•
Increased mortality risk , particularly due to lympho-proliferative complications
• Quality of life - equated with moderate angina
• “Disability” predominantly due to fatigue and cognitive
• “Limitations” :
 dry eyes (limits work- especially computer)
 dry mouth (limits sleep and social interactions around eating)
 extra-glandular manifestations, particularly neurologic
•
Expense of artificial tears and dental decay

Sjogren’s syndrome represents the interface of: a) Immune and exocrine secretory functions ( dryness ) b) Immune and neural function ( neuropathy/cognitive ) c) Immune and hypothalamic-adrenal axis ( autonomic ) d) Autoimmune proliferation and lymphoma e) Lupus-like features of vasculitis and immune complex

When we get “ flu symptoms ” of arthralgia, fatigue, cognitive dysfunction— it is a result of the cytokines/neurotransmitters released by the innate immune system.
When these reactions persist due to a vicious cycle perpetuated in genetically predisposed individual by the acquired immune system , the result is autoimmune disease .

1.
Innate and Acquired Immune Systems are targets for current therapy—including TNF, BAFF and IL-6 inhibitors, steroids, traditional DMARD’s and new oral agents (Jak and syk inhibitors).
2.
Functional circuit that controls immune and neural function comprises the new “frontier” for therapy from fibromyalgia to depression. The functional circuit is the link between cytokines and symptoms.

Take Home Lesson - 2
The two arms of the immune system mutually interact in the initiation and perpetuation of Sjogren’s Syndrome
Acquired System

(HLA-DR)-memory

Traditional T-cell and
B-cell and their cytokines

HLA-DR association with autoantibody production

Target of drugs such as
DMARDs and certain biologics
Innate System

(Adaptive, immediate) - HLA independent

Dendritic cells

Cytokines-particularly

Type I interferon
 Interferon-gamma

BAFF, IL-6, IL-17

Complement, CRP
Sensors of the innate system

Toll receptors (TLR)-pathogen motiffs

DAMP (damage recognition patterns)apoptosis

RIG-1 (retinoid inducible genes)

NOD/Card receptors-more than in colitis

Take Home Lesson 3:
The Functional Circuit
(Cytokines are not enough)
Control of tears or saliva flow are complex processes that involve both afferent nerve pathways that go to the midbrain and efferent nerves that modulate glandular function.
The midbrain signals are influenced by the cortical outflow and the hypothalamic axis.

Normal tearing or salivation secretion requires a functional unit
4. Stimulation of gland water mucin protein
1. Ocular or oral surface irritation
Nerves on mucosal water nutrients hormones
3. Stimulation of blood vessel
3. Cortical
Outflow
Tracts and
HPA
Afferent nerves
2. Midbrain of central nervous system
Lacrimatory or salivatory nuclei

The functional circuit involves known neural connections to the brain
*Pflugfelder SC, et. al. Dry Eye and Ocular Surface Disorders. NY: Dekker, 2004.

In Sjogren’s syndrome, the release of Ach and VIP by efferent nerves to the glands
--and the response of the glands to neural transmitters-are impaired by lymphocytes that enter the gland and release inflammatory factors lymphocytes
Focal lymphocytic infiltrates in the glands ocular and oral dryness
Gland dysfunction
• Autoantibodies
(anti-muscarinic antibody)
 Cytokines (type I IFN, g
-IFN)
• Metalloproteinases
(outside-inside signaling molecules)

In Sjogren’s, only 50% of the acini and ducts are destroyed.
Despite their retention of neural innervation, the residual glands do not function as a result of the inflammatory environment
Foci of lymphs
Sjogren’s Normal

The residual glandular cells are paralyzed by the local immune reaction.
Even though the acini/ducts are 50% present, their innervation and their receptors for neurotransmitters are present.

Thus, the interesting question is:
Why are the residual glandular elements not working?
This fundamental question of how immune and neural systems interact will be the “holy grail” of neuroscience for the next decade.

Although many complex interactions take place in the salivary gland, a characteristic type I interferon gene signature is noted repeatedly .
The relationship of autoantibody to SS-A/SS-B and type I interferon signature has recently been suggested.
This links our blood tests (SS-A) and clinical features.

