Approved: New Antimicrobial Stewardship Standard

The Joint Commission recently announced a new Medication Management (MM)
standard for hospitals, critical access hospitals, and nursing care centers. Stan￾dard MM.09.01.01 addresses antimicrobial stewardship and becomes effective
January 1, 2017.
Current scientific literature emphasizes the need to reduce the use of inap￾propriate antimicrobials in all health care settings due to antimicrobial resistance.
According to the World Health Organization (WHO): “Antimicrobial resistance
threatens the effective prevention and treatment of an ever-increasing range of
infections caused by bacteria, parasites, viruses and fungi.”1
The Centers for Disease
Control and Prevention (CDC) identified that 20%–50% of all antibiotics pre￾scribed in US acute care hospitals are either unnecessary or inappropriate.2
The
CDC has also stated: “Antibiotics are among the most commonly prescribed medi￾cations in nursing homes. Up to 70% of long-term care facilities’ residents receive
an antibiotic every year.”3
On June 2, 2015, The Joint Commission participated in the White House
Forum on Antibiotic Stewardship. The Joint Commission joined representatives
from more than 150 major health care organizations, food companies, retailers, and
animal health organizations at the forum to express commitment for implementing
changes over the next five years to slow the emergence of antibiotic-resistant bacte￾ria, detect resistant strains, preserve the efficacy of existing antibiotics, and prevent
the spread of resistant infections.4
Subsequently, The Joint Commission developed the antimicrobial steward￾ship standard for hospitals, critical access
hospitals, nursing care centers, ambula￾tory care organizations, and office-based
surgery practices and conducted a field
review in November and December
2015. Prior to and during the field
review, Joint Commission staff conducted
stakeholder calls on the proposed antimi￾crobial stewardship standard with several
governmental and professional organiza￾tions, including the Centers for Medicare & Medicaid Services (CMS), the CDC, and the Society for
Healthcare Epidemiology of America (SHEA).
There was significant support for the antimicrobial
stewardship standard for the hospital, critical access hospital,
and nursing care center accreditation programs. Additionally,
CMS is in the process of developing a Condition(s) of Par￾ticipation (CoP) on antimicrobial stewardship for the hospital
and nursing home settings, which therefore aligns the Joint
Commission’s standard with CMS’s plans for a CoP(s) in this
area. In the meantime, the antimicrobial stewardship standard
for Joint Commission–accredited ambulatory care organiza￾tions and office-based surgery practices is still in development.
The approved antimicrobial stewardship standard and
EPs are shown in the box that begins below and will also be
displayed on The Joint Commission website at http://www.
jointcommission.org/standards_information/prepublication_
standards.aspx. In addition, the requirements will be posted
in the fall 2016 E-dition® update and published in the 2017
Comprehensive Accreditation Manual for the Critical Access
Hospital, Hospital, and Nursing Care Center Accreditation
Programs.
Questions regarding the new antimicrobial stewardship
standard may be directed to Kelly Podgorny, DNP, CPHQ, RN,
project director, Department of Standards and Survey Methods,
The Joint Commission, at kpodgorny@jointcommission.org. P
References
1. World Health Organization. Antimicrobial Resistance. (Updated: Apr
2015.) Accessed May 27, 2016. http://www.who.int/mediacentre/
factsheets/fs194/en/#
2. Centers for Disease Control and Prevention. Core Elements of Hospital
Antibiotic Stewardship Programs. Accessed May 27, 2016. http://www.
cdc.gov/getsmart/healthcare/implementation/core-elements.html
3. Centers for Disease Control and Prevention. Antibiotic Use in Nursing
Homes. Nov 5, 2013. Accessed May 27, 2016. http://www.cdc.gov/
getsmart/healthcare/learn-from-others/factsheets/nursing-homes.html
4. The Joint Commission. Joint Commission Joins White House Effort to
Reduce Antibiotic Overuse. Jt Comm Perspect. 2015 Jul;35(7):4, 11.

