A comprehensive and comparative phenotypic analysis of the collaborative founder strains identifies new and known phenotypes

Mammalian Genome - The collaborative cross (CC) is a large panel of mouse-inbred lines derived from eight founder strains (NOD/ShiLtJ, NZO/HILtJ, A/J, C57BL/6J, 129S1/SvImJ, CAST/EiJ, PWK/PhJ, and...     

Responsiveness of cardiometabolic-related microbiota to diet is influenced by host genetics

Intestinal microbial community structure is driven by host genetics in addition to environmental factors such as diet. In comparison with environmental influences, the effect of host genetics on intestinal microbiota, and how host-driven differences alter host metabolism is unclear. Additionally, the interaction between host genetics and diet, and the impact on the intestinal microbiome and possible down-stream effect on host metabolism is not fully understood, but represents another aspects of inter-individual variation in disease risk. The objectives of this study were to investigate how diet and genetic background shape microbial communities, and how these diet- and genetic-driven microbial differences relate to cardiometabolic phenotypes. To determine these effects, we used the 8 progenitor strains of the collaborative cross/diversity outbred mapping panels (C57BL/6J, A/J, NOD/ShiLtJ, NZO/HILtJ, WSB/EiJ, CAST/EiJ, PWK/PhJ, and 129S1/SvImJ). 16s rRNA profiling of enteric microbial communities in addition to the assessment of phenotypes central to cardiometabolic health was conducted under baseline nutritional conditions and in response to diets varying in atherogenic nutrient (fat, cholesterol, cholic acid) composition. These studies revealed strain-driven differences in enteric microbial communities which were retained with dietary intervention. Diet–strain interactions were seen for a core group of cardiometabolic-related microbial taxa. In conclusion, these studies highlight diet and genetically regulated cardiometabolic-related microbial taxa. Furthermore, we demonstrate the progenitor model is useful for nutrigenomic-based studies and screens seeking to investigate the interaction between genetic background and the phenotypic and microbial response to diet.     

Germline Genetic Variation Modulates Tumor Progression and Metastasis in a Mouse Model of Neuroendocrine Prostate Carcinoma

Neuroendocrine (NE) differentiation has gained increased attention as a prostate cancer (PC) prognostic marker. The aim of this study is to determine whether host germline genetic variation influences tumor progression and metastasis in C57BL/6-Tg(TRAMP)8247Ng/J (TRAMP) mouse model of aggressive NEPC. TRAMP mice were crossed to the eight progenitor strains of the Collaborative Cross recombinant inbred panel to address this. Tumor growth and metastasis burden were quantified in heterozygous transgene positive F1 male mice at 30 weeks of age. Compared to wild-type C57BL/6J-Tg(TRAMP)824Ng/J males, TRAMP x CAST/EiJ, TRAMP x NOD/ShiLtJ and TRAMP x NZO/HlLtJ F1 males displayed significant increases in tumor growth. Conversely, TRAMP x WSB/EiJ and TRAMP x PWK/PhJ F1 males displayed significant reductions in tumor growth. Interestingly, despite reduced tumor burden, TRAMP x WSB/EiJ males had an increased nodal metastasis burden. Patterns of distant pulmonary metastasis tended to follow the same patterns as that of local dissemination in each of the strains. All tumors and metastases displayed positive staining for NE markers, synaptophysin, and FOXA2. These experiments conclusively demonstrate that the introduction of germline variation by breeding modulates tumor growth, local metastasis burden, and distant metastasis frequency in this model of NEPC. These strains will be useful as model systems to facilitate the identification of germline modifier genes that promote the development of aggressive forms of PC.     

Hindlimb casting decreases muscle mass in part by proteasome-dependent proteolysis but independent of protein synthesis