SS
SS SG biopsy with type I
IFN gene profile
SS SG biopsy with type I IFN
NML Non-SS sicca

Take home lesson-3
Homing receptors determine both glandular and extraglandular features
1.
Salivary glands normally lack lymphocytes, so their mere presence in an extraglandular tissues imply a lymphocyte aggressive process.
2. Homing to the gland tissue is due to specific receptors/ligands controlled by chemokines/cytokines.
3. Retention of lymphocytes in the tissue is due to specific ligands.
4. Their apoptosis or expansion is regulated through Fas pathways that are modulated by cytokines and bcl-2

Pathogenesis:
Take Home Lessons-4
1. Extraglandular manifestations are determined by lymphocyte homing to tissues-- factors that govern their retention in tissues and their apoptosis.
2. Factors governing their clonal expansion and lympho-proliferation lead to lymphoma-derived from B-cells themselves, T-cells, and dendritic cells.

1. Tissue Homing/Retention of lymphocytes is the key process for accumulation of glandular infiltrates, as virtually no mitotic cells are seen in the gland.
2. Subsequent migration from gland into efferent lymphatic defines re-circulating memory lymphocyte pool.

The endothelial cells attract T-cells by
ICAM’s and Chemokines
Sjogren’s Lip Biopsy
B

The endothelial cells release B-cell chemo-attractants
A B
Ref 63

Endothelial cells attract dendritic cells to home to the gland.

Take Home Points for Homing Receptors : a)Expressed by salivary glands in NOD.scid mice, so expression is independent of cytokines released from the lymphocytes.
b) Thus, the story of Sjogren’s syndrome is not a poor salivary gland that is “beaten up” by the lymphocytes-- but that the glands participate in the homing and pathogenesis of inflammatory cells and subsequent inflammation.

c) The problem with blockade of homing receptors
(whether Tsabri or Raptiva) has been the emergence of rare but devastating problems due to reactivation of papilloma viruses (such as JC) with clinical PML
(progressive multi-focal leukoencephalopathy).

Sjogren’s Syndrome – with parotid enlargement indicates lymphoproliferative tendency

Risk factors for lymphoma a) Germinal centers on minor salivary gland biopsy b) Low complement C4 c) MGUS (esp. IgM-K with RF activity) and mixed cryoglobulin
The T-cells and dendritic cells drive B-cell clonal expansion, particularly driven by BAFF, until a B-cell clone escapes to become a lymphoma.
Germinal Centers in Minor SG Biopsy

• X-chromosome location of Toll receptor;
• X-linked genes for apoptosis;
• X-linked genes for transcription promoter of pro-inflammatory loci including NF-K;
• X-linked control of metalloproteinase release under prolactin hormonal regulation.

factors.
Time course of autoimmune response*
1. Environmental stress is interpreted in context of genetic
2. Antibodies precede disease.
3. Presence of antibody does not mean disease.
Environmental
Stress
(virus-such as EBV )
(apoptotic fragment)
Innate
Immune system
(Toll receptor)
Autoantibodies
Type I IFN
Immune complex
Genetic
Factors
Acquired
Immune system
(HLA-DR)
T/B-cells
Disease
Manifestations
Time period of years
Ref. 32-33

In genome wide screens, association of IRF5 alleles and Stat 4, with predisposition to development of SS*
* Refs 36-38

• Gender - SS is a predominantly a disease of women.
• Onset and increase of dryness with menopause.
• Increased risk of Klinefelter (XXY) in male SS*
—Toll receptor translocation (BXB model).
• Aromatase knockout mouse gets SS.
• RbAp48--estrogen dependent apoptosis.
• DHEA and CRISP-role in glandular processing.
* Refs 34-40

• I want to stop for questions and see if you would like to break or stay for new approaches to therapy
• Thanks again for inviting mel.

a) Treatment of Dry Eyes and Mouth b) Treatment of Extraglandular
Manifestations --
• Lupus like symptoms-arthralgia, rash
• Neuropathy (central and peripheral)
• Cognitive and myalgia (fibromyalgia)
• Lymphoproliferative

1. Topical therapy of dry eyes and dry mouth: new targets include water transport, mucins, and topical small molecules such as jak 3.
2. Dry mouth symptoms may be “burning” mouth and require treatment as a local neuropathy.