Standard MM.09.01.01
The [critical access] hospital has an antimicrobial stewardship
program based on current scientific literature.
Elements of Performance for MM.09.01.01
1. Leaders establish antimicrobial stewardship as an orga￾nizational priority. (See also LD.01.03.01, EP 5)
Note: Examples of leadership commitment to an antimi￾crobial stewardship program are as follows:
l Accountability documents
l Budget plans
l Infection prevention plans
l Performance improvement plans
l Strategic plans
l Using the electronic health record to collect antimi￾crobial stewardship data
2. The [critical access] hospital educates staff and li￾censed independent practitioners involved in antimicro￾bial ordering, dispensing, administration, and monitor￾ing about antimicrobial resistance and antimicrobial
stewardship practices. Education occurs upon hire or
granting of initial privileges and periodically thereafter,
based on organizational need.
3. The [critical access] hospital educates patients, and
their families as needed, regarding the appropriate use
of antimicrobial medications, including antibiotics. (For
more information on patient education, refer to Stan dard PC.02.03.01)
Note: An example of an educational tool that can be
used for patients and families includes the Centers for
Disease Control and Prevention’s Get Smart docu￾ment, “Viruses or Bacteria—What’s got you sick? at
http://www.cdc.gov/getsmart/community/downloads/
getsmart-chart.pdf.
4. The [critical access] hospital has an antimicrobial stew￾ardship multidisciplinary team that includes the follow￾ing members, when available in the setting:
l Infectious disease physician
l Infection preventionist(s)
l Pharmacist(s)
l Practitioner
Note 1: Part-time or consultant staff are acceptable as
members of the antimicrobial stewardship multidisci￾plinary team.
Note 2: Telehealth staff are acceptable as members of
the antimicrobial stewardship multidisciplinary team.
5. D The [critical access] hospital’s antimicrobial steward￾ship program includes the following core elements:
l Leadership commitment: Dedicating necessary hu￾man, financial, and information technology resources.
l Accountability: Appointing a single leader respon￾sible for program outcomes. Experience with suc￾cessful programs shows that a physician leader is
effective.
l Drug expertise: Appointing a single pharmacist leader
responsible for working to improve antibiotic use.
l Action: Implementing recommended actions, such
as systemic evaluation of ongoing treatment need,
after a set period of initial treatment (for example,
“antibiotic time out” after 48 hours).
l Tracking: Monitoring the antimicrobial stewardship
program, which may include information on antibi￾otic prescribing and resistance patterns.
l Reporting: Regularly reporting information on the
antimicrobial stewardship program, which may
include information on antibiotic use and resistance,
to doctors, nurses, and relevant staff.
l Education: Educating practitioners, staff, and
patients on the antimicrobial program, which may
include information about resistance and optimal
prescribing. (See also IC.02.01.01, EP 1 and
NPSG.07.03.01, EP 5)
Note: These core elements were cited from the Centers
for Disease Control and Prevention’s Core Elements of
Hospital Antibiotic Stewardship Programs (http://www.
cdc.gov/getsmart/healthcare/pdfs/core-elements.pdf).
The Joint Commission recommends that organizations
use this document when designing their antimicrobial
stewardship program.
6. D The [critical access] hospital’s antimicrobial steward￾ship program uses organization-approved multidisci￾plinary protocols (for example, policies and procedures).
Note: Examples of protocols are as follows:
l Antibiotic Formulary Restrictions
l Assessment of Appropriateness of Antibiotics for
Community-Acquired Pneumonia
l Assessment of Appropriateness of Antibiotics for
Skin and Soft Tissue Infections
l Assessment of Appropriateness of Antibiotics for
Urinary Tract Infections
l Care of the Patient with Clostridium difficile (c.-diff)
l Guidelines for Antimicrobial Use in Adults
l Guidelines for Antimicrobial Use in Pediatrics
l Plan for Parenteral to Oral Antibiotic Conversion
l Preauthorization Requirements for Specific
Antimicrobials
l Use of Prophylactic Antibiotics
7. D The [critical access] hospital collects, analyzes, and
reports data on its antimicrobial stewardship program.
Note: Examples of topics to collect and analyze data
on may include evaluation of the antimicrobial steward￾ship program, antimicrobial prescribing patterns, and
antimicrobial resistance patterns.
8. D The [critical access] hospital takes action on im￾provement opportunities identified in its antimicrobial
stewardship program. (See also MM.08.01.01, EP 6)

