Segment-specific Changes Research Articles

Probiotics to HIV-Infected Immunological Nonresponders: Altered Mucosal Immunity and Microbial Diversity Restricted to Ileum.

HIV-infected immunological nonresponders (INRs) have increased risk of non-AIDS morbidity and compromised gut barrier immunity. Probiotics are widely used to improve health. We assessed the effects of probiotics in INRs with a comprehensive analysis of gut immunity and microbiome in terminal ileum and sigmoid colon. The study involved clinical intervention with five-strain probiotic capsules (1.2 × 1010 CFUs/d) for 8 weeks in 20 INRs with CD4+ T-cell counts <400 cells/µL and plasma HIV RNA <50 copies/mL for more than 3.5 years. Colonoscopy with sampling of gut biopsies from terminal ileum and sigmoid colon and fecal and blood sampling were performed before and after the intervention. Flow cytometry (cytokine production, immune activation, and exhaustion), ELISA (inflammation, microbial translocation, and enterocyte damage), and 16S rRNA sequencing analyses were applied. In the terminal ileum, increased alpha diversity, increased abundance of Bifidobacterium sp., and decreased frequencies of IL-22+ CD4+ T cells were observed. The increased abundance of Bifidobacterium sp. in the terminal ileum correlated with increased fraction of CD4+ T cells in the same compartment (r = 0.54, P = 0.05) and increased CD4/CD8 ratio in peripheral blood (r = 0.49, P = 0.05). There were no corresponding changes in the sigmoid colon and no changes in fecal microbiome. Probiotic intervention did not affect peripheral blood CD4 count, viral load, or soluble markers of inflammation and microbial translocation. Probiotics induced segment-specific changes in the terminal ileum but did not affect systemic CD4 counts in INRs. Further clinical studies are warranted to recommend probiotics to INRs.

Renal microvascular endothelial cell responses in sepsis-induced acute kidney injury.

Microvascular endothelial cells in the kidney have been a neglected cell type in sepsis-induced acute kidney injury (sepsis-AKI) research; yet, they offer tremendous potential as pharmacological targets. As endothelial cells in distinct cortical microvascular segments are highly heterogeneous, this Review focuses on endothelial cells in their anatomical niche. In animal models of sepsis-AKI, reduced glomerular blood flow has been attributed to inhibition of endothelial nitric oxide synthase activation in arterioles and glomeruli, whereas decreased cortex peritubular capillary perfusion is associated with epithelial redox stress. Elevated systemic levels of vascular endothelial growth factor, reduced levels of circulating sphingosine 1-phosphate and loss of components of the glycocalyx from glomerular endothelial cells lead to increased microvascular permeability. Although coagulation disbalance occurs in all microvascular segments, the molecules involved differ between segments. Induction of the expression of adhesion molecules and leukocyte recruitment also occurs in a heterogeneous manner. Evidence of similar endothelial cell responses has been found in kidney and blood samples from patients with sepsis. Comprehensive studies are needed to investigate the relationships between segment-specific changes in the microvasculature and kidney function loss in sepsis-AKI. The application of omics technologies to kidney tissues from animals and patients will be key in identifying these relationships and in developing novel therapeutics for sepsis.

Establishment of a novel probe-based RT-qPCR approach for detection and quantification of tight junctions reveals age-related changes in the gut barriers of broiler chickens.

Tight junctions (TJs) play a dominant role in gut barrier formation, therefore, resolving the structures of TJs in any animal species is crucial but of major importance in fast growing broilers. They are regulated in molecular composition, ultrastructure and function by intracellular proteins and the cytoskeleton. TJ proteins are classified according to their function into barrier-forming, scaffolding and pore-forming types with deductible consequences for permeability. In spite of their importance for gut health and its integrity limited studies have investigated the TJs in chickens, including the comprehensive evaluation of TJs molecular composition and function in the chicken gut. In the actual study sequence-specific probes to target different TJ genes (claudin 1, 3, 5, 7, 10, 19, zonula occludens 1 (ZO1), occludin (OCLN) and tricellulin (MD2)) were designed and probe-based RT-qPCRs were newly developed. Claudin (CLDN) 1, 5, ZO1 and CLDN 3, 7, MD2 were engulfed in multiplex RT-qPCRs, minimizing the number of separate reactions and enabling robust testing of many samples. All RT-qPCRs were standardized for chicken jejunum and caecum samples, which enabled specific detection and quantification of the gene expression. Furthermore, the newly established protocols were used to investigate the age developmental changes in the TJs of broiler chickens from 1–35 days of age in the same organ samples. Results revealed a significant increase in mRNA expression between 14 and 21days of age of all tested TJs in jejunum. However, in caecum, mRNA expression of some TJs decreased after 1 day of age whereas some TJs mRNA remained constant till 35 days of age. Taken together, determining the segment-specific changes in the expression of TJ- proteins by RT-qPCR provides a deeper understanding of the molecular mechanisms underpinning pathophysiological changes in the gut of broiler chickens with various etiologies.

