Atherosclerotic plaques are sites of chronic inflammation with diverse cell contents and complex immune signaling. Plaque progression and destabilization are driven by the infiltration of immune cells and the cytokines that mediate their interactions. Here, we attempted to compare the systemic cytokine profiles in the blood plasma of patients with atherosclerosis and the local cytokine production, using ex vivo plaque explants from the same patients. The developed method of 41-plex xMAP data normalization allowed us to differentiate twenty-two cytokines produced by the plaque that were not readily detectable in free circulation and six cytokines elevated in blood plasma that may have other sources than atherosclerotic plaque. To verify the xMAP data on the putative atherogenesis-driving chemokines MCP-1 (CCL2), MIP-1α (CCL3), MIP-1β (CCL4), RANTES (CCL5), and fractalkine (CX3CL1), qPCR was performed. The MIP1A (CCL3), MIP1B (CCL4), FKN (CX3CL1), and MCP1 (CCL2) genes were expressed at high levels in the plaques, whereas RANTES (CCL5) was almost absent. The expression patterns of the chemokines were restricted to the plaque cell types: the MCP1 (CCL2) gene was predominantly expressed in endothelial cells and monocytes/macrophages, MIP1A (CCL3) in monocytes/macrophages, and MIP1B (CCL4) in monocytes/macrophages and T cells. RANTES (CCL5) was restricted to T cells, while FKN (CX3CL1) was not differentially expressed. Taken together, our data indicate a plaque-specific cytokine production profile that may be a useful tool in atherosclerosis studies.

BACKGROUND: The efficacy of on-demand HIV pre-exposure prophylaxis (PrEP) for men in sub-Saharan Africa has not been evaluated, and the on-demand PrEP dosing requirement for insertive sex remains unknown.
METHODS: HIV-negative males 13-24 years, requesting voluntary medical male circumcision (VMMC), were enrolled into an open-label randomised controlled trial (NCT03986970), and randomised 1:1:1:1:1:1:1:1:1 to control arm or one of eight arms receiving emtricitabine-tenofovir disoproxil fumarate (F/TDF) or emtricitabine-tenofovir alafenamide (F/TAF) over one or two days, and circumcised 5 or 21 h thereafter. The primary outcome was foreskin p24 concentrations following ex vivo HIV-1BaL challenge. Secondary outcomes included peripheral blood mononuclear cell (PBMC) p24 concentration, and drug concentrations in foreskin tissue, PBMCs, plasma and foreskin CD4+/CD4-cells. In the control arm, post-exposure prophylaxis (PEP) activity of non-formulated tenofovir-emtricitabine (TFV-FTC) or TAF-FTC was assessed with ex vivo dosing 1, 24, 48 or 72 h post-HIV-1 challenge.
RESULTS: 144 participants were analysed. PrEP with F/TDF or F/TAF prevented ex vivo infection of foreskins and PBMCs both 5 and 21 h after PrEP dosing. There was no difference between F/TDF and F/TAF (p24day15 geometric mean ratio 1.06, 95% confidence interval: 0.65-1.74). Additional ex vivo dosing did not further increase inhibition. In the control arm, PEP ex vivo dosing was effective up to 48 post-exposure diminishing thereafter, with TAF-FTC showing prolonged protection compared to TFV-FTC. Participants receiving F/TAF had higher TFV-DP concentrations in foreskin tissue and PBMCs compared with F/TDF, irrespective of dose and sampling interval; but F/TAF did not confer preferential TFV-DP distribution into foreskin HIV target cells. FTC-TP concentrations with both drug regimens were equivalent and ∼1 log higher than TFV-DP in foreskin.
CONCLUSIONS: A double dose of either F/TDF or F/TAF given once either 5 or 21 h before ex vivo HIV-challenge provided protection across foreskin tissue. Further clinical evaluation of pre-coital PrEP for insertive sex is warranted.
BACKGROUND: EDCTP2, Gilead Sciences, Vetenskapsrådet.