Applicable to Nursing Care Centers
Effective January 1, 2017
Medication Management (MM)
Standard MM.09.01.01
The organization has an antimicrobial stewardship program
based on current scientific literature.
Elements of Performance for MM.09.01.01
1. Leaders establish antimicrobial stewardship as an orga￾nizational priority. (See also LD.01.03.01, EP 5)
Note: Examples of leadership commitment to an antimi￾crobial stewardship program are as follows:
l Accountability documents
l Budget plans
l Infection prevention plans
l Performance improvement plans
l Strategic plans
l Using the electronic health record to collect antimi￾crobial stewardship data
2. The organization educates staff and licensed inde￾pendent practitioners involved in antimicrobial order￾ing, dispensing, administration, and monitoring about
antimicrobial resistance and antimicrobial stewardship
practices. Education occurs upon hire or granting of
initial privileges and periodically thereafter, based on
organizational need.
3. The organization educates residents, and their families
as needed, regarding the appropriate use of antimi￾crobial medications, including antibiotics. (For more
information on patient and resident education, refer to
Standard PC.02.03.01)
Note: An example of an educational tool that can be
used for patients and families includes the Centers for
Disease Control and Prevention’s Get Smart docu￾ment, “Viruses or Bacteria—What’s got you sick? at
http://www.cdc.gov/getsmart/community/downloads/
getsmart-chart.pdf.
4. The organization has an antimicrobial stewardship mul￾tidisciplinary team that includes the following members,
when available in the setting:
l Infectious disease physician
l Infection preventionist(s)
l Pharmacist(s)
l Practitioner
Note 1: Part-time or consultant staff are acceptable as
members of the antimicrobial stewardship multidisci￾plinary team.
Note 2: Telehealth staff are acceptable as members of
the antimicrobial stewardship multidisciplinary team.
5. D The organization’s antimicrobial stewardship pro￾gram includes the following core elements:
l Leadership commitment: Demonstrate support and
commitment to safe and appropriate antibiotic use
in your facility.
l Accountability: Identify physician, nursing, and phar￾macy leads responsible for promoting and oversee￾ing antibiotic stewardship activities in your facility.
l Drug expertise: Establish access to consultant
pharmacists or other individuals with experience or
training in antibiotic stewardship for your facility.
l Action: Implement policy or practice changes to
improve antibiotic use.
l Tracking: Monitor and measure the use of antibiotic
use and at least one outcome from antibiotic use in
your facility.
l Reporting: Regularly reporting information on the
antimicrobial stewardship program, which may
include antibiotic use and resistance, to physicians
and other practitioners, nurses, and relevant staff.
l Education: Provide resources to physicians and
other practitioners, nursing staff, residents, and
families about antibiotic resistance and opportunities
for improving antibiotic use. (See also IC.02.01.01,
EP 1)
Note: These core elements were cited from the Centers
for Disease Control and Prevention’s The Core Ele￾ments of Antibiotic Stewardship for Nursing Homes
(http://www.cdc.gov/longtermcare/prevention/antibiotic￾stewardship.html). The Joint Commission recommends
that nursing care centers use this document when
designing their antimicrobial stewardship program.
6. D The organization’s antimicrobial stewardship pro￾gram uses organization-approved multidisciplinary
protocols (for example, policies and procedures).
Note: Examples of protocols are as follows:
l Antibiotic Formulary Restrictions
l Assessment of Appropriateness of Antibiotics for
Community-Acquired Pneumonia
l Assessment of Appropriateness of Antibiotics for
Skin and Soft Tissue Infections
l Care of the Long Term Care Patient with a Urinary
Tract Infection
l Care of the Patient with Clostridium difficile (c.-diff)
l Facility Guidelines for Antimicrobial Use in Adults
l Plan for Parenteral to Oral Antibiotic Conversion
l Preauthorization Requirements for Specific
Antimicrobials
7. D The organization collects, analyzes, and reports data
on its antimicrobial stewardship program.
Note: Examples of topics to collect and analyze data
on may include evaluation of the antimicrobial steward￾ship program, antimicrobial prescribing patterns, and
antimicrobial resistance patterns.
8. D The organization takes action on improvement op￾portunities identified in its antimicrobial stewardship
program. (See also MM.08.01.01, EP 6)