1. Poor correlation of symptoms and objective findings of both dryness and neuropathic symptoms.
2. This poor correlation is the greatest challenge since it involves cortical perception of discomfort
3. The neuro-endocrine circuit in Sjogren’s may provide insight into “fibromyalgia”

• Systemic Manifestations for lupus like symptoms
DMARDs
Hydroxychloroquine
Methotrexate
Leflunomide
Small molecules-Jak3 and Jak ½
Filomodulin (MS approved)
Biologic Agents
Anti-CD20 rituximab and new variants
Anti-BAFF (Benlysta)
Anti-CD22 (Eprumazab)
Taci-Ig and
ICOS
Homing receptors

1. Topical Ocular Dryness
“smart artificial tears”
Mucin
Androgen
Micro-iRNA
Anti-IL-17*
Jak 3 inhibitor*
Metalloproteinase inhibitor
* Currently cyclosporin is water insoluble and irritating

New Approaches to Dryness-2
Oral Agents are better than pilocarpine or cevimeline
… since the gland is not destroyed but is paralyzed by cytokines and metalloproteinases
•
Improved secretagogues
(new muscarinic agents in trial)
•
Anti-cytokine therapy has modest effect only in patients with early disease

New Approaches to Dryness-3
Novel methods of water conservation
• 1. Transport water across the conjunctiva (diquafasol) p2Y2 purine receptor agonist
2. Decrease evaporative loss
(muc 3, muc 5A, lipid)
3.Decrease water reabsorption through membranes of eye by blocking trans-epithelial salt
(and water) channels that drain orbit (compound P552-02) p2Y2 receptor directly transport water across conjunctiva
Tear film
Membranes at the base of the orbit are a major site of ‘water exit” (in addition to the puncta)

Electrical Stimulation with intra-oral device
Strietzel et al (2011) Arth Rheum pg. 63—Abstract misleading
Results: “The active intervention performed better than sham for some secondary outcome measures for dryness frequency. No statistical significance for the parameters oral discomfort, sleeping difficulty, resting salivary flow rate, and stimulated salivary flow rate.”

Reminiscent of Electrical Stimulation (Salitron) in 1980’s
Stellar (1988); Daniels (1992) a) Approved by FDA as a “device” b) Denied by insurance due to “efficacy” c) Price precluded use by patients d) About the same benefit as use of a vibrator
(used intra-oral) or electric toothbrush to stimulate tongue and buccal mucosa
Importance of mechanical stimulation, including the use of lozenges

• Rheumatologists only have one drug: steroids.
• The training of a rheumatologists is how to get the patient to a lower dose of steroids or off them entirely.

• Traditional DMARDs- alone and combination.
• Hydroxychloroquine—works on “antigen processing” by raising pH of antigen loading compartment.
• Hydroxychloroquine is a weak diprotic base that diffuses into compartment for loading and raises the pH of the “endosome.”
• This effect prevents the “loading” of low affinity (autoantigens) onto nascent DR molecules.
• Also, affects the ability to bind to Toll receptors in the lysosome.
• Using this model, new through-put screening of new and better drugs

• Traditional DMARDs- What is new with methotrexate?
• Alone or in combination with hydroxychlorquine to taper steroids;
• Methotrexate polyglutamate may predict efficacy and toxicityhowever, methotrexate polyglutamate levels are still in trial in
SLE and SS;
• Most exciting are the reports of new SNPs to predict methotrexate responsive patients in RA;
• (all the SNPs are in the de novo adenosine pathway).

Systemic Therapies-2
Leflunomide and mycophenolic acidboth have mechanism of action that are similar and analogous to methotrexate
• Methotrexate works on de novo synthesis for purine ribonucleotide pathway.
• Leflunomide and Mycophenolic acid on de novo pyrimidine ribonucleotide pathway.
• These ribonucleotides serve as energy source (mostly for glycosylation) that is required for cell division.
• Unless, adequate rUMP, impaired G1-S transition.