The hypothesis of the present study was that rats subjected to short-term unilateral hindlimb immobilization would incur skeletal muscle wasting and concomitant alterations in protein synthesis, controllers of translation, and indexes of protein degradation. Rats were unilaterally casted for 1, 3, or 5 days to avoid complications associated with other disuse models. In the casted limb, gastrocnemius wet weight decreased 12% after 3 days and thereafter remained constant. In contrast, the contralateral control leg displayed a steady growth rate over time. The rate of protein synthesis and translational efficiency were unchanged in the immobilized muscle at day 5. The total amount and phosphorylation state of regulators of translational initiation and elongation were unaltered. The mRNA contents of polyubiquitin and the ubiquitin ligases muscle atrophy F-box (MAFbx)/Atrogin-1 and muscle RING finger 1 (MuRF1) were elevated in immobilized muscle at all time points, with peak expression occurring at day 3. Daily injection of the type II glucocorticoid receptor antagonist RU-486 did not prevent decreases in gastrocnemius wet weight nor increases in mRNA for MAFbx/Atrogin-1 and MuRF1. However, in vivo administration of the proteasome inhibitor Velcade prevented 53% of wet weight loss associated with 3 days of immobilization. These data suggest that the loss of skeletal muscle mass in this model of disuse appears to be glucocorticoid independent, can be partially rescued with a potent proteasome inhibitor, and is associated with enhanced mRNA expression of multiple factors that contribute to ubiquitin- proteasome-dependent degradation and are likely to control the remodeling of immobilized skeletal muscle during atrophy.     

Atrolnc-1在制动诱导小鼠后肢肌萎缩中的作用研究*

目的: 探讨Atrolnc-1在制动诱导小鼠后肢肌萎缩中的作用。方法: 将雄性C57BL/6小鼠随机分为对照组(Control)和制动组(Immobilization),每组10只。对照组不作任何实验处理,制动组小鼠右侧后肢装入自制塑料制动器固定。2周后分离其腓肠肌,用苏木素-伊红(HE)染色并观察腓肠肌形态学改变,测定肌纤维横截面积。采用实时荧光定量PCR(QRT-PCR)检测肌肉萎缩F-box蛋白(Atrogin-1)及肌萎缩特异性长链非编码RNA Atrolnc-1的变化。蛋白免疫印迹(WB)检测Atrogin-1、肌肉环状指蛋白1(MuRF-1)、胞浆及胞核p-NF-κB蛋白的表达。结果: 制动2周后小鼠腓肠肌萎缩。与对照组相比,制动组小鼠腓肠肌湿重减少(P＞0.05),腓肠肌湿重/体重千分比明显降低(P＜0.05);HE染色可见制动组骨骼肌大量肌纤维缩小或溶解,肌纤维横纹排列紊乱,间质见炎症细胞浸润;肌纤维横截面积减少(P＜0.01)。QRT-PCR及WB结果显示,Atrolnc-1表达上升(P＜0.01),胞浆p-NF-κB蛋白表达减少(P＜0.01),但胞核p-NF-κB蛋白表达升高(P＜0.01),同时Atrogin-1(P＜0.01)与MuRF-1(P＜0.01)表达均升高。结论: 制动诱导小鼠腓肠肌萎缩,可能与Atrolnc-1激活NF-κB入核,促进MuRF-1表达增加有关。     

A ketogenic amino acid rich diet benefits mitochondrial homeostasis by altering the AKT/4EBP1 and autophagy signaling pathways in the gastrocnemius and soleus

Muscle biology is important topic in diabetes research. We have reported that a diet with ketogenic amino acids rich replacement (KAAR) ameliorated high-fat diet (HFD)-induced hepatosteatosis via activation of the autophagy system. Here, we found that a KAAR ameliorated the mitochondrial morphological alterations and associated mitochondrial dysfunction induced by an HFD through induction of the AKT/4EBP1 and autophagy signaling pathways in both fast and slow muscles. The mice were fed with a standard HFD (30% fat in food) or an HFD with KAAR (HFD^KAAR). In both the gastrocnemius and the soleus, HFD^KAAR ameliorated HFD-impaired mitochondrial morphology and mitochondrial function, characterized by decreased mitofusin 2, optic atrophy 1, peroxisome proliferator-activated receptor (PPAR) γ coactivator-1α and PPARα levels and increased dynamin-related protein 1 levels. The decreased levels of phosphorylated AKT and 4EBP1 in the gastrocnemius and soleus of HFD-fed mice were remediated by HFD^KAAR. Furthermore, the HFD^KAAR ameliorated the HFD-induced autophagy defects in the gastrocnemius and soleus. These findings suggest that KAAR may be a novel strategy to combat obesity-induced mitochondrial dysfunction, likely through induction of the AKT/4EBP1 and autophagy pathways in skeletal muscle.     