Ex vivo culture of human atherosclerotic plaques: A model to study immune cells in atherogenesis

Ex vivo culture of human atherosclerotic plaques: A model to study immune
cells in atherogenesis

Abstract
Background and aims: The mechanisms that drive atherosclerotic plaque progression
and destabilization in humans remain largely unknown. Laboratory models are
needed to study these mechanisms under controlled conditions. The aim of this study
was to establish a new ex vivo model of human atherosclerotic plaques that
preserves the main cell types in plaques and the extracellular components in the
context of native cytoarchitecture.
Methods: Atherosclerotic plaques from carotid arteries of 28 patients undergoing
carotid endarterectomy were dissected and cultured. At various time-points, samples
were collected and analysed histologically. After enzymatic digestion, single cells
were analysed with flow cytometry. Moreover, tissue cytokine production was
evaluated.
Results: We optimised the plaque dissection protocol by cutting plaques into circular
segments that we cultured on collagen rafts at the medium–air interface, thus
keeping them well oxygenated. With this technique, the relative presence of T and B
lymphocytes did not change significantly during culture, and the sizes of lymphocyte
subsets remained stable after day 4 of culture. Macrophages, smooth muscle cells,
and fibroblasts with collagen fibres, as well as both T and B lymphocyte subsets and
CD16 natural killer cells, remained largely preserved for 19 days of culture, with a
continuous production of inflammatory cytokines and chemokines.
Conclusions: Our new model of ex vivo human atherosclerotic plaques, which
preserves the main subsets of immune cells in the context of tissue cytoarchitecture,
may be used to investigate important aspects of atherogenesis, in particular, the
functions of immune cells under controlled laboratory conditions.

Introduction
Atherosclerosis and its cardiac and cerebral complications are the leading
causes of death from cardiovascular diseases. For a long time, accumulation of
modified lipoproteins within the arterial wall was considered to be the main cause of
atherosclerotic disease [1]. More recently, however, cells of various types, such as
smooth muscle cells, macrophages, and T cells, have been found to play an
important role in atherosclerotic plaque formation [2]. Moreover, a currently accepted
theory of atherogenesis emphasizes the role of immune system activation caused by
oxidized lipoproteins, which activate endothelial cells (as do other foreign agents
within the vascular wall) [3,4]. It is thought that immune cells are attracted by
chemokines, which are produced by activated endothelial cells, and migrate into the
subendothelium, where they proliferate, leading to atherosclerotic plaque progression
[2,5–7].
The role of immune cells in the growth of plaques has been confirmed in
experimental models on immunodeficient mice, as well as from the presence of
autologous antibodies against oxidized low density lipoproteins in atherosclerotic
plaques [8,9]. In our earlier work, we demonstrated T lymphocyte activation in human
atherosclerotic plaques in comparison with the blood of the same patients [10], thus
providing further evidence for the involvement of the immune system in
atherogenesis.
Despite plentiful evidence for the critical role of the immune system in
atherosclerosis, many important aspects of this phenomenon remain unknown. The
lack of this knowledge is in part due to limitations on access to human atherosclerotic
plaques in vivo, while animal models are often not adequate because of differences

