*Fecal Microbiota Transplantation to Preserve Residual Beta Cell Function In Patients With Newly-Diagnosed Type 1 Diabetes Mellitus: The FMT-Preserve-DM1-trial*

The incidence of Type 1 diabetes mellitus (T1D) has tripled in the last thirty
years, and T1D is associated with a lifelong increase ofconsiderable morbidity
and mortality compared to healthy subjects. In fact, T1D diagnosed in childhood
leads to an almost 20 year loss of life-expectancy, more than most childhood
cancers. Notwithstanding decades of intensive research in animals,
theenvironmental factors driving T1D are still unknown and therapeutic
strategies have invariably failed to halt disease progression.

As the increased T1D incidence is primarily observed in subjects who are not
genetically predisposed, environmental factorsincluding altered diet,
antibiotic use as well as mode of birth have been suggested to play a role, and
these factors have invariablybeen linked to changes in the gut microbiome.
Indeed, an altered composition of the fecal microbiota composition was observed
inadolescent T1D patients. Moreover, an increased amount of pathogenic
bacterial species has been observed in fecal samples ofT1D patients at the time
of diagnosis. Interestingly, this altered fecal microbiota is already present
before the clinical onset of T1D and is related to islet autoantibodies.

Interestingly, non-obese diabetic (NOD) mouse studies suggested that
interaction of intestinal microbes with the innate immunesystem is a critical
factor in developing T1DM [16], most likely by inducing an altered T-helper
cell type 17 (Th17) population in thesmall-intestinal lamina propria. One of
the current hypotheses linking the gut microbiome to immunological tone is
production ofmicrobial metabolites such as the short-chain fatty acids (SCFAs).
Production of these compounds is indeed altered in T1D, and thebest known SCFA
butyrate is known to stabilize T-cell function in mice. Furthermore, irritation
of the pancreatic duct by microbiota inthe proximal gut may contribute to beta
cell inflammation. By introducing beneficial fecal microbiota to the proximal
gut, theorganisms that alter immunological tone and irritate the pancreatic
duct may be attenuated, resulting in improved beta cell functionand
restoration. Thus, intestinal microbiota, their metabolites and their
associated gut immune system alterations, may eitherpromote or protect from
beta cell autoimmunity. We hypothesize that if one is able to shape the (small)
intestinal microbiota withfecal microbiota transplantation (FMT) it may be
possible to stabilize or even reverse β-cell destruction, reducing exogenous
insulinneeds and subsequently associated complications in T1D.

FMT is a promising treatment for T1D, not only because of potential efficacty,
but also because it is a safe procedure, that in our institute has been
performed >500 times without any serious adverse events. In short, a fecal
microbiota suspension is deliveredthrough a duodenal tube after large bowel
lavage. This procedure is usually repeated 3 times with a 2-month interval.
Using anextensive screening protocol for infectious agents in accordance with
European guidelines, to date no infections attributable to theprocedure have
been recorded in our group. The FMT procedure itself is tasteless and odorless
and in general generates few side-effects outside of the discomfort of placing
a duodenal tube. Based on this notion, we initiated in 2013 a randomized pilot
trial using repetitive donor (healthy donor) vs. autologous (own) FMTon
residual β-cell function in new-onset T1D (DIMID trial, publiced in Gut 2020).
Newly diagnosed male/female patientswith T1D were included and randomized.
Moreover, healthy aged matched males/females were used as donors. Surprisingly,
autologous FMT in 10 new onset T1D subjects had a significant (p<0.01) effect
on preserving residual β-cell functionas determined by Sustacal Boost (Nestle
HS) stimulated C-peptide AUC0-120min response after 12 months, whereas donor
FMT in10 new onset T1D had a less obvious beneficial effect and showed overall
a similar β-cell decline as seen in other trials withplacebo use. We have found
several changes induced by both donor and autologous FMT on gut microbiome
composition andidentified several bacterial strains and plasma metabolites and
T-cell signatures that predicted response to FMT.