Previous studies on highly HIV-1-exposed, yet persistently seronegative women from the Punwami Sex Worker cohort in Kenya, have shed light on putative protective mechanisms, suggesting that mucosal immunological factors, such as antiproteases, could be mediating resistance to HIV-1 transmission in the female reproductive tract. Nine protease inhibitors were selected for this study: serpin B4, serpin A1, serpin A3, serpin C1, cystatin A, cystatin B, serpin B13, serpin B1 and α-2-macroglobulin-like-protein 1. We assessed in a pilot study, the activity of these antiproteases with cellular assays and an ex vivo HIV-1 challenge model of human ecto-cervical tissue explants. Preliminary findings with both models, cellular and tissue explants, established an order of inhibitory potency for the mucosal proteins as candidates for pre-exposure prophylaxis when mimicking pre-coital use. Combination of all antiproteases considered in this study was more active than any of the individual mucosal proteins. Furthermore, the migration of cells out of ecto-cervical explants was blocked indicating potential prevention of viral dissemination following amplification of the founder population. These findings constitute the base for further development of these mucosal protease inhibitors for prevention strategies.

BACKGROUND: In view of continued development of new oncological approaches, there is a high demand for personalized tumor therapy. However, fast and effective functional platforms for the prediction of individual patient response to drug therapy are largely unavailable. Various promising approaches have already been described for three-dimensional cell culture models, which represent cellular complexity and almost identical structures of the original tumor tissue.
OBJECTIVE: Based on a case report, we show the capability and results of a novel test system using patient-derived microtumors (PDMs) and autologous tumor-infiltrating lymphocytes (TILs) for the prediction of response to cancer therapy.
METHODS: We established PDMs and TILs from primary tumor tissue of a renal cell carcinoma metastasis. Using immunohistochemistry and multiplex florescence-activated cell sorting (FACS ) analyses, the PDMs and TILs were characterized regarding to histology and immunophenotype. Tumor-specific cytotoxicity of standard of care and investigational compounds were assessed. The results were compared to the patient\'s individual in vivo response to therapy.
CONCLUSIONS: The cytotoxicity assay of PDMs and TILs showed a significant therapeutic response (p = 0.0004) to therapy with a programmed cell death protein 1 (PD-1) inhibitor and lenvatinib compared to the control. The in vitro results correlated positively with the in vivo data. In the future, patient-derived models could predict response to cancer therapy and may help to optimize treatment decision-making.
UNASSIGNED: HINTERGRUND: Die personalisierte Tumortherapie rückt immer mehr in den Fokus onkologischer Behandlungsstrategien. Schnelle und effektive Plattformen zur Vorhersage des individuellen Ansprechens eines Patienten auf eine medikamentöse Tumortherapie sind jedoch weitestgehend nicht verfügbar. Für dreidimensionale (3D-)Zellkulturmodelle wurden bereits verschiedene vielversprechende Ansätze beschrieben, bei denen sich Eigenschaften und Strukturen der Modelle dem des Originaltumors in hohem Maße gleichen.
UNASSIGNED: Wir zeigen anhand eines Fallberichts exemplarisch die möglichen Potenziale und Ergebnisse eines neuartigen Testsystems unter der Verwendung von patientenabgeleiteten Mikrotumoren (PDMs) und autologen tumorinfiltrierenden Lymphozyten (TILs) auf.
UNASSIGNED: Aus dem Tumorfrischgewebe einer klarzelligen Nierenzellkarzinommetastase wurden PDMs und TILs isoliert. Es wurden histologische und immunphänotypische Charakterisierungen der PDMs und TILs durchgeführt. Des Weiteren wurde funktionelle In-vitro-Substanztestungen mit typischen Medikamenten/-kombinationen durchgeführt. Die Ergebnisse wurden mit dem In-vivo-Therapieansprechen des Patienten verglichen.
UNASSIGNED: Im durchgeführten Zytotoxizitätsassay der Nierenzellkarzinommetastasen-PDMs und TILs zeigt sich ein signifikantes Therapieansprechen (p = 0,0004) auf die Therapie mit einem PD-1-Inhibitor („programmed death 1“) und Lenvatinib im Vergleich zur Kontrolle. Die erhobenen Ergebnisse in der Zellkultur korrelierten positiv mit den In-vivo-Daten, welche ebenfalls ein Ansprechen beim Patienten zeigten. Es besteht die Möglichkeit, dass patientenabgeleitete Modellsysteme ein Therapieansprechen vorhersagen könnten und daraus in Zukunft gegebenenfalls auch eine optimierte Therapieentscheidung abgeleitet werden könnte.

