Damir Janigro, PhDFlocel, Inc.Cleveland, OH

Blood-brain barrier and immunity in TBI

Flocel Inc., a sales pitch

• Started in 2005• First biotech to focus exclusively on BBB research• Developed, patented and manufactured in vitro models of the BBB based on intravascular flow• Expanded into serum markers of BBB failure and immune consequences of TBI

BrainEndothelium

(BBB)

Glucose

Proteins

UNDERLYING MECHANISMS OF BBB MEASUREMENTS

PETtracers

MRItracers

BrainEndothelium

(BBB)

Serum proteins (e.g., albumin)

MRITracer, (e.g., Gd++)

UNDERLYING MECHANISMS OF CONTRAST/CSF SAMPLING

The mammalian blood-brain barrier:Scoring BBB function

1

100

20

Is the BBB really important?

What happens if your barrier is leaky?

When would you like it to be leaky?

When is it best to have it intact?

What is your “BBB score”?

BBB dysfunction

Seizures

Measuring the BBB the hard way…

Measuring BBB function Radiologic determination

Measuring BBB function Morphological determination

• Large molecules are restricted to the cerebral vasculature

• Regions exists where a BBB is lacking (hypothalamic nuclei)

How to detect BBB changes in vivo?

Non invasive markers of BBB function

Gradient for vectorial

trans-BBBflux

Emmi and JanigroJ. Neuroscience, 2000

S100b as marker of BBB function

0.25 mmS100b

Osmotic BBBD

Osmotic opening of the human BBB

• The human Blood-brain barrier disruption (BBBD) protocol represents an efficient approach in the treatment of primary brain lymphomas.

• Blood-brain barrier disruption (BBBD) is performed using a hyperosmolar bolus of mannitol to temporarily shrink the endothelial cells that form the barrier allowing chemotherapic drugs (Methotrexate, MTX) injected subsequently to reach the tumor more efficiently.

• BBBD is an angiographic procedure. While patients are under general anesthesia, a catheter is inserted into the femoral artery and finally placed into the carotid or vertebral artery. Mannitol + MTX or MTX alone are then administered.

BBB disruption precedes seizures

Conclusions• Peripheral markers of BBB function are brain protein that

appear in blood after BBB disruption

• Use of these marker may overcame the obstacles of traditional means of BBB measures such as contrast MRI, CSF sampling, etc.

• BBB disruption can cause seizures in non-epileptic individuals

• Seizures are preceded by BBB disruption in epileptics

Death

TBI - concussion

Ding / m-TBI

Normal

S100BSerumlevels

Serum S100B, ding injuries and mTBI

S100B sensitivity for m-TBI

Approximately 300,000 concussions happen annually with the majority occurring in high school and college football players. Up to 40% of football participants suffer a concussion or pathological repetitive head hits on an annual basis, the majority of these going unreported (Cantu et al., 2009; Pellman et.al., 2004)

A recent NFL showed that 50% of players returned to the same game after sustaining a concussion (Pellman et.al. 2004). Evaluation of brain damage remains an unmet issue

Figure 3 Riddell’s Sideline Response System. Left: Helmet-mounted accelerometers, antenna that receives impact data real-time, laptop computer with software that displays impact information numerically, as well as graphically (Right)

Biomarkers as signaling molecules

• Biomarkers may contain or belong to Damage-Associated Molecular Patterns– Hyaluronic acid– HMGB1– ATP– S100B– Heat shock proteins

Besides of being clinically useful, are biomarkers biologically active?

What happens to S100B when it shows up in serum?

IP injection of GREEN fluorescent S100B Organ-specific levels of exogenous S100B

ICC for (RED) endogenous S100B

CA2

Neuronal cell bodies

Exogenous S100B Endogenous S100B

Sem. t.

Stroma

Sertoli cells

A1 A2

B1 B2

StromaSem. t.

Sertoli cellsSem. t.

CD4+

S100B+

S100B+Alexa S100B

Alexa S100B

50 µm

BR

AIN

TES

TIS

mRNA

mRNA

50 µm

50 µm

50 µm

200 µmA1 B1 C1

C2A2 B2

50 µm

Alexa S100B

100 µm

Skin Thymus Lymph nodemRNA mRNA mRNA

CD4+ S100B+

Spleen CD4+ lymphocytestake up circulating S100B

Prognostic significance of BBB markers

• Besides of playing a role in the detection of acute events, what can markers of BBBD predict?

• Do markers of BBBD leave a “trail” that can be detected even long after the initial event is over?

Prognostic significance of BBB markers• S100B (and GFAP) trigger an autoimmune response

characterized by autoreactive IgGs in serum

• This autoimmune response may lead to structural (as seen on DTI) and behavioral changes

• Are these immune changes persistent during the life time?