Effects of varying periods of immobilization of a limb on the morphology of a peripheral nerve

Immobilization of a hind limb of the cat in the resting position was found to result in morphological changes in the nerve supplying the medial head of the gastrocnemius muscle. A reduction in the diameter of the larger myelinated fibres (greater than 12 micrometers) concomitant with a reduction in the mean fibre diameter of the nerve were observed. This reduction in the mean fibre diameter of the nerve was found to be directly proportional to the period of immobilization. Prolonged immobilization beyond 8 weeks, resulted in the splitting of the myelin lamellae, formation of myelin rings and figures in some of the larger axons in addition to an abundant increase in collagen. These structural changes indicate a definite neural involvement in the disuse atrophy of the muscle as a result of immobilization extending for a period of 8 weeks and above.     

Lethal Monkeypox Virus Infection of CAST/EiJ Mice Is Associated with a Deficient Gamma Interferon Response

Monkeypox virus (MPXV) is endemic in Africa, where it causes disease in humans resembling smallpox. A recent importation of MPXV-infected animals into the United States raises the possibility of global spread. Rodents comprise the major reservoir of MPXV, and a variety of such animals, even those native to North America, are susceptible. In contrast, common inbred strains of mice, including BALB/c and C57BL/6, are greatly resistant to MPXV. However, several inbred strains of mice derived from wild mice, including CAST/EiJ, exhibit morbidity and mortality at relatively low inoculums of MPXV. Elucidating the basis for the susceptibility of CAST/EiJ mice could contribute to an understanding of MPXV pathogenicity and host defense mechanisms and enhance the value of this mouse strain as a model system for evaluation of therapeutics and vaccines. Here we compared virus dissemination and induced cytokine production in CAST/EiJ mice to those in the resistant BALB/c strain. Following intranasal infection, robust virus replication occurred in the lungs of both strains, although a relatively higher inoculum was required for BALB/c. However, while spread to other internal organs was rapid and efficient in CAST/EiJ mice, the virus was largely restricted to the lungs in BALB/c mice. Gamma interferon (IFN-γ) and CCL5 were induced in lungs of BALB/c mice concomitant with virus replication but not in CAST/EiJ mice. The importance of IFN-γ in protection against MPXV disease was demonstrated by the intranasal administration of the mouse cytokine to CAST/EiJ mice and the resulting protection against MPXV. Furthermore, C57BL/6 mice with inactivation of the IFN-γ gene or the IFN-γ receptor gene exhibited enhanced sensitivity to MPXV.     

Lower skeletal muscle mass in male transgenic mice with muscle-specific overexpression of myostatin

Mutations in the myostatin gene are associated with hypermuscularity, suggesting that myostatin inhibits skeletal muscle growth. We postulated that increased tissue-specific expression of myostatin protein in skeletal muscle would induce muscle loss. To investigate this hypothesis, we generated transgenic mice that overexpress myostatin protein selectively in the skeletal muscle, with or without ancillary expression in the heart, utilizing cDNA constructs in which a wild-type (MCK/Mst) or mutated muscle creatine kinase (MCK-3E/Mst) promoter was placed upstream of mouse myostatin cDNA. Transgenic mice harboring these MCK promoters linked to enhanced green fluorescent protein (EGFP) expressed the reporter protein only in skeletal and cardiac muscles (MCK) or in skeletal muscle alone (MCK-3E). Seven-week-old animals were genotyped by PCR of tail DNA or by Southern blot analysis of liver DNA. Myostatin mRNA and protein, measured by RT-PCR and Western blot, respectively, were significantly higher in gastrocnemius, quadriceps, and tibialis anterior of MCK/Mst-transgenic mice compared with wild-type mice. Male MCK/Mst-transgenic mice had 18-24% lower hind- and forelimb muscle weight and 18% reduction in quadriceps and gastrocnemius fiber cross-sectional area and myonuclear number (immunohistochemistry) than wild-type male mice. Male transgenic mice with mutated MCK-3E promoter showed similar effects on muscle mass. However, female transgenic mice with either type of MCK promoter did not differ from wild-type controls in either body weight or skeletal muscle mass. In conclusion, increased expression of myostatin in skeletal muscle is associated with lower muscle mass and decreased fiber size and myonuclear number, decreased cardiac muscle mass, and increased fat mass in male mice, consistent with its role as an inhibitor of skeletal muscle mass. The mechanism of gender specificity remains to be clarified.     