Take home lesson for Methotrexate, Azathioprine, Leflunomide, mycophenolic acid
• All work by inhibiting synthesis of ribonucleotides that serve as an energy source (de novo synthesis pathway) required for G1-S transition of maturation.
• This pathway links p52 and p21 driven apoptosis; p52 is the “sensor” for adequate ribonucleotide level.
• Ribonucleotide synthesis as an energy source for cell membrane synthesis and glycosylation.
• In future, it is likely that we can use SNPs to predict response to these agents based on their enzyme polymorphisms.

• Traditional steroids:
• Prednisolone and methylprednisolone)—cheap and work but side effects
• In general, the issue with steroids is the dose
(less than prednisone 7.5 and duration of therapy)
• Development of “soft steroids”—lotemax-like for effect on NFKb ; this was the basis of p38 map kinase
• Novel I KKB inhibitors that lack effect on weight, bone, etc.

•
Biologic Agents
—the new “holy target” based on success in RA; however, biologics have been disappointing in SLE and SS (we will deal with these later in talk).
•
Cytotoxics such as cyclophosphamide—although we worry about cyclophosphamide, we need to ask how much is actually justified if we use carefully and limit cycles.
• We worry about marrow depletion but yet hematologists use it to mobilize “stem cells” into the periphery.
• In order to “cure” immune disease—we must reset the repertoire using cyotkines and growth factors. There will probably be a role for cyclophosphamide in this process.

Available biologic therapy for Sjogren’s-1
•
TNF antagonists- one initial report of success with infliximab*
•
Repeat studies not replicate early success
•
Repeat infliximab study (multi-center, more patients)*
•
Etanercept*
•
*Refs 43-47

• B-cell depletion efficient in periphery;
• Decreased lymphocytes in gland biopsy;
• Surprisingly, little change in serum
BAFF or IgG levels;
• Well tolerated.

Rituximab failed the two pivotal
FDA trials in lupus— both renal and non-renal
• These trials were poorly designed.
• The steroid dose was too high.
• The patients were too heterogeneous.
• The drug worked, but so did the placebo
(standard of care).
• The finding of several cases of PML
(progressive neurodystrophy due to JC virus).

Rituximab Most Consistent Role for
Hematologic Features in SS
*lymphadenopathy,
*pseudolymphoma,
*thrombocytopenia
* mixed cryoglobulin
*low grade lymphoma
As it will not have “FDA label,” it will be an off-label use, and expense in US will limit its use-- as insurance will not cover.

1. Only small changes in tear/saliva flow and only in patients with early disease;
2. Changes in salivary gland biopsy with improvement in foci score;
3. B-cell depletion as expected;
4. Change in T-cell repertoire (CD25+ T-reg) in some patients.

Important lesson about biologics from Rituximab
When you deplete B-cells (rituximab):
1.
Create an excess of circulating BAFF in comparison to the number of B-cells that bear BAFF-Receptor;
2.
This excess of ligand stimulates a round of cell division not only of B-cells but of B-cells;
3.
Any round of cell division leads to activation induced cell death (AICD) and opportunity to re-shape the repertoire.

will depend on changing the repertoire-a) T-regs to modulate auto-immune cells; b) Alteration of homing receptors; c) Regeneration of damaged target organs.

• Higher Affinity for B-cells;
• Had Fc receptor for complement and B-cell depletion;
• Clinical trials halted due to increased infection (although mostly at non-US sites).

• Anti-BAFF (Benlysta) antibody;
• Approved for SLE by FDA;
• The SS subset of SLE (SS-A) did not show significant improvement compared to SLE cohort;
• Although the Benlysta subset did better than with no treatment, the patient and physician Global
Assessment was not significantly different from placebo and marginally different than low dose.

• Anti-CD22 (Epratuzumab)—another B-cell marker;
• Initial clinical trials in SS (and SLE) plagued by production of a uniform product (problems in glycosylation), so 6 different lots needed with interruption of protocol;
• New Drug Manufacturer and preliminary studies indicate safety;
• Any FDA approval for SS will require increased saliva and tears. Expect at best, results similar to rituximab.