Rapid and Concomitant Gut Microbiota and Endocannabinoidome Response to Diet-Induced Obesity in Mice.

The intestinal microbiota and the expanded endocannabinoid (eCB) system, or endocannabinoidome (eCBome), have both been implicated in diet-induced obesity and dysmetabolism. These systems were recently suggested to interact during the development of obesity. We aimed at identifying the potential interactions between gut microbiota composition and the eCBome during the establishment of diet-induced obesity and metabolic complications. Male mice were fed a high-fat, high-sucrose (HFHS) diet for 56 days to assess jejunum, ileum, and cecum microbiomes by 16S rRNA gene metataxonomics as well as ileum and plasma eCBome by targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS). The HFHS diet induced early (3 days) and persistent glucose intolerance followed by weight gain and hyperinsulinemia. Concomitantly, it induced the elevation of the two eCBs, anandamide, in both ileum and plasma, and 2-arachidonoyl-glycerol, in plasma, as well as alterations in several other N-acylethanolamines and 2-acylglycerols. It also promoted segment-specific changes in the relative abundance of several genera in intestinal microbiota, some of which were observed as early as 3 days following HFHS diet. Weight-independent correlations were found between the relative abundances of, among others, Barnesiella, Eubacterium, Adlercreutzia, Parasutterella, Propionibacterium, Enterococcus, and Methylobacterium and the concentrations of anandamide and the anti-inflammatory eCBome mediator N-docosahexaenoyl-ethanolamine. This study highlights for the first time the existence of potential interactions between the eCBome, an endogenous system of multifunctional signaling lipids, and several intestinal genera during early and late HFHS-induced dysmetabolic events, with potential impact on the host capability of adapting to increased intake of fat and sucrose.IMPORTANCE The intestinal microbiota and the expanded endocannabinoid system, or endocannabinoidome, have both been implicated in diet-induced obesity and dysmetabolism. This study aims at identifying the potential interactions between these two fundamental systems-which form the gut microbiota-endocannabinoidome axis-and their involvement in the establishment of diet-induced obesity and related metabolic complications. We report here time- and segment-specific microbiome disturbances as well as modifications of intestinal and circulating endocannabinoidome mediators during high-fat, high-sucrose diet-induced glucose intolerance and subsequent obesity and hyperinsulinemia. This highlights the involvement of, and the interaction between, the gut microbiota and the endocannabinoidome during metabolic adaptation to high-fat and high-sucrose feeding. These results will help identifying actionable gut microbiome members and/or endocannabinoidome mediators to improve metabolic health.

Absorptive transport of amino acids by the rat colon.

