By

Prof. Dr. Mona El Samahy

Head of The Diabetes Clinic
Children’s Hospital, Ain Shams University
 Diabetes Mellitus (DM)

• A group of metabolic diseases characterized by

hyperglycemia, resulting from defects in insulin
secretion, action or both.

• The chronic hyperglycemia of diabetes is associated with

long-term damage, dysfunction and failure of various
organs, especially the eyes, kidneys, nerves, heart and
blood vessels.

• It is the most common endocrine – metabolic disorder of

childhood and adolescence.
Etiology and Pathogenesis
Pathogenesis of type 1 diabetes
 The clinical and biological characteristics of
different subtypes of type 1 diabetes

Type 1a Type 1b Type 1c

Signs of anti-islet + - -
autoimmunity
Duration of symptoms 8 months 7 months < 1 week
Before diagnosis
Ketosis, ketoacidosis frequent frequent constant
at diagnosis
Blood glucose levels ↑↑ ↑↑ ↑↑↑
at diagnosis
HbAlc at diagnosis ↑↑ ↑↑ Normal or
slightly elevated
The war on Diabetes Mellitus
 The war on DM

• Starts by attempts to identify environmental

determinants of DM in the young which
focus to identify dietary, infectious, and
other environmental triggers of type 1a DM
in genetically at risk children.
• Identification of such triggers could lead to the
study and implementation of primary
prevention.
 1- Primary Prevention

Through

Avoidance Immunization
of dietary against an
offender infectious trigger
 TRIGR STUDY
• The trial to reduce IDDM in the
genetically at risk is ongoing.
• Its focus is to determine whether delayed
exposure to or avoidance of certain
food proteins will reduce the risk of
developing type 1 DM in infants at
Genetic Risk.
• Type 1a DM is the autoimmune form whose major genetic
risk traits are defined on certain HLA DR/DQ alleles.
• The genes mutation will not be correctable by avoidance of
dietary or infectious agents.
• However, insights gained from these mutations may
facilitate ultimate prevention strategies such as
immunization with specific segments or epitopes of the
insulin molecule to limit autoimmune self aggression.

The results of primary prevention trials are still unknown

(June, 2008)
 2- Secondary Prevention
• Diabetes Prevention Trial (DPT-1) for type 1 DM used Insulin
as a possible means to suppress the immune response.

• Other intervention studies (Blocking progression of auto-

immunity) by:
• CTLA-4Ig : block T-cell activation
• Rituximab (anti-CD20) : block B-cell from presenting
antigens
• GAD vaccine : interfers with presenting islet cell
antigens
• Anti-CD3 : deletes activate T-cells
 2- Secondary Prevention

Even if these studies identify one or several

magic bullets for arresting or preventing the
progression of DM, we would still be
confronted with the problem of curing or
reversing established disease.
Insulin has been the gold standard
therapy for DM since its discovery.
It remains the only pharmacologic
therapy for type 1DM
 Before Insulin….What’s After?

Before insulin was

discovered in 1921,
everyone with type
1 diabetes died
within weeks to
years of its onset
JL on 12/15/22 and 2 mos later
Insulin Therapy in Type 1 Diabetes
is a must………
 Ideal Insulin Regimens Type 1 DM
The challenge is to
Come as close as possible to normoglycemia and reduce hypoglycemia.

Minimizes nocturnal
hypos Optimize
FBS

Minimizes late morning and

afternoon hypos
Basal-Bolus Regimen
 Although molecularly produced
insulin administered 4 or 5 times per
day can provide a physiologic
regulation, yet it is not able to prevent
diabetic complications that account for
the morbidity and mortality of diabetic
patients.
 Also insulin does not eliminate the
type1 DM hallmark : BETA- CELL
SPECIFIC AUTOIMMUNITY.

In other words

Insulin is not a cure

 A successful cure must meet
the following criteria:

1. Either replace or maintain the functional

integrity of the natural insulin-
producing tissue.
2. Must at least control the autoimmunity
or eliminate it altogether.
3. Easy to apply to a large number of
patients
 Criterion 1
Has been partially realized by allogenic islet
transplantation.
Development of Insulin-Secreting Pancreatic-Like Cells From
Mouse Embryonic Stem Cells.
 Criterion 2

Has been partially realized using

monoclonal antibodies specific for T-cell
surface proteins.