1 Anti-CD22 antibody : initial results inconclusive and repeat trials in progress*
2 Antibody to IFN-a (Medi 545) in SLE*
(Dan Wallace at ACR (2007) and recruiting in Japan)
3 Antibody to type II (gamma) IFN (fontolizumab)
5.
Antibody to BAFF-R, April and TACI-Ig*
6.
Benlysta (free BAFF) and Ly2127399 (Lilly’s anti-
BAFF)(membrane and free)
7.
Bortezomib (protesome inhibitor)
*Refs 60-61 and clinicaltrials.gov

• Raptiva (stopped)
• Thalidomide (stopped)
• DHEA—past NIH trial vs. SLE trial
• Mycophenolic Acid
• Rituxan and biogeneric

• The molecular biology of phosphofingolimod is thought to lie in its activity at one of the five sphingosine-1-phosphate receptors
Stromal cell has
Sphingosine receptor
Lymphocyte is retained in the lymph node until the sphingosine ligand is
“removed”

Fingolimod-2
(recently approved for multiple sclerosis)
• It can sequester lymphocytes in lymph nodes, preventing them from moving to the central nervous system for autoimmune responses in multiple sclerosis, and was originally proposed as a anti-rejection medication indicated posttransplantation.
• It has been reported to stimulate the repair process of glial cells and precursor cells after injury.
• Fingolimod has also been reported to be useful in murine lupus and Sjogren’s.

Problem will again be the risk of PML
— a rare but devastating complication
• May be acceptable in life threatening diseases such as multiple sclerosis;
• Unclear if the FDA will approve such drugs for “quality of life” issues such as dryness or fatigue;
• The global assessment by physician and patient has been the huge limitation in FDA approval.

Fingolimod provides rationale for new therapies that interfere with homing
3.
When the homing receptor encounters vascular adhesive molecules, the lymphocyte enters tissue.
CD4+
B cell
Blood
2.
Lymphs migrate through blood to tissues.
1 .
T- and B-cells have surface “homing receptors” when generated in node or marrow.
4. Pearl:
Failure to bind to homing receptor in 72 hours leads to obligate apoptosis of the lymphocyte.
This is why we do not become one large lymph node.

Caution:
PML
(progressive multifocal leukoencephalopathy)
• Due to reactivation of polyoma virus (JC) in CNS
• We have seen with agents: that alter homing:
– Natalazumab (Tsabri)
– Efalizumab (Raptiva), and
• B-cell depleting agents (Rituximab);
• SS and SLE patients already handle polyoma poorly, as evidenced by higher frequency pap smear abnormalities.

1. Neuropathy—peripheral and central;
2. Chronic fatigue and vague cognitive impairment;
3. Lymphoproliferation;
4. Accelerated cardiovascular complications.

• Poor correlation between symptoms and objective findings:
– Eye pain- does not correlate with tear flow;
– Mouth pain-not correlate with saliva;
– Peripheral neuropathy-not correlate with nerve biopsy;
– Cognitive-not correlate with acute phase reactants.

Fatigue
Cognitive
Dry eyes and dry mouth
Nerve pain
Fibromyalgia:
The elephant in the Room

• We will need to learn a new vocabulary about the perception of pain and how it is modulated by cytokines.
• The key term is the “plasticity” of the nervous system. How the perception of pain is modulated by cytokines of the
“stress axis.”

Neuroplasticity in Pain Processing 1-3
100
80
60
Cytokines alter pain perception
Hyperalgesia
3
Pain state
Allodynia
Normal
40
20
0 innocuous noxious
Stimulus Intensity
1. Woolf CJ, Salter MW. Science.
2000;288:1765-1768.
2. Basbaum AI, Jessell TM. The perception of pain. In: Kandel ER, et al, eds.
Principles of Neural Science.
4 th ed. 2000:479.
3. Cervero F, Laird JMA. Pain.
1996;68:13-23.