The capacity of the colon to absorb microbially produced amino acids (AAs) and the underlying mechanisms of AA transport are incompletely defined. We measured the profile of 16 fecal AAs along the rat ceco-colonic axis and compared unidirectional absorptive AA fluxes across mucosal tissues isolated from the rat jejunum, cecum, and proximal colon using an Ussing chamber approach, in conjunction with 1H-NMR and ultra-performance liquid chromatography-mass spectrometry chemical analyses. Passage of stool from cecum to midcolon was associated with segment-specific changes in fecal AA composition and a decrease in total AA content. Simultaneous measurement of up to 16 AA fluxes under native luminal conditions, with correction for endogenous AA release, demonstrated absorptive transfer of AAs across the cecum and proximal colon at rates comparable (30-80%) to those across the jejunum, with significant Na+-dependent and H+-stimulated components. Expression profiling of 30 major AA transporter genes by quantitative PCR revealed comparatively high levels of transcripts for 20 AA transporters in the cecum and/or colon, with the levels of 12 exceeding those in the small intestine. Our results suggest a more detailed model of major apical and basolateral AA transporters in rat colonocytes and provide evidence for a previously unappreciated transfer of AAs across the colonic epithelium that could link the prodigious metabolic capacities of the luminal microbiota, the colonocytes, and the body tissues.NEW & NOTEWORTHY This study provides evidence for a previously unappreciated transfer of microbially generated amino acids across the colonic epithelium under physiological conditions that could link the prodigious metabolic capacities of the luminal microbiota, the colonocytes, and the body tissues. The segment-specific expression of at least 20 amino acid transporter genes along the colon provides a detailed mechanistic basis for uniport, heteroexchange, Na+-cotransport, and H+-cotransport components of colonic amino acid absorption.

Epididymitis: ascending infection restricted by segmental boundaries

Is the regionalization of epididymitis related to epididymal segmentation? We show for the first time that luminal ascent of bacteria is strictly gated by epididymal segment boundaries, involving ductal constriction adjacent to the infected area. The epididymal duct is a continuous, unbranched tube, coiled into segments that are divided by connective tissue septa. Sonographic analysis indicates that swelling associated with epididymitis is predominant in the cauda region. Epididymal segmentation has never been investigated in the context of pathological alterations. We analyzed segment-specific changes in the epididymal duct in a mouse model and in men. In the mouse epididymitis model (3 days post-infection, injection of bacteria into the lumen of the vas deferens), two Escherichia coli strains were tested: a uropathogenic strain CFT073 (UPEC, n = 7) and a fecal non-pathogenic strain NPEC470 (NPEC, n = 5). Two control groups: phosphate-buffered saline, sham-treated animals (n = 4) and untreated mice (n = 8). In addition, segmentation was verified by ex vivo injection of dye into the interstitial spaces of untreated mouse epididymides. Histological findings were compared with specimens from epididymitis patients (n = 10, age range 14-78, median 60 years) who underwent surgical intervention; control: samples from patients without epididymitis (n = 16, age range 38-87, median 73 years). We investigated the ascending infections by detailed histological analysis in correlation with local infection status in a mouse epididymitis model. As a proof of concept, rare patient material from two archives was analyzed: epididymides from patients who underwent surgical intervention for persisting epididymitis, and for control, histologically normal epididymides from men who underwent orchiectomy for therapy of prostatic carcinoma. Luminal ascent of E. coli in mice was strictly gated by epididymal segment boundaries. In the mouse model, both strains of E. coli were detected exclusively in the distal cauda segment associated with damage of the epithelium and muscle layer. Ductal constriction occurred in the non-infected upstream segments of infected area, putatively blocking further luminal ascent of bacteria in UPEC-infected animals. Corresponding histological and morphological changes were found in epididymitis patients. The caput region was found to be unaffected in patients and the mouse model. Patient samples represented advanced cases of epididymitis that made surgical intervention necessary. Our data demonstrate the impact of epididymal segmentation, presumably a protective response mechanism against infectious invasion and bacterial ascent, during epididymitis and affirm the importance of rapid intervention. This work was supported by grants from the State of Hessen (LOEWE-MIBIE) and the DFG (KFO 181). The authors have no conflicts of interest to declare. No clinical trial involved.

Estrogen synthesis in the spinal dorsal horn: a new central mechanism for the hormonal regulation of pain

The data summarized here suggest the existence of a new central pathway for the hormonal regulation of pain. These data mainly collected in quail, a useful model in neuroendocrinology, demonstrate that numerous neurons in the superficial laminae of the spinal cord express aromatase (estrogen-synthase). Chronic and systemic blockade of this enzyme in quail alters nociception within days, indicating that the slow genomic effects of sex steroids on nociception classically observed in mammals also occur in birds and require aromatization of androgens into estrogens. However, by contrast with these slow effects, acute intrathecal inhibition of aromatase in restricted spinal cord segments reveals that estrogens can also control nociception much faster, within 1 min, presumably through the activation of a nongenomic pathway and in a manner that depends on an immediate response to fast activation/deactivation of local aromatase activity. This emergent central and rapid paracrine mechanism might permit instantaneous and segment-specific changes in pain sensitivity; it draws new interesting perspectives for the study of the estrogenic control of pain, thus far limited to the classical view of slow genomic changes in pain, depending on peripheral estrogens. The expression of aromatase in the spinal cord in other species and in other central nociception-related areas is also briefly discussed.