Prevention of Disuse Muscle Atrophy by Dietary Ingestion of 8-Prenylnaringenin in Denervated Mice

Flavonoids have attracted considerable attention in relation to their effects upon health. 8-Prenylnaringenin (8-PN) is found in the common hop (Humulus lupulus) and assumed to be responsible for the health impact of beer consumption. We wanted to clarify the effects of prenylation on the physiological functions of dietary flavonoids by comparing the effects of 8-PN with that of intact naringenin in the prevention of disuse muscle atrophy using a model of denervation in mice. Consumption of 8-PN (but not naringenin) prevented loss of weight in the gastrocnemius muscle further supported by the lack of induction of the protein content of a key ubiquitin ligase involved in muscle atrophy, atrogin-1, and by the activation of Akt phosphorylation. 8-PN content in the gastrocnemius muscle was tenfold higher than that of naringenin. These results suggested that, compared with naringenin, 8-PN was effectively concentrated into skeletal muscle to exert its preventive effects upon disuse muscle atrophy. It is likely that prenylation generates novel functions for 8-PN by enhancing its accumulation into muscle tissue through dietary intake.     

Deficiency of inducible nitric oxide synthase attenuates immobilization-induced skeletal muscle atrophy in mice

The present study examined the effects of inducible nitric oxide synthase (iNOS) deficiency on skeletal muscle atrophy in single leg-immobilized iNOS knockout (KO) and wild-type (WT) mice. The left leg was immobilized for 1 wk, and the right leg was used as the control. Muscle weight and contraction-stimulated glucose uptake were reduced by immobilization in WT mice, which was accompanied with increased iNOS expression in skeletal muscle. Deficiency of iNOS attenuated muscle weight loss and the reduction in contraction-stimulated glucose uptake by immobilization. Phosphorylation of Akt, mTOR, and p70S6K was reduced to a similar extent by immobilization in both WT and iNOS KO mice. Immobilization decreased FoxO1 phosphorylation and increased mRNA and protein levels of MuRF1 and atrogin-1 in WT mice, which were attenuated in iNOS KO mice. Aconitase and superoxide dismutase activities were reduced by immobilization in WT mice, and deficiency of iNOS normalized these enzyme activities. Increased nitrotyrosine and carbonylated protein levels by immobilization in WT mice were reversed in iNOS KO mice. Phosphorylation of ERK and p38 was increased by immobilization in WT mice, which was reduced in iNOS KO mice. Immobilization-induced muscle atrophy was also attenuated by an iNOS-specific inhibitor N(6)-(1-iminoethyl)-l-lysine, and this finding was accompanied by increased FoxO1 phosphorylation and reduced MuRF1 and atrogin-1 levels. These results suggest that deficiency of iNOS attenuates immobilization-induced skeletal muscle atrophy through reduced oxidative stress, and iNOS-induced oxidative stress may be required for immobilization-induced skeletal muscle atrophy.     

Quantitative trait loci associated with blood pressure of metabolic syndrome in the progeny of NZO/HILtJ × C3H/HeJ intercrosses

In a previous study in 15 inbred mouse strains, we found highest and lowest systolic blood pressures in NZO/HILtJ mice (metabolic syndrome) and C3H/HeJ mice (common lean strain), respectively. To identify the loci involved in hypertension in metabolic syndrome, we performed quantitative trait locus (QTL) analysis for blood pressure with direction of cross as a covariate in segregating F₂ males derived from NZO/HILtJ and C3H/HeJ mice. We detected three suggestive main-effect QTLs affecting systolic and diastolic blood pressures (SBP and DBP). We analyzed the first principle component (PC1) generated from SBP and DBP to investigate blood pressure. In addition to all the suggestive QTLs (Chrs 1, 3, and 8) in SBP and DBP, one suggestive QTL on Chr 4 was found in PC1 in the main scan. Simultaneous search identified two significant epistatic locus pairs (Chrs 1 and 4, Chrs 4 and 8) for PC1. Multiple regression analysis revealed three blood pressure QTLs (Bpq10, 100 cM on Chr 1; Bpq11, 6 cM on Chr 4; Bpq12, 29 cM on Chr 8) accounting for 29.4% of blood pressure variance. These were epistatic interaction QTLs constructing a small network centered on Chr 4, suggesting the importance of genetic interaction for development of hypertension. The blood pressure QTLs on Chrs 1, 4, and 8 were detected repeatedly in multiple studies using common inbred nonobese mouse strains, implying substantial QTL independent of development of obesity and insulin resistance. These results enhance our understanding of complicated genetic factors of hypertension in metabolic diseases. Eri Nishihara, Shirng-Wern Tsaih, Chieko Tsukahara and Sarah Langley contributed equally to this work.     