The Pain Roadmap: Peripheral and
Central Nervous System Landmarks
• The neural processing of pain involves the:
– peripheral nervous system
– spinal cord
(central nervous system)
– brain (central nervous system)
Central
Nervous
System
Peripheral Nervous
System
Brain image courtesy of ATI

Brain Regions that May Modulate Pain and
Emotion 1-4
Regions Found on Functional MRI
Both
Pain
Somatosensory Cortex
Insular Cortex
Prefrontal Cortex
Thalamus
Hippocampus

1. Topical therapy and ability to stimulate saliva or tears remains inadequate.
2. Treatment of extraglandular manifestations such as arthritis, rashes, hemolytic anemia, or lymphomas is rapidly improving.
3. The treatment of the neuro-endocrine manifestations (cognitive impairment and fatigue) remains inadequate.

I would be happy to entertain any questions now or later.
The slides are available to you for your use at
RobertFoxMD@mac.com

Rituximab (anti-CD20)*
1.
Multiple studies of small number of patients and single center trials
•
•
•
2.
General Conclusion-a) useful in extra glandular manifestations including:
-- mixed cryoglobulinemia
-- pseudolymphoma (glands or lung)
-- hemolytic anemia and thrombocytopenia b) results in tear/saliva flow not significant except in group of patients with early SS, where increase saliva was statistically increased but still modest improvement and biopsies showed improvement;
3.
Among SLE patients treated with anti-CD20, SS-A subset responded less frequently and had shorter remissions;
4.
After an NIH multi-center trial, company is not pursuing indication.
•
*Refs 47-59

There may be many different triggers in the genetically predisposed individual…
1. Defective apoptosis of glandular cells and clearance of these autoantigens;
2. Viral infection including EBV (in Caucasion and Japanese) and Coxsackie (in Greek patients);
3. Other viral infections (examples of Hep C, HIV and HTLV-1) can mimic SS;
4. Activation of endogenous retroviral fragments.
Ref 1

Anti-SS A antibody
(associated with HLA-DR3) binds to SS-A
SS-A hYRNA
(ds RNA)
To the innate immune system (dendritic cells), hYRNA is a double-stranded RNA and looks like a viral RNA that binds to a specific Toll receptor.

Salivary gland dendritic cells bind to the Fc g receptor to internalize the immune complexes containing SS-A/hYRNA
1.
Immune complex antibody to SS-A
2.
Fcg
R
3.
Toll 3 receptor is in located in the cytoplasm hYRNA
(ds RNA)
SS-A
Plasmacytoid
Dendritic
Cell
4. IFN
Type 1

--The Vicious Cycle -of innate and acquired leads to IFN type I
(links genetic and autoantibody response)
6.
B-cell
Anti-body response
Anti-SS-A in HLA-DR3 pre-disposed female
4.
Dendritic Cell with
Toll Receptor and Fc-g Receptor
3.
Toll receptor
Fc-gamma R
5.
IFN-a
2.
Immune
Complex containing…
______
SS-A/hYRNA
1.Apoptotic Cell

:
• a) Alteration of T-cell subsets (especially appearance of
CD25+/FoxP3 T-regs) after treatment indicates a role in “rebooting the computer.”
•
•
• b) Lymphocytes remain present in the SG biopsy, probably due to BAFF secreted by dendritic cells.
•
*Refs 57-59

Dryness results in the clinical appearance of keratoconjunctivitis sicca (KCS) characteristic of Sjogren’s syndrome
The upper lid literally sticks to the surface epithelial surface and pulls surface mucin layers off.
The Rose Bengal dye retention is like
“rain water pooling in a street pothole”
This test can be done at bedside and allows
“triage” and rapid referral of patients to Ophthalmology

For example
IL-17 plays a key role in decreased secretion of water, proteins and mucin required in tears and saliva

Sjogren’s Syndrome- Cervical Dental Caries

The Body’s 2 Distinct But Interconnected
Immune Systems
ACQUIRED
HLA-DR4 – dependent:
T cells respond to peptide antigens and generate memory cells
Lymphyocytes
(Type 2 interferon signature)
Beutler B et al. Blood Cells Mol Dis . 1998;24:216-230.
INNATE
HLA-DR – independent:
Dendritic cells respond to specific structures found on bacteria and apoptotic
Products (Toll receptors)
Dendritic Cells
(Type 1 interferon signature)