The association between the use of the injectable contraceptive depot medroxyprogesterone acetate and HIV-1 susceptibility has been addressed mainly in respect to the changes occurring in the female genital mucosa and blood. However, one of the main sites of HIV-1 pathogenesis is lymphoid organs. To investigate the immunoregulatory effect of medroxyprogesterone acetate (MPA) at this site, human tonsillar tissue explants were infected ex vivo with either a CCR5 (BaL) or CXCR4 (LAI) HIV-1 variant and the release of p24gag and cytokines was measured in culture supernatant. The response to MPA was compared with that elicited by treatment with progesterone (P4) and dexamethasone (DEX), which selectively binds the glucocorticoid receptor, in donor-matched explant cultures. MPA treatment reduced the replication of both tested HIV-1 strains as well as the production of the mediators of inflammation IL-1β, IL-17A and CCL5, but not CCL20, in a similar way to DEX, whereas P4 had no effect on HIV-1 replication. The magnitude of both MPA and DEX-mediated responses was proportional to the length of exposure and/or administered dose. Blockage of the progesterone and glucocorticoid receptors with mifepristone abolished all observed changes in HIV-1 and cytokine production, and was associated with increased IL-22 levels in HIV-infected explants. Our data indicate that elevated doses of MPA may affect the immune responses in lymphoid tissue in a glucocorticoid-like fashion with an immediate impact on local HIV-1 replication.

The long-term success of visual rehabilitation in patients with severe conjunctival scarring is reliant on the reconstruction of the conjunctiva with a suitable substitute. The purpose of this study is the development and investigation of a re-epithelialized conjunctival substitute based on porcine decellularized conjunctiva (PDC).
PDC was re-epithelialized either with pre-expanded human conjunctival epithelial cells (PDC + HCEC) or with a human conjunctival explant placed directly on PDC (PDC + HCEx). Histology and immunohistochemistry were performed to evaluate epithelial thickness, proliferation (Ki67), apoptosis (Caspase 3), goblet cells (MUC5AC), and progenitor cells (CK15, ΔNp63, ABCG2). The superior construct (PDC + HCEx) was transplanted into a conjunctival defect of a rabbit (n = 6). Lissamine green staining verified the epithelialization in vivo. Orbital tissue was exenterated on day 10 and processed for histological and immunohistochemical analysis to examine the engrafted PDC + HCEx. A human-specific antibody was used to detect the transplanted cells.
From day-14 in vitro onward, a significantly thicker epithelium and greater number of cells expressing Ki67, CK15, ΔNp63, and ABCG2 were noted for PDC + HCEx versus PDC + HCEC. MUC5AC-positive cells were found only in PDC + HCEx. The PDC + HCEx-grafted rabbit conjunctivas were lissamine-negative during the evaluation period, indicating epithelial integrity. Engrafted PDC + HCEx showed preserved progenitor cell properties and an increased number of goblet cells comparable to those of native conjunctiva.
Placing and culturing a human conjunctival explant directly on PDC (PDC + HCEx) enables the generation of a stable, stratified, goblet cell-rich construct that could provide a promising alternative conjunctival substitute for patients with extensive conjunctival stem and goblet cell loss.

Tumour-promoting inflammation is involved in colorectal cancer (CRC) development and therapeutic resistance. However, the antibiotics and antibacterial drugs and signalling that regulate the potency of anticancer treatment upon forced differentiation of cancer stem-like cell (CSC) are not fully defined yet. We screened an NIH-clinical collection of the small-molecule compound library of antibacterial/anti-inflammatory agents that identified potential candidate drugs targeting CRC-SC for differentiation. Selected compounds were validated in both in vitro organoids and ex vivo colon explant models for their differentiation induction, impediment on neoplastic cell growth, and to elucidate the mechanism of their anticancer activity. We initially focused on AM404, an anandamide uptake inhibitor. AM404 is a metabolite of acetaminophen with antibacterial activity, which showed high potential in preventing CRC-SC features, such as stemness/de-differentiation, migration and drug-resistance. Furthermore, AM404 suppressed the expression of FBXL5 E3-ligase, where AM404 sensitivity was mimicked by FBXL5-knockout. This study uncovers a new molecular mechanism for AM404-altering FBXL5 oncogene which mediates chemo-resistance and CRC invasion, thereby proposes to repurpose antibacterial AM404 as an anticancer agent.