Increased physical activity cosegregates with higher intake of carbohydrate and total calories in a subcongenic mouse strain

C57BL/6 J (B6) and CAST/EiJ (CAST), the inbred strain derived from M. musculus castaneus, differ in nutrient intake behaviors, including dietary fat and carbohydrate consumption in a two-diet-choice paradigm. Significant quantitative trait loci (QTLs) for carbohydrate (Mnic1) and total energy intake (Kcal2) are present between these strains on chromosome (Chr) 17. Here we report the refinement of the Chr 17 QTL in a subcongenic strain of the B6.CAST- ^{D17Mit19-D17Mit91} congenic mice described previously. This new subcongenic strain possesses CAST Chr 17 donor alleles from 4.8 to 45.4 Mb on a B6 background. Similar to CAST, the subcongenic mice exhibit increased carbohydrate and total calorie intake per body weight, while fat intake remains equivalent. Unexpectedly, this CAST genomic segment also confers two new physical activity phenotypes: 22% higher spontaneous physical activity levels and significantly increased voluntary wheel-running activity compared with the parental B6 strain. Overall, these data suggest that gene(s) involved in carbohydrate preference and increased physical activity are contained within the proximal region of Chr 17. Interval-specific microarray analysis in hypothalamus and skeletal muscle revealed differentially expressed genes within the subcongenic region, including neuropeptide W (Npw); glyoxalase I (Glo1); cytochrome P450, family 4, subfamily f, polypeptide 1 (Cyp4f15); phospholipase A2, group VII (Pla2g7); and phosphodiesterase 9a (Pde9a). This subcongenic strain offers a unique model for dissecting the contributions and possible interactions among genes controlling food intake and physical activity, key components of energy balance.     

Diet-induced obesity in genetically diverse collaborative cross mouse founder strains reveals diverse phenotype response and amelioration by quercetin treatment in 129S1/SvImJ,PWK/EiJ,CAST/PhJ,and WSB/EiJ mice

Significant evidence suggests protective effects of flavonoids against obesity in animal models, but these often do not translate to humans. One explanation for this disconnect is use of a few mouse strains (notably C57BL/6 J) in obesity studies. Obesity is a multifactorial disease. The underlying causes are not fully replicated by the high-fat C57BL/6 J model, despite phenotypic similarities. Furthermore, the impact of genetic factors on the activities of flavonoids is unknown. This study was designed to explore how diverse mouse strains respond to diet-induced obesity when fed a representative flavonoid. A subset of Collaborative Cross founder strains (males and females) were placed on dietary treatments (low-fat, high-fat, high-fat with quercetin, high-fat with quercetin and antibiotics) longitudinally. Diverse responses were observed across strains and sexes. Quercetin appeared to moderately blunt weight gain in male C57 and both sexes of 129S1/SvImJ mice, and slightly increased weight gain in female C57 mice. Surprisingly, quercetin dramatically blunted weight gain in male, but not female, PWK/PhJ mice. For female mice, quercetin blunted weight gain (relative to the high-fat phase) in CAST/PhJ, PWK/EiJ and WSB/EiJ mice compared to C57. Antibiotics did not generally result in loss of protective effects of quercetin. This highlights complex interactions between genetic factors, sex, obesity stimuli, and flavonoid intake, and the need to move away from single inbred mouse models to enhance translatability to diverse humans. These data justify use of genetically diverse Collaborative Cross and Diversity Outbred models which are emerging as invaluable tools in the field of personalized nutrition.     