Site-specific effects of dietary salt intake on guanylin and uroguanylin mRNA expression in rat intestine.

Guanylin and uroguanylin are newly discovered intestinal peptides that have been shown to affect NaCl transport in both the intestine and kidney. The present study tests the hypothesis that guanylin and uroguanylin mRNA expression in each major region of the intestine is regulated by NaCl intake. Semiquantitative multiplex RT-PCR analysis was used to determine the molecular expression of guanylin and uroguanylin in the duodenum, jejunum, ileum, and colon in rats maintained on low (LS), normal (NS), or high (HS) NaCl intake for 4 days. LS intake reduced the expression of uroguanylin, and to a lesser degree, guanylin mRNA in all intestinal segments compared to NS intake. The duodenum was the site of the greatest decrease for both. In contrast, HS intake significantly increased the expression of guanylin mRNA only in the duodenum and jejunum and had minimal effect on uroguanylin mRNA. The minimum time required for altered gene expression was determined by delivering an oral NaCl challenge directly to the gastrointestinal tract by oro-gastric administration to LS or NS animals. In LS rats, NaCl oro-gastric administration significantly increased mRNA expression of both peptides in all intestinal segments. Furthermore, the increases in guanylin and uroguanylin mRNA were detected within 4 h and plateaued by 8 h. Conversely, acute oro-gastric administration of the same NaCl solution to NS rats caused elevations of guanylin mRNA only in the duodenum and jejunum, and of uroguanylin mRNA only in the ileum and colon. In conclusion, the data demonstrate that variations in NaCl intake lead to intestinal segment-specific changes in guanylin and uroguanylin mRNA expression.

Sperm structural and motility changes during aging in the Brown Norway rat.

The Brown Norway rat provides a useful model to study aging of the male reproductive tract because of the selective age-dependent pathological changes that are found in the testis, epididymis, and prostate. In the testis, there is a clear age-dependent decrease in both steroidogenesis and spermatogenesis. In the epididymis, some striking segment-specific changes occur at the histological and biochemical levels prior to the major loss of spermatogenesis. We hypothesized that formation of spermatozoa in the testis and maturation of spermatozoa in the epididymis (ie, acquisition of motility and loss of the cytoplasmic droplet) may be altered during aging. Changes in the morphology of spermatozoa were assessed by light and electron microscopy. Using computer-assisted sperm analysis, the motility parameters of spermatozoa obtained from the caput and cauda epididymidis of young and old Brown Norway rats were compared. In old animals, we also compared the motility of spermatozoa from epididymides adjacent to regressed testes with those from epididymides adjacent to nonregressed testes. There was a marked increase with age in the number of spermatozoa with abnormal flagellar midpieces; the nature of these defects did not change with age. In caput epididymidis, the percentage of motile sperm was similar in young and old rats. In contrast, the percentage of motile spermatozoa was significantly decreased in cauda epididymidis of old rats; spermatozoa from the regressed testis side had altered motility characteristics. Furthermore, in the cauda epididymidis on the regressed testis side of aged Brown Norway rats, the proportion of spermatozoa that retained their cytoplasmic droplet was markedly elevated. Some of these effects are likely due to changes taking place in spermatozoa during the process of spermatogenesis in the testis (eg, formation of the flagellum), whereas others could occur during sperm maturation in the epididymis (eg, acquisition of motility). The multiple effects of aging on sperm morphology, the acquisition of motility, and the shedding of the cytoplasmic droplet clearly indicate that the quality of spermatozoa is affected by aging.