in structures of arterial walls [11,12]. In vitro laboratory-controlled systems are
required for the study of atherosclerotic plaque formation and rupture. Several such
models with cells of only one or two types cultured together have been suggested
[13,14]; however, none of them faithfully reproduces the whole range of intercellular
interactions within human atherosclerotic plaques [15,16].
Here, we describe a new ex vivo model of human atherosclerotic plaques,
which preserves the main cell types of plaques in vivo, together with the general
tissue cytoarchitecture. We think that this model may prove useful for investigation of
immune cell function in atherogenesis and for development of novel therapeutic
approaches to atherosclerosis treatment.
Materials and methods
For a detailed description of the Materials and methods see Supplementary Data.
Patients
We collected atherosclerotic plaques from carotid arteries of 28 patients with
peripheral artery disease, undergoing carotid endarterectomy because of extended
atherosclerosis (19 men and 9 women; mean age ± standard deviation = 65.4 ± 8.5
years). The degree of carotid artery stenosis varied from 65% to 90% (median
90.0%, interquartile range (IQR) 73.8% to 90.0%). Ten patients suffered from
transient ischemic attack or stroke within 5 years before surgery, and more than half
of all plaque specimens (60.7%) were ruptured, as determined from macroscopic
evaluation. All patients’ characteristics are presented in Supplementary Table1.
This protocol was approved by the A.I. Yevdokimov Moscow State University
of Medicine and Dentistry Ethics Committee. All the participants provided written
informed consent.

Tissue processing
Our work was based on the pioneer work of Dr. Hoffman [17,18], who
developed the technique of histoculture that makes possible maintenance of blocks
of mammalian tissues for weeks at the air–liquid interface.
According to the protocol, surgical atherosclerotic plaque samples were
dissected and divided into three parts: one part of the material was fixed in 4%
formaldehyde (Pierce, Thermo Fisher Scientific, Waltham, MA, USA, cat. 28908) and
embedded in paraffin for histological examination, the second part was digested with
an enzymatic cocktail into a single-cell suspension for flow cytometry. The third part
was dissected, placed on a wetted collagen sponge raft (Pfizer, New York, NY, USA,
cat. 0315-08) at the medium–air interface, and cultured. After one day of culture, and
then every 3rd day, culture medium was collected and replaced with fresh medium.
Every 3rd day, several tissue blocks were analysed histologically and by means of
flow cytometry.

Histology
Histology, histochemistry, and immunohistochemistry were performed
according to standard techniques. We focused on several cell types that could not be
properly isolated from plaques by enzymatic treatment and thus were not analysed
with flow cytometry. In particular, we assessed fibroblasts and collagen tissue,
macrophages, and smooth muscle cells, using Masson’s Trichrome staining (Agilent
Technologies, Santa Clara, CA, USA, cat. AR17392-2), antibodies against CD68
(clone KP1, Agilent Technologies) and α-smooth muscle actin (α-SMA) (clone 1A4,
Agilent Technologies), respectively


Statistical analyses

The data obtained in the present study were not normally distributed, according
to the Shapiro-Wilk test, and are presented as medians and IQR. Since distributions
were not normal, for comparison of two independent groups we used the Mann￾Whitney rank test, and for dependent groups we used the Wilcoxon matched pair
test, Friedman ANOVA, and Kendall test. To assess between-group effects, we used
a multiple comparisons rank test. For the age distribution, we made the assumption
of its normality. Statistical analysis was performed with Statistica 10.0 (Statsoft,
Tulsa, OK, USA) and SPSS Statistics 21.0 (IBM, Armonk, NY, USA). Values of p
<0.05 were considered statistically significant.


Results
Histology of ex vivo plaques
Initially, we separated atherosclerotic plaques from normal artery tissue and
dissected them into ~2-mm cubic blocks for culture, similarly to what was
successfully used earlier to culture various human tissues ex vivo [17,19]. However,
analysis of stained histological sections showed that the viability of cultured tissue

