Clinical Trial Details
— Status: Recruiting
Administrative data
| NCT number |
NCT04620824 |
| Other study ID # |
0640 |
| Secondary ID |
|
| Status |
Recruiting |
| Phase |
|
| First received |
|
| Last updated |
|
| Start date |
August 3, 2018 |
| Est. completion date |
December 31, 2025 |
Study information
| Verified date |
October 2023 |
| Source |
University of Leicester |
| Contact |
Marco Oggioni |
| Phone |
0 |
| Email |
mro5[@]leicester.ac.uk |
| Is FDA regulated |
No |
| Health authority |
|
| Study type |
Observational
|
Clinical Trial Summary
The human immune response to bacterial blood stream infection (sepsis) and systemic viral
infection are fairly well understood, but we lack details on the most early phases. Better
knowledge of these events would be important for the prevention and treatment of severe
bacterial or viral disease. From models of infection, we have data showing that bacteria
replicate in a specific type of cells in the spleen from where the bacteria then seed to the
blood to cause blood stream infection. In order to gain more relevant data for humans, we
have developed a spleen perfusion model using pig organs. This model confirms our previous
work and most importantly will now allow us to study these events in human organs.
In order to move novel treatment strategies into human trials, we propose to test early
events during infection using human spleens and the cells and tissues derived thereof. This
research is expected to provide data on the relevance of the early events in bacterial and
viral disease, in particular on the role of immune cells. The research includes work with
human cells and tissue obtained from human spleens. In these settings, we will test
pharmacological prevention and treatment of severe bacterial or viral infection (sepsis). The
expected outcome of this work on human organs and tissue is expected to provide evidence that
allows to move new treatment options into clinical trial.
This study is a preclinical research project and does not involve any intervention on
patients, but it involves human tissue. The source of the human splenic tissue for our
research will be spleen tissue removed during radical surgery. The present application
relates exclusively to the ethical approval for the use of spleen tissue removed during
radical surgery and discarded. The utilisation of the spleen or splenic tissue for research
purposes does not alter in any way the surgical procedures at any stage and importantly the
research will involve only anonymity samples.
Description:
This is a preclinical research project and no patient intervention is planned. The project
makes use of anonymised human tissue samples discarded during radical surgery. The spleen
organ samples included in this project will be from patients undergoing elective surgery for
a lesion in the pancreas in the HPB Unit of the Leicester General Hospital. The sample size
of 100 (100 enrolled subjects for 42 spleens) has been determined by power calculations and
is in line with the number of spleens actually removed by elective pancreas surgery over the
past years. No change in the surgical procedure or recruiting results from this study and the
use of the samples. All the experimental work carried out on the spleen samples will have no
effect on the patient.
Experiments include ex vivo organ perfusion and infection with bacteria or viruses. Biopsies
of the perfused 42 spleens are processed for in vitro tissue slice and macrophage cell
culture. The resulting primary cell cultures and tissue slices will be stored frozen for
future research. All in vitro studies on the 42 organs, and the tissues and cells derived
from them, will evaluate the effect of therapeutic interventions (antibodies, receptor
antagonists, antibiotics and other molecules) on the capacity of splenic cells to clear
invasive bacteria or viruses. The experiments will be carried out each time a spleen sample
will be available.
