Researchers at KU Leuven and UZ Leuven performed orthotopic intestinal transplantations in inbred Lewis rats, a syngeneic Lewis-to-Lewis setup deliberately chosen to eliminate immunological rejection as a confounding variable. By using genetically identical donors and recipients, the team could isolate the purely physiological consequences of transplantation itself: the severing of lymphatic vessels, the loss of autonomic innervation from the brain and spinal cord, and the ischemia-reperfusion injury that occurs when the gut is deprived of blood during procurement and then flooded with oxygenated blood upon reperfusion. Thirteen transplants were completed, with donor procedures lasting on average 48 minutes, cold ischemia times of about an hour, and recipient operations of roughly two and a half hours.

The centerpiece of the study was an elegant tracer technique based on fluorescently labeled dextrans, non-digestible sugar polymers that can be given by oral gavage and then tracked in the bloodstream by measuring plasma fluorescence. The trick lies in molecular weight. A small 4-kilodalton dextran tagged with fluorescein (FITC4) crosses the intestinal lining mainly through the paracellular route, squeezing between epithelial cells through the tight junctions that normally seal the barrier. A bulky 70-kilodalton dextran tagged with rhodamine (TRITC70) can only enter via the transcellular pathway, riding on energy-dependent endocytosis through the enterocytes themselves. Because the two tracers report on structurally distinct absorption mechanisms, comparing their blood concentrations at 3, 6, and 24 hours after gavage allows researchers to dissect which pathway has been altered.

The timing of the blood draws also maps onto gut geography. In rats, small intestinal transit takes roughly two to six hours, so the 3-hour measurement reflects uptake in the small bowel, the 6-hour point blends small intestinal and colonic absorption, and the 24-hour sample captures late colonic permeability. As an added bonus, the team could follow the red-colored 70-kilodalton tracer in the feces, timing its excretion as a functional readout of total gastrointestinal motility. Each animal served as its own control, receiving the dextran cocktail two days before transplantation and again four weeks afterward, with paired statistical comparisons throughout.

The motility results were, on the surface, reassuring. Fecal excretion of the large tracer took 9.58 hours on average before transplant and 11.02 hours four weeks after, a difference that did not reach statistical significance. Despite the complete severing of sympathetic and parasympathetic nerves, leaving the graft dependent on its intrinsic enteric nervous system and circulating hormones, the transplanted bowel kept food moving at essentially its normal pace. Histology reinforced this picture of preserved wiring: the numbers of ganglia and neurons in both the myenteric and submucosal plexuses were indistinguishable from baseline four weeks after surgery, echoing earlier reports of preserved neuronal markers and even partial reinnervation lasting hundreds of days in similar models.

Absorption told a very different story. Plasma levels of the small paracellular tracer FITC4 were more than threefold higher in transplanted animals at 3 hours (0.72 versus 0.23 micrograms per milliliter, p = 0.041) and remained elevated at 6 hours (p = 0.031). The total exposure over time, quantified as the area under the plasma concentration curve, was nearly four times greater after transplantation (8.84 versus 2.39 microgram-hours per milliliter, p = 0.029). In stark contrast, the large transcellular tracer showed no significant change at any time point, and even trended downward at 24 hours. In other words, the graft became selectively leakier between its cells while the active, cell-crossing machinery of absorption carried on undisturbed.

What could drive this selective permeability shift? The histological analysis pointed toward a structural explanation. Villus length grew from 350 to 491 micrometers and villus width from 79 to 99 micrometers, pushing the calculated absorptive surface area per villus from 0.087 to 0.153 square millimeters, a 76 percent expansion. Because paracellular flux is passively driven by concentration gradients across the intercellular space, more lining means more potential leakage, whereas transcellular transport is self-limited by the slower, energy-hungry machinery of the enterocytes. Consistent with remodeling of the intercellular compartment, occludin immunohistochemistry revealed a significant increase in tight-junction protein staining after transplantation. The authors also raise the possibility of graft hypercontractility at the local level: accelerated small-bowel transit could shorten mucosal contact time, penalizing the slower transcellular route more than the passive one, and could even functionally mimic a shortened bowel, triggering the compensatory mucosal growth the team observed.

One cell population did not fare so well. Paneth cells, the antimicrobial peptide-secreting sentinels of the crypts that also nurture intestinal stem cells and reinforce the epithelial barrier, dropped significantly from 44 to 32 cells per high-power field four weeks after transplant. Crypt-to-villus ratios were unchanged, suggesting the overall architecture of renewal remained intact, but the persistent Paneth cell deficit mirrors findings in ischemia-reperfusion injury and in human inflammatory conditions such as Crohn’s disease and graft-versus-host disease. Whether this depletion reflects lingering regeneration after reperfusion injury or a stable new steady state of the graft remains an open question with potential implications for barrier defense and microbiome interactions.