blocks of this size decreased over 8–12 days, and cultured blocks contained only ~20
live cells per 100 mg of tissue (Supplementary Fig.3).
We thought that the tissues might be damaged during dissection into small
blocks. Therefore, to diminish tissue injury during preparation, we modified our
protocol and instead of dissecting into small blocks, we sliced tissue into ring-shaped
2-mm thick segments, and with a diameter depending of the carotid artery size (Fig.
1). To verify tissue viability, every 3rd day several dissected segments were analysed
histologically and by means of flow cytometry.
Analysis of histological sections showed that the dissection of plaques into
large circular segments significantly increased cell survival: tissues were preserved
for 19 days. For histological evaluation, tissue segments were stained with
hematoxylin and eosin, Masson’s Trichrome, anti-CD68, and anti-α-SMA antibodies
and their morphology was assessed as described in Materials and methods. We
found that these plaque segments retained their gross morphology and appeared
viable for more than 19 days of culture. In particular, the integrity of the endothelium
and internal elastic membrane, which are most sensitive to the culture conditions,
were preserved over 19 days of culture without a significant increase in the necrotic
core area (Fig. 2).
Furthermore, in 6 plaques, we quantified areas reacting with aniline blue, anti￾CD68, and anti-α-SMA at days 0, 4, 7, and 19 (Fig. 3). We identified macrophages,
fibroblasts, and smooth muscle cells, along with the endothelium, until day 19 of
culture. We found no statistically significant changes (p >0.05) in the fraction of these
cells during the entire culture period (Table 1).

In plaque samples from 16 donors, we assessed tissue viability using flow
cytometry by analysing immune cells extracted from plaque tissue. We compared
flow cytometry results at day 0 with those at days 4, 7, and 19. Towards this goal, we
digested plaque segments with an enzymatic cocktail containing collagenase XI and
desoxyribonuclease I, washed isolated cells, and stained them with live/dead staining
and monoclonal antibodies against CD45, CD3, CD19, CD4, CD8, and CD16. Two
plaques were excluded from the analysis because of a low cellularity at day 0 (lower
than 500 live cells per 100 mg of tissue).
Analysis of tissue at day 0 revealed a median of 6,286.0, IQR [3,172.1–
12,918.9] lymphocytes per 100 mg of plaque tissue. The absolute numbers and
percentages of B lymphocytes among all lymphocytes at day 0 were significantly
lower than those of T lymphocytes (24.6 [7.8–55.3] cells/100 mg vs. 5,694.1
[2,226.7–11,726.6] cells/100 mg; 0.4% [0.1%–0.5%] vs. 89.6% [84.3%–91.2%],
p=0.001). At day 0, among T lymphocytes, the fraction of CD4+CD8- cells was larger
than the fraction of CD4-CD8+ cells (51.6% [43.8%–58.9%] vs. 39.9% [30.0%–
45.1%], p=0.041). The median amount of CD16 NK cells at day 0 was 58.6 [18.7–
360.9] per 100 mg of plaque tissue (Fig. 4). Consecutive flow cytometry after day 0
was performed in plaques from 8 patients, four of which were cultured until day 19.
We showed that,after a decrease during the first 4 days of culture (n=8, 4,125.8
[2,771.4–6,286.0] cells/100 mg at day 0 vs. 2,619.3 [1,360.8–3,712.2] cells/100 mg
at day 4, p=0.036), the amounts of lymphocytes stabilized and did not change
significantly until the 7th day of culture (n=8, 1,249.6 [445.5–3,706.0] cells/100 mg;
p=0.161). A similar pattern was found in T cells, with a stabilization of their amounts
after a reduction during the first 4 days of culture (n=8, 3,546.5 [2,194.2–5,694.1]
cells/100 mg at day 0 vs. 2,123.4 [1,210.5–3,280.1] cells/100 mg at day 4, p=0.036
vs. 949.5 [375.1–2,378.3] cells/100 mg at day 7, p=0.124). In addition, we found no