Criterion 3

Has yet to be realized

New Approach to
(fulfill the 3 criteria)
 A non-insulin based therapy,
With a focus on
Cell-Based Immunomodulation
Hoping for cure
 All proposals of treatment in the
past and recently are depending on
discoveries in understanding the
immune pathophysiology of
destruction of pancreatic β cells that
results in DM.
 Nature of autoimmunity in type 1a DM

• The major affectors of β-cell destruction

are T cells reactive to β-cell-specific
antigens.
• A strong genetic predisposition is the key
for genetic susceptibility loci that
affect genesis, function and survival of
immune cell subsets including T cells and
dendritic cells.
 Type 1 Diabetes as immune destruction of beta
cells

T-cell T-cell

Inflammatory APC
cell
(IL1, TNF, NO)
Beta cell
Fas antigens
Cytokines
Cytotoxic
Th1 (IL2, IFN) Beta cell
T-cell

Beta cell
death

B-cell
Type 1
diabetes
Antibodies
(ICA, IAA, IA2, GAD65)
 The epitope spreading phenomenon i.e.
expansion of newly recognized antigens
observed in the islet inflammation is due to:

Islet-reactive T cells Generation and survival

that were generated of T cells activated in
in the thymus early the periphery by these
in ontogeny new antigens
 There is a pathologic vicious circle of continuous
presentation of old and new antigens, collected by the
dendritic cells (DC) from the newly destroyed β-cells to
naive T cells in the pancreatic lymph nodes  eventually
go back to the pancreas to kill other β-cells.

This vicious circle does not allow the recovery of the

insulin secreting cells, even when the physiologic
homeostasis process tries to substitute the lost cells
with new cells.
 Therefore, an approach developed to stop
autoimmunity in already diabetic patients,
could possibly facilitate the recovery of
autologous insulin production.

So, safe induction of an autoimmunity – free

status might became a new promising
therapy for type 1a DM.
In the past 20 years, researches have made
many promises to cure type 1 DM. Only
recently it has been possible to clinically
implement a limited number of
successes.
Autologous Bone Marrow Transplantation.

• Autologous transplantation of hematopoietic stem cell

enriched BM was used to treat type 1 DM.

• The effects were limited to simple postponement of

DM recurrence, i.e.: just delayed by the time
necessary for the transplanted BM to reorganize
itself and to reestablish all of its immunocompetent
cell subpopulations.
 Autologous Bone Marrow Transplantation.

• The autologous BM did not change the patient's

genetic characteristics under which tolerance for
the insulin – producing beta cells was not achieved
in the first place.

So, Autoimmunity easily recurred.

 BMT can encourage
beta cell regeneration
Regeneration of beta cells
in diabetic NOD mice
A) Infiltration of T cells destroys
beta cells
B) After BMT infusion, signs of
autoimmune destruction
diminish
C) Insulin-producing cells start to
appear (red)
D) 4 mo after BMT infusion, see
proliferation of insulin-producing
cells (red)
Another approach was developed to
modify the genetic characteristics of a
patient BM-derived stem cells in vitro
which will result in T-cells or (APC) that
cannot initiate an autoimmune response.
(maybe transferable to clinical trials in the near
future)
This was achieved by:
• Transduction of BM-derived stem cells with
protective (non diabetogenic) HLA transgenes.
• Infusion of the engineered cells will culminate in
repopulation of the thymus with protective
HLA-expressing APCs.
• This in turn will decrease the probability that T –
cells with diabetogenic T cell receptors mature
and exit into the periphery.
• It is postulated that the Rest period from the
autoimmune destructive process permits
regeneration of endogenous β cells from
pancreatic stem cells within the pancreatic
ducts or islets.
• The suppression of the immune environment
by several means was conclusively shown
to be the mechanism that resulted in
recovery from type 1a DM in NOD mouse.
Two novel recent mechanisms involving
cell based immunomodulation aimed at
producing a cure for type 1a DM :

1. Diabetes-suppressive autologous DC.

2. Diabetes-suppressive microsphere vaccine

1. Diabetes-suppressive autologous DC.

The first clinically adapted immunoregulatory

cell therapeutic
 Why dendritic cells?
• DC are the body's sentinels largely
responsible for host surveillance against
micro-environmental anomalies
including pathogen invasion, infection,
and damaged tissue architecture, while
coordinating the mechanisms of self
tolerance.
 Why dendritic cells?
• DC continously traffic throughout all body
tissues sampling molecules from their
surroundings, where it is believed they
maintain potentially autoreactive
immune cells in quiescence either
directly or via indirect regulatory
immune cell networks.
 When DC encounter local disruption of tissue
architecture & elevated proinflammatory signal from
infected cells:

Undergo maturation Migrate away from the

through a series of site of danger and into
internal changes. the closest LNs.

Within the LNs, the DC, as a powerful APC,

initially interacts using its class I or class II
MHC / peptide complex with the TCR
present on a naïve T cell. This will constitute
the so-called first signal for T-cell activation.
• To bring a T cell to full activation, a
subsequent contact between receptors on both
APC & T cell (Co receptors) is necessary.
• Co-stimulatory molecules present on the APC
with their counterparts on the T cell interact
to further stabilize the signal of activation
between the two cells, thus providing
the second signal.
• Absence of this co-stimulatory molecule binding
and consequently lack of secondary signal
generation has been shown to lead to
impaired activation of the responding T cell,
eventually bring it to functional anergy or
apoptosis.