Legionnaires\' disease is a severe pneumonia caused by inhalation of Legionella pneumophila. Although powerful infection models ranging from monocellular host systems to mammals were developed, numerous intra- and extracellular interactions of L. pneumophila factors with human lung tissue structures remain unknown. Therefore, we developed and applied a novel infection model for Legionnaires\' disease comprising living human lung tissue explants (HLTEs). This model allows analyzing Legionella infections at a unique level of complexity and narrows the gap between current infection models and postmortem histopathology analyses of infected patients. Here we describe the infection of tumor-free pulmonary tissue samples from patients undergoing lobe- or pneumectomy because of lung cancer. The method comprises bacterial cultivation, preparation of HLTEs, and infection of HLTEs. The infected tissue samples allow to characterize tissue damage, bacterial localization, dissemination and growth kinetics, and the host\'s molecular response.

Introduction: The pannexin-1 (Panx1) channels are found in many cell types, and ATP released from these channels can act on nearby cells activating purinergic P2X7 receptors (P2X7R) which lead to inflammation. Although Panx1 and P2X7R are implicated in the process of inflammation and cell death, few studies have looked at the role they play in inflammatory bowel disease in human. Hence, the aim of the present study was to investigate the function of Panx1 and P2X7R in an ex vivo colitis model developed from human colonic mucosal explants. Materials and Methods: Healthy human colonic mucosal strips (4 × 10 mm) were incubated in carbogenated culture medium at 37°C for 16 h. Proinflammatory cytokines TNFα and IL-1β (each 10 ng/mL) were used to induce colitis in mucosal strips, and the effects of Panx1 and P2X7R on cytokines-induced tissue damage were determined in the presence of the Panx1 channel blocker 10Panx1 (100 μM) and P2X7R antagonist A438079 (100 μM). The effects of 10Panx1 and A438079 on cytokines-enhanced epithelial permeability were also studied using Caco-2 cells. Results: Histological staining showed that the mucosal strips had severe structural damage in the cytokines-only group but not in the incubation-control group (P < 0.01). Compared to the cytokines-only group, crypt damage was significantly decreased in groups receiving cytokines with inhibitors (10Panx1, A438079, or 10Panx1 + A438079, P < 0.05). The immunoreactive signals of tight junction protein zonula occludens-1 (ZO-1) were abundant in all control tissues but were significantly disrupted and lost in the cytokines-only group (P < 0.01). The diminished ZO-1 immunoreactivity induced by cytokines was prevented in the presence of 10Panx1 (P = 0.04). Likewise, 10Panx1 significantly attenuated the cytokines-evoked increase in paracellular permeability of Caco-2 cells. Although the inhibition of P2X7R activity by A438079 diminished cytokines-induced crypt damage, its effect on the maintenance of ZO-1 immunoreactivity and Caco-2 epithelial cell integrity was less evident. Conclusion: The blockade of Panx1 and P2X7R reduced the inflammatory cytokines-induced crypt damage, loss of tight junctions and increase in cell permeability. Thus, Panx1 and P2X7R may have roles in causing mucosal damage, a common clinical feature of inflammatory bowel disease.

The circadian clock generates and regulates many daily physiological, metabolic and behavioral rhythms as well as acute responses to various types of stresses including those induced by anticancer treatment. It has been proposed that modulatory function of the clock may be used for improving the therapeutic efficacy of established anti-cancer treatments. In order to rationally exploit this mechanism, more information is needed to fully characterize the functional status of the molecular clock in tumors of different cellular origin; however, the data describing tumor clocks are still inconsistent. Here we tested the status of clock in two models of tumors derived from connective tissue: sarcomas spontaneously developed in p53-deficient mice and human fibrosarcoma cells grown as xenografts in immunocompromised severe combined immunodeficient (SCID) mice. We show that both types of tumors retain a functional clock, which is synchronized in phase with normal tissues. We also show that spontaneously developed tumors are not only oscillating in the context of an organism where they receive hormonal and metabolic signals but continue oscillating ex vivo in tissue explants demonstrating that tumors have functional clocks capable of timing all their functions. We also provide evidence that similar to liver, tumors can be synchronized by food availability independent of the central pacemaker in the suprachiasmatic nuclei (SCN). These data provide the basis for the design of anticancer therapies that take into account the circadian metabolic and physiological patterns of both the tumor and normal tissues.