The detailed technical description of the experimental strategy including the strategy for
storage and discarding of the human tissue will be as follows:
1. Whole organ ex vivo perfusion: This work is done to stabilise the organ, infect the
organ and then to derive from it tissue slices and cells for primary cell culture. We
have successfully set up a normo-thermic ex vivo porcine spleen perfusion model. This
expertise allowed us to define the parameters for organ collection, transport and
perfusion of porcine spleens for up to six hours and importantly develop and infection
model. Infection showed conservation of capacity to remove bacteria from blood and good
filtering capacity and, essential for this application, that also in the whole organ
model specific subsets of macrophages are permissive to bacterial intracellular
replication. In this experimental series we propose to perfuse whole explanted human
spleens. In contrast to pigs, we will use for perfusion of the human organ synthetic
medium in order to neutralise or minimise acquired immunity. Histology, confocal
microscopy, FACS analysis aRNAseq of pathogens and host cells and bacterial, parasite
and viral enumeration will be the main methodologies to analyse the perfused organ. The
aim of this part of the work is to demonstrate that at the whole organ level bacteria
and viruses are able to replicate in splenic tissue macrophages pinpointing attention of
anti-infective treatments to the prevention of these early steps potentially preventing
invasive bacterial and viral disease. Samples from the 42 whole perfused organs will be
the source of tissue slice cultures and primary cell cultures.
2. Primary cell cultures: From the 42 human spleens we will derive samples for primary
culture of human tissue macrophages and other immune cells. Using the methodology in use
for cells from mice and pigs, we will set up primary cultures of human splenic immune
cells and in particular macrophages. Immune cells including macrophages and neutrophils
will be purified by different methodologies including by percoll gradient
centrifugation, FACS and magnetic separation and cultured for up to seven days. Cultures
will be tested for multiple parameters including cytokines, surface markers, anybody
production and gene expression. To test interaction with bacteria or viruses, the
cultured cells will be infected with a panel of bacterial and viral pathogens. Upon
other methodology, confocal microscopy, FACS and RNAseq will be used monitor behaviour
of bacterial, parasitic and viral pathogens and host cells. Interventions with
cytokines, chemokines, drugs, antibiotics and other compounds will be carried out to
test tractability of the system. With the aim in mind to identify targets for
intervention, the cell culture models are intended to provide detailed quantifiable
parameters of the interaction of bacteria or viruses and immune cells in particular
tissue macrophages in a controlled in vitro culture system. This is intended to allow
the design of medium-throughput assays to test strategies modifying initiation of
invasive disease. Cells will be stored in liquid nitrogen for further research.
3. Organotypic slice cultures: From the 42 human spleens we will derive, in addition to
cells, also tissue samples for organotypic slice cultures. With the methodology well set
up for brain slices and described for spleen, we will culture organotypic human spleen
slice cultures. These organ slices are superior to cell cultures, as they maintain the
multicellular spatial microarchitecture of the organ, which is particularly important
when analysing tissue macrophages. As for brain slice cultures, we will infect the
slices with bacterial or viral pathogens and monitor their migration within the tissue,
the infection of cells and the reaction of the single host cells. The use of tissue
slice culture should allow to specifically study the targeting and
replication-permissive nature of specific subsets of immune cells including macrophages.
A goal of the objective is to allow for detailed development of infectious foci in the
spleen including time laps confocal microscopy of infected spleen slices. Testing in
this model cytokines and other host derived molecules are expected to show the potential
of interventions targeting specific subsets of immune cells including macrophages.
Tissue slices will be stored in liquid nitrogen for further research.
Sample transfer between units: The 42 planned organ perfusion will be performed at the
laboratories of the HBP unit of the Leicester General Hospital. In selected cases organ
perfusion might be performed at the Department of Genetics and Genome Biology of the
University of Leicester. The relative HBA form has been obtained (Hazardous Biological Agents
form GEN/45 Cell lines and primary human cell and tissue culture, date 02/10/2017). During
and after perfusion, tissue samples for tissue slice culture and primary cell culture will be
taken. These tissue samples will be transferred to the Department of Genetics and Genome
Biology or Dept. of Infection and Immunity and Inflammation for the scheduled research work
and will be stored there in accordance with regulations relative to the Human Tissue Act.
Stored tissue slices and primary cell cultures may then be transferred for immunological
studies to the University of Nottingham for experimental work detailed above for cell
cultures. Conditions for handling, storage and transport are defined by the HBA form GEN45
and transfer will be regulated by an MTA.