The immunological consequence of flooding the small intestine with large bowel
microbiota by FMT may be an importantimmunological event. Based on our pilot
data, we therefore formulate a paradigm shift, in which we hypothesize that the
molecular mimicry against microbiome-associated antigens that drives T1D can be
exhausted by challenging the immune system though FMT administered in the
duodenum. Exhausted T-cells are ineffective T-cells that express high levels of
the so-called check-pointproteins that inhibit immunological responses. Slow
progression of T1D is linked to more exhausted CD8 cells in infiltrated
islets[23], while increased circulating exhausted T cells predicted response to
anti-CD3 therapy in T1D.

We hypothesize that the beneficial effect of autologous FMT in newly diagnosed
T1D individuals can be maximized by using fecal microbiotica from individuals
with T1D and a durable highly preserved beta cell fraction, as these
individuals likely carry a microbiome with an overrepresentation of the
microbiota that dampen the beta cell directed immune response. Our aim is to
use FMTs to shift the *permanent honeymoon-phase* from a rare to the default
phenotype. This will be, if achieved, a major breakthrough for the treatment of
T1D.
*

In this study we will investigate whether changes in gutmicrobiota composition
induced by fecal transplantation from *preserved* type 1 diabetes mellitus,
administered through a small intestinal tube, has beneficial effects on
residual beta cell function and immune status in newly type 1 diabetes
mellitus. A parallel objective is to see which small (duodenal biopsy) and
large intestinal (fecal samples) microbiota predict these clinical changes.

This is a double blind randomised clinical trial. Patients will be treated with
infusion of a placebo or allogenic feces by duodenal tube after bowel lavage.
Newly diagnosed type 1 diabetes mellitus patients will be randomized to the
following 2 treatment arms:
1. Multiple allogenic Type 1 diabetes donor fecal infusions at 0, 8 and 16
weeks.
2. Multiple placebo infusions at 0, 8 and 16 weeks.

At baseline, 6, 9 and 12 months residual beta cel function, microbial
composition, and immune celfunction will be characterised.

Patients will be treated with infusion of allogenic feces by duodenal tube
after bowel lavage.
Type 1 diabetes mellitus patients will be randomized to the following 2
treatment arms:
1. Multiple allogenic Type 1 diabetes donor fecal infusions at 0, 8 and 16
weeks.
2. Placebo infusions at 0, 8 and 16 weeks.

Participant may benefit in terms of helping to further unravel the relation
between the gut microbiome and residual beta cell function.The DIMID study was
promising, suggesting that manipulation of the gut microbiome may indeed
preserve the residual beta cell mass. This study will investigate if FMT is
more beneficial when individudal with type 1 diabetes and a highly preserved
beta cell mass are used. Participants may benefit from an increased redisual
beta cell function, which is associated with a lower risk of diabetic
complications and hypoglycemia. In the long therm, the FMT procedure may be
refined to a probiotic formation or a combination of potent microbial
metabolites andantigens that induce immunotolerance. Therefore, the risks
described below outweigh the potential gain from this study.

For the mixed meal test, morning long- and short acting insulin must be
withheld. This carries a small risk of hypo- andhyperglycemia. The study
participants will be carefully instructed and no major risk is expected in the
context of this procedure,especially given the current use of continuous
glucose-monitoring technologies. The placing of the intravenous cannula in our
studycan be an unpleasant experience for the subjects and may result in (self
limiting) bruising.

Gastroscopy is a procedure associated with discomfort, but when participants
are well fasted is very safe. The required fasting isassociated with a small
risk of hypoglycemia, for which participants will be adequately instructed. In
the age of continuous glucosemonitoring this risk is also very low.
In our centre, FMT procedures have not been associated with major adverse
events, and donors are extensively screened tomitigate the risk of potential
infections by the FMT procedure.