Segment-Specific Changes with Age in the Expression of Junctional Proteins and the Permeability of the Blood-Epididymis Barrier in Rats1

In aging Brown Norway rats, there is a striking increase in the number of halo cells in the epididymis; this reflects an activation of the immune system. As the blood-epididymis barrier should protect from immunological attack, we hypothesized that there would be changes in the structure and function of this barrier with age. To test this hypothesis, we assessed the immunocytochemical localization of occludin, ZO-1, and E-cadherin, as well as the lanthanum nitrate permeability of the blood-epididymis barrier, in the epididymides of Brown Norway rats aged 3, 18, and 24 mo. In the initial segment, occludin, ZO-1, and E-cadherin immunostaining was observed at the apico-lateral junction between principal cells in the 3-mo-old animals; with increasing age, occludin and ZO-1 reactivity decreased, while E-cadherin staining increased along the lateral membrane between principal cells. In the caput, corpus, and cauda epididymidis, occludin, ZO-1, and E-cadherin immunostaining showed segment-specific and age-dependent differences in their staining patterns. The most dramatic changes were seen in the corpus epididymidis with age; the intense E-cadherin cytoplasmic staining that was observed at 3 mo was absent by 24 mo, and no occludin or ZO-1 reactivity was observed in older animals. The greatest penetration of lanthanum nitrate across the blood-epididymis barrier and in the lumen was seen in the aging corpus epididymidis, while there was no barrier permeability in the initial segment or cauda epididymidis of the aged animals. Taken together, these data indicate that there are segment-specific decreases in the structural and functional integrity of the blood-epididymis barrier with age, most notably in the corpus epididymidis.

Segment-specific morphological changes in aging Brown Norway rat epididymis.

In aging Brown Norway rats, both spermatogenesis and steroidogenesis decrease. Little is known about changes in the epididymis during aging. However, since the two major components entering the epididymis from the testis change, we hypothesized that epididymal histology would be affected by advancing age. The epididymides of Brown Norway rats ranging in age from 3 to 24 mo were prepared for light and electron microscopy. Striking quantitative and qualitative changes were noted. There was an age-dependent increase in the thickness of the basal membrane and in the number of halo cells. There were also major segment-specific changes in the appearance of cells along the epididymis with age. At 12 mo, basal cells in the initial segment emitted pseudopods into the basement membrane. By 18 mo, in the caput epididymidis, clear cells were filled with lysosomes; these cells frequently showed bulging protrusions into the lumen. In the corpus epididymidis, the cytoplasm of principal cells had numerous large lysosomes both below and above the nucleus; apical cells were usually occupied by one giant membranous lysosome. In the proximal cauda, clear cells became filled with dense lysosomes, and principal cells presented large clear vacuoles; debris from spermatozoa was found in the larger vacuoles. In summary, aging of the epididymis was accompanied by the emergence of characteristic features of aging and activation of the immune system. Furthermore, there were many cell- and segment-specific changes. Finally, these changes were not related to the presence of spermatozoa, often preceding their disappearance, thus indicating that there may be an intrinsic mechanism of aging in epididymal epithelial cells.

Effects of cold exposure and hibernation on renal Na,K-ATPase of the jerboa Jaculus orientalis.

Changes in activity and abundance of renal Na,K-ATPase were evaluated during cold exposure and hibernation of the jerboa Jaculus orientalis by measuring the hydrolytic activity, the number of units and the transport activity of Na,K-ATPase in isolated nephron segments. As compared to controls, jerboas exposed to cold (6 degrees C) for 4-5 weeks displayed mild diuresis, decreased urinary osmolality and increased kaliuresis. In cold-exposed jerboas, Na,K-ATPase hydrolytic activity was reduced in the medullary thick ascending limb and enhanced in the cortical and outer medullary collecting duct, whereas it was not altered in other nephron segments. The number of Na,K-ATPase units and the activity of Na,K-pump, determined by [3H]-ouabain binding and by ouabain-sensitive rubidium uptake respectively, changed in parallel with the hydrolytic activity in the medullary thick ascending limb and cortical collecting duct. The maximal rate of activity (Vmax) of Na,K-ATPase was not modified further during hibernation. Thus, cold exposure, but not the onset of hibernation, induces segment-specific changes in the abundance and activity of Na,K-ATPase units which are likely to be related to the entry into hibernation, but not to the maintenance of some renal functions during deep hibernation.