The authors are candid about the limits of their pilot study. Only six animals completed the tracer experiments, the four-week follow-up may be too short to capture long-term adaptation such as lymphatic reconnection, which typically occurs within four to ten weeks, and dextran permeability does not directly predict the absorption of real nutrients or drugs, which travel mainly through transcellular pathways shaped by many additional factors. The design also omitted immunosuppression, a central feature of clinical intestinal transplantation, and lacked a sham surgery group to separate graft-specific effects from the stress of major abdominal surgery alone.

Even so, the study delivers a methodological gift to the field: a paired, in vivo technique that simultaneously quantifies gastrointestinal transit, paracellular permeability, and transcellular permeability in the same animal before and after transplantation. The central message, that a denervated, re-plumbed gut can maintain normal motility while quietly remodeling its absorptive lining toward greater paracellular flux, reframes how clinicians might think about the persistent enteric dysfunction seen in human recipients. If the same pathway-specific shift occurs in patients, it could inform nutritional strategies, drug dosing, and targeted therapies aimed at tight-junction regulation, and it sets the stage for longer studies probing how the transplanted intestine continues to adapt, and perhaps compensate, in the months and years after it finds a new home.

Subject of Research: Long-term motility and permeability changes in rat intestinal grafts after transplantation

Article Title: Changes in rat intestinal motility and absorption following intestinal transplantation: an experimental study

Article References: Dubois, A., Leenders, A., De Hertogh, G., Rustichelli, A., Boelhouwer, C., Wylin, T., Heedfeld, V., Bonaccorsi-Riani, E., Pirenne, J., Vanuytsel, T., Farré, R., & Ceulemans, L. J. (2026). Changes in rat intestinal motility and absorption following intestinal transplantation: an experimental study. Transplant International, 39, Article 17141. https://doi.org/10.3389/ti.2026.17141

Image Credits: AI Generated

DOI: 10.3389/ti.2026.17141

Keywords: intestinal transplantation, intestinal permeability, paracellular transport, transcellular absorption, fluorescent dextrans, gastrointestinal motility, enteric nervous system, tight junctions, occludin, Paneth cells, villus remodeling, ischemia-reperfusion injury

The intestinal epithelial barrier is the body’s frontline customs office, a single layer of cells that decides what passes from the gut lumen into the bloodstream and what stays out. Its defining feature is the tight junction, a belt of protein complexes that seals the gaps between neighboring epithelial cells. These junctions perform what biologists call a gate-and-fence function: they permit the selective passage of water, ions, and nutrients through the spaces between cells while blocking bacteria and their toxins. When tight junctions fail, the result is the condition popularly known as leaky gut, in which unwanted molecules slip through and provoke inflammatory responses. Disrupted barrier function is implicated in inflammatory bowel disease, metabolic disorders, and a growing list of chronic conditions.

The key players at these junctions are the tight junction proteins, including occludin and members of the claudin family. Occludin, the first of these proteins to be identified, stabilizes the structural framework of the junction. Claudin-2, one of several tissue-specific isoforms, is associated with permeability properties of the intestinal epithelium, and its behavior is subtle: depending on context, it can either loosen or remodel the barrier. Another important player is lipocalin-2, an antibacterial, iron-sequestering protein of innate immunity that participates in the barrier’s defensive repertoire and is often induced by inflammatory stimuli. The inflammatory cytokine tumor necrosis factor alpha, meanwhile, can undermine the barrier by triggering the internalization of occludin, raising permeability.

Probiotic lactobacilli have repeatedly been shown to bolster this system. Certain strains of Lactiplantibacillus plantarum increase the production and correct localization of tight junction proteins by upregulating the genes that encode them, thereby reducing permeability and guarding against inflammation. Researchers have also explored postbiotics, the cell-free supernatants left behind after bacterial growth, which contain the bioactive metabolites of probiotic cultures without the living cells themselves. What has remained murky is how the food matrix itself contributes when probiotics are delivered as part of a fermented food, rather than as a purified supplement. The new study set out to probe exactly that boundary.

The team, led by Jihen Missaoui with colleagues at the University of Bari Aldo Moro in Italy and the University of Monastir in Tunisia, worked with two lactobacilli previously isolated from traditional zgougou: Lactiplantibacillus paraplantarum A1 and Lactiplantibacillus plantarum A2. They prepared these bacteria in three forms: living cell biomass from liquid culture, cell-free supernatant from the same cultures, and zgougou juice fermented with both strains together as a starter consortium. For comparison, they also prepared zgougou juice that had fermented spontaneously, relying on whatever microbes happened to be present. Each preparation was applied to monolayers of Caco-2 cells, a human colon cancer cell line that, when grown for 21 days on Transwell inserts, differentiates into a faithful in vitro model of the intestinal epithelium.