Mapping of genetic modifiers of Eya1 bor/bor in CAST/EiJ and BALB/cJ that suppress cochlear aplasia and associated deafness

Mice homozygous for the hypomorphic allele Eya1 ( bor ) exhibit cochlear aplasia, with associated deafness, and renal hypoplasia, similar to Branchio-Oto-Renal syndrome (BOR) in humans. Although much is known about the genetics of the disease, little is known about the factors that modify its phenotypic expression. We have recently detailed two modifier loci (Mead1 and Mead2) in a C3HeB/FeJ-Eya1 ( bor/+ ) x C57BL/6 J intercross that suppress the ear-related phenotypes in our hypomorphic mutants. In this study we report corroborating evidence for our initial finding with the identification of two modifier loci mapping to the same region in CAST/EiJ and BALB/cJ. Furthermore, we describe an additional locus (Mead3) on chromosome 19 in CAST/EiJ, within which the previously cloned suppressor Nxf1 resides. The suppression effect on cochlear coiling was studied on congenic line(s) for each protective allele. The penetrance and suppressor strength of these alleles vary by strain and locus. Eya1 ( bor/bor ) hypomorphs, when homozygous for each of the three protective alleles (CAST/EiJ, C57BL/6 J, or BALB/cJ) at the Mead1 or Mead2 locus, exhibit completely penetrant suppression of cochlear agenesis. At the Mead1 locus, the C57BL/6 J and BALB/cJ alleles have comparable strengths. At the Mead2 locus, the C57BL/6 J and CAST/EiJ alleles have comparable strengths. In contrast, mice with genotype Eya1 ( bor/bor )Mead3(CAST/CAST) exhibit incomplete penetrance (50%) and a wide range of cochlear coiling (1/4-1(1/2) turns). The identification of these additional modifier alleles could provide crucial clues for evaluating the candidate genes.     

Acute antibody-directed myostatin inhibition attenuates disuse muscle atrophy and weakness in mice

Counteracting the atrophy of skeletal muscle associated with disuse has significant implications for minimizing the wasting and weakness in plaster casting, joint immobilization, and other forms of limb unloading, with relevance to orthopedics, sports medicine, and plastic and reconstructive surgery. We tested the hypothesis that antibody-directed myostatin inhibition would attenuate the loss of muscle mass and functional capacity in mice during 14 or 21 days of unilateral hindlimb casting. Twelve-week-old C57BL/10 mice were subjected to unilateral hindlimb plaster casting or served as controls. Mice received subcutaneous injections of saline or a mouse chimera of anti-human myostatin antibody (PF-354, 10 mg/kg; n = 6-9) on days 0 and 7 and were tested for muscle function on day 14, or were treated on days 0, 7, and 14 and tested for muscle function on day 21. Hindlimb casting reduced muscle mass, fiber size, and function of isolated soleus and extensor digitorum longus (EDL) muscles (P < 0.05). PF-354 attenuated the loss of muscle mass, fiber size, and function with greater effects after 14 days than after 21 days of casting, when wasting and weakness had plateaued (P < 0.05). Antibody-directed myostatin inhibition therefore attenuated the atrophy and loss of functional capacity in muscles from mice subjected to unilateral hindlimb casting with reductions in muscle size and strength being most apparent during the first 14 days of disuse. These findings highlight the therapeutic potential of antibody-directed myostatin inhibition for disuse atrophy especially within the first 2 wk of disuse.     

Zonula Occludens-1, Occludin and E-cadherin Expression and Organization in Salivary Glands with Sj?gren’s Syndrome

Sjögren’s syndrome (SS) is a chronic inflammatory autoimmune disorder that causes secretory dysfunction of the salivary glands leading to dry mouth. Previous studies reported that tight junction (TJ) proteins are down-regulated and lose polarity in human minor salivary glands with SS, suggesting that TJ structure is compromised in SS patients. In this paper, we utilized the NOD/ShiLtJ mouse with the main goal of evaluating this model for future TJ research. We found that the organization of apical proteins in areas proximal and distal to lymphocytic infiltration remained intact in mouse and human salivary glands with SS. These areas looked comparable to control glands (i.e., with no lymphocytic infiltration). TJ staining was absent in areas of lymphocytic infiltration coinciding with the loss of salivary epithelium. Gene expression studies show that most TJs are not significantly altered in 20-week-old NOD/ShiLtJ mice as compared with age-matched C57BL/6 controls. Protein expression studies revealed that the TJ proteins, zonula occludens-1 (ZO-1), occludin, claudin-12, as well as E-cadherin, do not significantly change in NOD/ShiLtJ mice. Our results suggest that ZO-1, occludin and E-cadherin are not altered in areas without lymphocytic infiltration. However, future studies will be necessary to test the functional aspect of these results.