• This is indeed the outcome of many

immunosuppressive strategies aimed at co-
stimulation blockade.
• Many investigations support the concept that
functionally immature DC (characterized by
low to absent co-stimulation) lead to Immune
hyporesponsiveness.
• Exogenous administration of functionally
immature DC achieves long term and stable
allograft survival in a variety of mouse and
rat models and prevents a number of
autoimmune diseases.
 Recently

In vitro administration of nuclear factor-kappa

β (NFκB) decays to DC as well as direct
targeting of CD40, CD80 and CD86 with
antisense oligodeoxyribonucleotides (AS-ODN)
reduces co-stimulatory molecule levels
producing functionally immature DC capable
of preventing or reversing new-onset DM in the
NOD mouse.
• Numerous clinical trials have safely
used DC-based treatments for
cancer therapy providing the basis
for clinical adaptation of DC
administration for Type 1aDM ttt.
• A National institutes of Health funded protocol
approved by FDA is currently underway phase I
clinical trial with an adult (18 yr or older) cohort
documented with Type 1aDM of at least 15-yr
duration.

• Leukocytes are obtained from the patient by apheresis

and DC are generated in vitro and engineered in
Good manufacturing practice facilities with the
addition of AS-ODN.
• These DC which express low levels of CD40, CD80 and
CD86 are injected into the patient by ID route at an
anatomical site proximal to the pancreas.

• DC will migrate to the nearest LNs where they will start

to interrupt the vicious circle that maintains islet-
specific inflammation i.e., insulitis.

• This therapeutic approach should be more successful

when DC injections start close to the clinical onset of
DM.
 IN THE PANCREAS

• DC acquire β-cell specific antigens from

apoptotic cells, leading to the eventual display
of these antigens to T cells in the pancreas-
draining LNs.
• The lack of co-stimulatory molecules will result in
anergizing signal to the T cells, and interrupt
the T cell mediated anti-beta-cell epitope
spreading phenomenon.
• The cessation of the autoimmune diabetogenic insult
should be sufficient to:
 Promote rescue of still present insulin producing
β cells and/or
 Promote neogenesis of other insulin producing
cells in the pancreas, even after the disease onset.

• This trial is underway at (6/2008) & once safety has

been demonstrated, a phase II efficacy trial
will start, involving new onset diabetic patients.
2. Diabetes Suppressive microsphere
vaccine
 Diabetes Suppressive microsphere vaccine

• In spite of the promise of the previous study,

many cumbersome logistical requirements to
generate these diabetes-suppressive DC have
been encountered.

• This may limit the future enrollment of new onset

diabetic children in the efficacy phase of the
trial.
Difficulties:
• Leukopheresis takes 2 or 3 hours to provide sufficient
precursors cells to generate the number of DC
necessary for six to eight injections.
• The obtained DC should be exposed to AS-ODN in GMP
facilities in which the laboratory practices are
frequently difficult to reproduce. GMP facilities are
frequently located far away from the clinic where the
patients are treated.
• Many DC are lost during the freezing/thawing procedures.
• In an effort to avoid these steps, an alternative
method to stabilize DC immaturity directly
in vivo using microparticle carriers of
immunomodulating agents like AS-ODN
(microsphere delivery system) have been
done.
• This microparticle carriers (microsphere
delivery system) is directed against 3 key
costimulatory molecules:

CD40
Essential for normal
CD80
immune response
CD86
• When injected in vivo, these microspheres
attract DC, which phagocytose the
microsphere particles  the microsphere
particles are deformulated inside the DC
 AS-ODN release  block the
expression of the genes to which they are
targeted.
• This proof technique reverse or prevent type
1aDM in mice
• This microsphere delivery system in
which AS-ODN have been
incorporated, is named Baxter
Healthcare's PROMAXX®.

• The inert PROMAXX microsphere

technology has been shown to be
safe and effective in human trials.
• More importantly, when administered
in vivo, this technology is neutral
with respect to DC maturation
state compared with the known
immunostimulatory properties of
other microsphere formulations.
• Other polyplex formulations have an inherent
capacity to induce the upregulation of co-
stimulatory proteins at the DC surface,
whereas the PROMAXX technology does
not.
• This neutrality on DC maturation is a critical
criterion in adopting microsphere chemistry
for immunosuppressive objectives where DC
are involved as mediators.
• They predict that once all preclinical studies
are completed, a phase I/II trial can be
initiated.

• The microspheres are simple to manufacture

to clinical grade on a large scale and do
not involve the cumbersome logistics for
DC based therapy.
 This novel microsphere
formulation represents the first
diabetes suppressive and reversing
nucleic acid vaccine that confers an
immunoregulatory phenotype to
endogenous DC.
• How long is a single course
of microsphere particles
effective?

• Does response wane with

repeated injection?

These questions remain to be answered.

But, if it's effective NEW ERA in
treating type 1 DM will appear.
The light at the end
of the tunnel for

Diabetes Cure