To gauge barrier integrity, the researchers measured transepithelial electrical resistance, or TEER, a standard technique in which a small current is passed across the cell layer; higher resistance means tighter junctions and a less permeable barrier. As a positive control, they challenged cells with lipopolysaccharide, a component of the outer membrane of E. coli that reliably disrupts tight junctions. The LPS treatment produced the expected time-dependent response, with resistance dropping significantly within 30 minutes before partially recovering. Against this baseline, the effects of the bacterial preparations emerged clearly, though not uniformly.

Exposure to the biomass of L. paraplantarum A1 raised TEER values significantly at early time points, an effect that depended on both the dose and the dilution used. The cell-free supernatant of the same strain increased resistance after six hours, and the biomass and supernatant of L. plantarum A2 produced significant boosts at 30 minutes. The most striking result, however, came from the fermented food itself. When the monolayers were treated with zgougou juice fermented by the two lactobacilli together, diluted tenfold, TEER values climbed to between roughly 690 and 963 ohms per square centimeter at every time point measured, dramatically higher than the untreated control. Spontaneously fermented juice produced a smaller but still significant effect at one time point. The combination of the two strains, embedded in the pine seed matrix, outperformed anything the bacteria achieved alone.

The molecular story behind these measurements proved equally intriguing. Using reverse transcription quantitative PCR, the team measured the expression of genes encoding occludin, claudin-2, lipocalin-2, and TNF alpha after 24 hours of exposure. Both bacterial biomasses and the driven-fermented juice significantly increased TNF alpha gene expression, yet an ELISA assay of the culture medium detected no corresponding increase in the protein itself. The authors attribute this discrepancy to post-transcriptional regulatory mechanisms that can block cytokine synthesis even when the gene is actively transcribed, a reminder that messenger RNA levels do not always translate into protein output.

More encouraging were the tight junction results. Biomass and cell-free supernatant of both lactobacilli significantly upregulated the genes for occludin and lipocalin-2, with the strongest effects seen at the tenfold dilution. The fermented zgougou juice, whether driven by the starter cultures or fermented spontaneously, significantly increased expression of both claudin-2 and occludin, with the spontaneous juice producing the strongest upregulation. The apparent tension between rising claudin-2 expression and rising TEER, which might seem contradictory given claudin-2’s association with permeability, is resolved by context: the functional impact of claudin-2 depends on the overall composition of the junction complex, and its overexpression here likely signals junction remodeling and epithelial homeostasis rather than a leaky barrier.

The authors hypothesize that the observed effects arise from a synergy between the bioactive compounds of Aleppo pine seeds, which are rich in linoleic and alpha-linolenic acids, flavonoids, and phenolics with antioxidant and anti-inflammatory activity, and the metabolic activity of the fermenting lactobacilli. Vegetable matrices may also protect probiotic bacteria during their passage through the stomach and small intestine, releasing them in the colon where they can interact directly with the host. The study is the first to show that fermented zgougou juice can upregulate genes encoding tight junction proteins in an intestinal cell model, and it underscores how traditional fermented foods, from kefir to kimchi, may modulate gut permeability through the combined action of microbes, their metabolites, and plant phytochemicals. The researchers caution that the effects are strain-dependent and shaped by the host and its resident microbiota, and they call for untargeted metabolomics to identify the specific compounds responsible, followed by animal studies to confirm whether the barrier-reinforcing effect survives the journey from the dish to the organism.

Subject of Research: Effects of Tunisian fermented zgougou and probiotic lactobacilli on intestinal epithelial barrier integrity in Caco-2 cell models

Article Title: Impact of Tunisian Fermented Zgougou and Related Lactobacilli on Caco‐2 Cell Barrier Integrity: Upregulation of Key Tight Junction Proteins

Article References: Missaoui, J., Liso, M., Lataoui, M., Limongelli, R., Minervini, F., Achour, L., & De Angelis, M. (2026). Impact of Tunisian Fermented Zgougou and Related Lactobacilli on Caco‐2 Cell Barrier Integrity: Upregulation of Key Tight Junction Proteins. Food Science & Nutrition, 14(10), Article e72387. https://doi.org/10.1002/fsn3.72387

Image Credits: AI Generated

DOI: 10.1002/fsn3.72387

Keywords: zgougou, Aleppo pine seeds, fermented foods, lactobacilli, probiotics, gut barrier, tight junction proteins, occludin, claudin-2, Caco-2 cells, TEER, postbiotics