, malaise and eye symptoms.

• Results: Highly significant improvement in treated group over placebo.

• Significant symptom improvement was seen in headache, nasal and throat symptoms together with general malaise. Temperature was moderately reduced. Cough and eye symptoms showed little improvement. The extract was well tolerated.– Phytomedicine. 2002 Oct;9(7):589-97.

Whey Protein

Whey Protein• Enhances immune function, scavenges free

radicals, and exhibits antimicrobial activity.• Actions partially due to lactoferrin a peptide

fraction, and its ability to bind excretion-related iron.

• Lactoferrin is present in exocrine excretions, nasal discharges, sputum, saliva, etc. Also in polymorphonuclear neutrophils.

• Actions are also anti-inflammatory and viral inhibitory.

conclusion• Relationship between immunity and

inflammation.• Role of abnormal macrophage function

in this• Beauchamp/ Pasteur• The use of herbs and natural substances in modulating these functions