significant changes in the amounts of B cells during the first days of culture (n=8,
12.2 [5.3–35.3] cells/100 mg at day 0 vs. 5.6 [3.3–37.2] cells/100 mg at day 4 vs.
7.9 [0.0–11.4] cells/100 mg at day 7, p=0.798). Furthermore, both T and B cells were
also preserved in plaque tissues during 19 days of culture, although their ratio
changed slightly because of the decrease in the number of T lymphocytes (n=4,
7,673.4 [3,789.0–14,813.2] cells/100 mg vs. 2,594.5 [1,926.8–7,569.0] cells/100 mg
for T cells, and 25.8 [5.3–57.9] cells/100 mg vs. 31.0 [12.2–91.2] cells/100 mg for B
cells). CD16 NK cells were found at day 19 as well: the median cell count at the last
day of culture constituted 44.9 [21.9–233.0] cells/100 mg (Fig. 5). We presume that
the initial fall in T cell count may originate from the intense effect of tissue dissection,
while the statistically significant decrease in the amounts of B cells and CD16 NK
cells may not have been revealed because of the small size of these cell subsets.
These changes were followed by a subsequent system stabilization. This was also
evident by the lack of significant changes (p=0.417) in the fraction of dead cells,
which even at day 19 remained at the level of on average 11.2% [10.1%–14.6%].
Importantly, the initial drop in T cell count was not accompanied by significant
changes in the fraction of T cells among all lymphocytes (n=8, 89.6% [76.3%–91.2%]
at day 0 vs. 86.2% [81.1%–90.8%] at day 4, p=0.779; vs. 86.0% [77.5%–86.9%] at
day 7, p=0.674) (Fig. 6A). The decrease in T cell numbers during the first days of
culture was predominantly associated with the reduction of the fraction of CD4 T cells
(n=7, 58.0% [43.8%–63.6%] at day 0 vs. 42.0% [31.4%–49.2%] at day 4, p=0.018;
vs. 40.8% [21.6%–48.7%] at day 7, p=0.018), accompanied by a minor rise in the
fraction of CD8 T cells (n=7, 36.0% [30.0%–47.5%] at day 0 vs. 45.2% [35.3%–
48.6%] at day 4, p=0.063; vs 44.4% [37.9%–55.1%] at day 7, p=0.018) (Figure 6B).
As a result of these changes, the CD4+CD8-/CD4-CD8+ ratio decreased slightly
during culture. As of the 19th day of culture, the fraction of CD4 T cells was reduced

with a concurrent increase in the fraction of CD8 T cells (49.7% [40.2%–57.6%] vs.
42.3% [27.8%–51.9%] for CD4 T cells, and 43.6% [36.1%–53.8%] vs. 50.8%
[40.9%–61.9%] for CD8 T cells) (Figure 7). Nevertheless, both CD4 and CD8 T cells
were also preserved in culture for at least 19 days.
Cytokine production by plaques ex vivo
We analysed the concentrations of cytokines and chemokines released by six
cultured plaques and accumulated in the culture medium from day 1 to day 4 and
from day 16 to day 19 when the medium was changed. We found that in our system
plaques produce substantial amounts of interleukin (IL)-1α, IL-6, IL-8, IL-16, IL-18, IL-
21, IL-22, eotaxin, interferon-λ, granulocyte macrophage colony-stimulating factor
(GM-CSF), macrophage-CSF, tumor necrosis factor (TNF)-α, transforming growth
factor (TGF)-β, growth related oncogene (GRO)-α, interferon gamma-inducible
protein-10, monocyte chemoattractant protein-1, monokine induced by gamma
interferon, macrophage inflammatory protein (MIP)-1α, MIP-1β, and RANTES. In
contrast, the concentrations of the other measured cytokines and chemokines were
lower than the detection limit of the Luminex platform for these analytes.
Within the panel of cytokines and chemokines that were detectable in the
culture medium, we found no significant changes during culture in most of the
cytokines, except for IL-16 and several cytokines whose concentration decreased, in
particular IL-8 (n=6, from 25,698.7 [14,817.3–52,553.3] pg/ml on day 4 to 4,273.1
[2,695.6–6,274.2] pg/ml on day 19), and to a lesser extent GM-CSF, TNF-α, and
GRO-α. At the same time, the concentrations of other cytokines (including eotaxin,
TGF-β, and MIP-1β) increased during culture (Supplementary Table 2).


DI