10%

25%

75%

90%

50%

99%

1%

10%

25%

75%

90%

50%

99%

1%

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

2.2

2.4

2.6

2.8

S100

B A

uto-

Ant

ibod

y (A

U)

SVID +SVID -

Antigen unmaskingCytokine Release

InflammationTREG dysregulation

No autoantibody generation

Induction of autoantibodies

Mechanical BBB disruption and cell

damage

Other Risk Factors• Ageing• Genetics• Vascular Disease• ROS

Chronic inflammation“Silent period”

Traumatic brain injury

ImmunosuppressantEarly intervention

Progressive neurodegeneration and

Alzheimer’s disease

Biomarkers

Why a BBB?

• No room for extracellular space– Large diameter fibers, myelinated fiber tracts– Poor management of water or osmotic shifts

• Lack of lymphatic drainage• Rigid skull, fairly inflexible dura mater

• Isolate CNS from systemic influences– Potassium and pH homeostasis are more rigidly controlled

in CNS compared to blood– Hormonal segregation– Controlled uptake of nutrient, clearance of waste

"NPH MRI 272 GILD" by © Nevit Dilmen.

Is there a mechanism of lymphatic drainage in the CNS?The “glymphatic” vessels

UCHL1 vs.QALBS100B vs.QALB Combined vs.QALB

These findings rule out glymphatic clearance as means of S100B and UCHL1 appearance

Conclusions

• Markers of BBB function are useful in the diagnosis of mTBI and other neurological conditions

• Protein released across a leaky BBB have an intrinsic autoimmune potential that depends of IL-6 and TGF-B levels in blood

• “Glymphatics” may be a new source of BBB or brain markers

• Understanding clearance of brain waste may be important to interpret the meaning of peripheral markers

Acknowledgements

Nicola Marchi, PhDTom Masaryk, MDPeter Rasmussen, MD

Gerald Grant, MDJeff Bazarian, MD MPH

NIH NINDSNIH NHLBINIH NICHDNIH NIMHNIH NCATSAmerican Epilepsy FoundationDiasorinELEKTAVaccinexAmerican Heart Association

Normal phenotype

AutoimmunephenotypeAnti-S100Bantibodies

Figure 1 The functional anatomy of the inflammatory reflex

Pavlov, V. A. & Tracey, K. J. (2012) The vagus nerve and the inflammatory reflex—linking immunity and metabolism Nat. Rev. Endocrinol. doi:10.1038/nrendo.2012.189

Figure 2 Molecular mechanisms of cholinergic control of inflammation

Pavlov, V. A. & Tracey, K. J. (2012) The vagus nerve and the inflammatory reflex—linking immunity and metabolism Nat. Rev. Endocrinol. doi:10.1038/nrendo.2012.189

GRAY MATTER AUTOANTIGEN accession MW MEAN DIFFERENCE CTE vs. CONTROL

histone H2A.J 29553970 14 1.3

histone H4 4504301 11 0.8

cystatin-B 4503117 11 0.4

plakoglobin 12056468 82 0.4

MAP-1B 31563518 14 0.3

synaptophysin 27764867 34 0.15

plectin isoform 1 41322916 532 0.1

protein S100B 5454034 11 0.1

WHITE MATTER AUTOANTIGEN accession MW MEAN DIFFERENCE CTE vs. CONTROL

neuronal membrane glycoprotein M6-a isoform 2 3.88E+08 27 1.1

vesicle-associated membrane protein 2 1.72E+08 13 0.6

dermcidin preproprotein 16751921 11 0.4

ATP synthase subunit gamma 50345988 33 0.1

ATP synthase subunit d, mitochondrial isoform a 5453559 18 0.09

plasma membrane calcium-transporting ATPase 4 48255957 134 0.09

calcineurin subunit B type 1 4506025 19 0.06

Table 1: Most significant autoantigens isolated by antigen retrieval from brain IgGs in CTE brain. These were selected based on the criteria listed below under Milestone 1 in the experimental approach.

Figure 1 The functional anatomy of the inflammatory reflex

Pavlov, V. A. & Tracey, K. J. (2012) The vagus nerve and the inflammatory reflex—linking immunity and metabolism Nat. Rev. Endocrinol. doi:10.1038/nrendo.2012.189

Conclusions

• The brain is affected by sub-concussive episodes in a way that is comparable to mTBI

• Markers of diagnostic value (S100B leakage in blood) exist and teach about early changes after impacts occur

• Delayed consequences of sub-concussive heads are immunologic and treating inflammation may be a better, more effective way to prevent CNS disease

Diagnostic significance of BBB markers• Excellent negative

predictive value for TBI sequelae

• Detection of repeated sub concussive head hits in sports

• Sensitivity comparable to spinal fluid albumin/IgGs, CT and MRI contrast scans

• Low positive predictive value for any neurological disease since BBB dysfunction is a leitmotif in several pathologies