Achalasia alters physiological networks depending on sex.

Achalasia is a rare esophageal motility disorder for which the etiology is not fully understood. Evidence suggests that autoimmune inflammatory infiltrates, possibly triggered by a viral infection, may lead to a degeneration of neurons within the myenteric plexus. While the infection is eventually resolved, genetically susceptible individuals may still be at risk of developing achalasia. This study aimed to determine whether immunological and physiological networks differ between male and female patients with achalasia. This cross-sectional study included 189 preoperative achalasia patients and 500 healthy blood donor volunteers. Demographic, clinical, laboratory, immunological, and tissue biomarkers were collected. Male and female participants were evaluated separately to determine the role of sex. Correlation matrices were constructed using bivariate relationships to generate complex inferential networks. These matrices were filtered based on their statistical significance to identify the most relevant relationships between variables. Network topology and node centrality were calculated using tools available in the R programming language. Previous occurrences of chickenpox, measles, and mumps infections have been proposed as potential risk factors for achalasia, with a stronger association observed in females. Principal component analysis (PCA) identified IL-22, Th2, and regulatory B lymphocytes as key variables contributing to the disease. The physiological network topology has the potential to inform whether a localized injury or illness is likely to produce systemic consequences and the resulting clinical presentation. Here we show that immunological involvement in achalasia appears localized in men because of their highly modular physiological network. In contrast, in women the disease becomes systemic because of their robust network with a larger number of inter-cluster linkages.

Increased synthesis and intestinal expression of IL-39 in patients with inflammatory bowel disease.

IL-39 (Interleukin-39) is a heterodimeric cytokine composed of IL-23p19 and EBI3 (Epstein-Barr virus-induced gene 3) subunits. Despite the evidence that correlates the role of IL-39 in regulating inflammation, its expression in the intestinal microenvironment of IBD (inflammatory bowel disease) patients is still unknown. Thus, this work was focused on characterizing relative mRNA (messenger RNA) IL-39 expression and intestinal synthesis in IBD patients. This study includes 37 patients diagnosed with ulcerative colitis (UC), 15 with Chron's disease (CD), and 22 controls. Gene expression of IL-39 subunits (IL-23p19/EBI3) was measured by RT-PCR (real time polymerase chain reaction). Intestinal synthesis was evaluated by immunohistochemistry and serum levels by ELISA. Statistical analysis was done using Prism GraphPad V6. Relative mRNA IL-39 expression was increased in patients with active UC and active CD compared to the remission UC, remission CD, and control group. High levels of relative mRNA expression of IL-39 (IL-23p19 subunit) were associated with histological activity. IHQ analysis showed increased IL-39 production in mucosa, submucosa, muscular, and serosa layer of patients with active disease. IL-39 serum production was increased in patients with UC. IL-39 gene's upregulation was found in patients with active IBD and was associated with severe histological activity in UC. This is the first report regarding the role of IL-39 in patients with IBD. The findings suggest that IL-39 might play a role as an inflammatory mediator in active IBD and could be considered a new alternative in treating this condition.

Differential expression of TOB/BTG family members in patients with plaque psoriasis: cross-sectional study.

TOB/BTG is a family of antiproliferative proteins that play an important role in the regulation of immune responses, acting as lymphocyte activators and macrophage-mediated cytotoxicity. No previous studies have explored their role in patients with psoriasis. The aim of this study was to characterize the expression of TOB/BTG family and their co-localization in skin from patients with psoriasis. This is an exploratory, observational, and cross-sectional study that included 24 plaque psoriasis patients and 15 controls. Gene expression of TOB/BTG family was determinate by RT-PCR. Protein products of TOB/BTG were evaluated by immunohistochemistry and compared with control skin tissues. Holm-Sidak's multiple comparisons test was performed. TOB/BTG family mRNA levels and protein expression were significantly decreased in psoriatic skin tissue compared to non-inflammatory control skin tissue. Double-positive cell TOB1/2, BTG1,2 and BTG4/CD16 expressions were found in normal control skin tissues through epidermis and dermis (p < 0.001) and lesser percentage in patients with mild, almost absent in moderate-severe plaque psoriasis. This is the first report of the TOB/BTG family gene and protein expression in skin tissues by a CD16 + subpopulation in plaque psoriasis. TOB/BTG family protein might represent a new therapeutic target among immune-mediated inflammatory diseases.

Prolactin Inhibits or Stimulates the Inflammatory Response of Joint Tissues in a Cytokine-dependent Manner.

The close association between rheumatoid arthritis (RA), sex, reproductive state, and stress has long linked prolactin (PRL) to disease progression. PRL has both proinflammatory and anti-inflammatory outcomes in RA, but responsible mechanisms are not understood. Here, we show that PRL modifies in an opposite manner the proinflammatory actions of IL-1ß and TNF-α in mouse synovial fibroblasts in culture. Both IL-1ß and TNF-α upregulated the metabolic activity and the expression of proinflammatory factors (Il1b, Inos, and Il6) via the activation of the nuclear factor-κB (NF-κB) signaling pathway. However, IL-1ß increased and TNF-α decreased the levels of the long PRL receptor isoform in association with dual actions of PRL on synovial fibroblast inflammatory response. PRL reduced the proinflammatory effect and activation of NF-κB by IL-1ß but increased TNF-α-induced inflammation and NF-κB signaling. The double-faceted role of PRL against the 2 cytokines manifested also in vivo. IL-1ß or TNF-α with or without PRL were injected into the knee joints of healthy mice, and joint inflammation was monitored after 24 hours. IL-1ß and TNF-α increased the joint expression of proinflammatory factors and the infiltration of immune cells. PRL prevented the actions of IL-1ß but was either inactive or further increased the proinflammatory effect of TNF-α. We conclude that PRL exerts opposite actions on joint inflammation in males and females that depend on specific proinflammatory cytokines, the level of the PRL receptor, and the activation of NF-κB signaling. Dual actions of PRL may help balance joint inflammation in RA and provide insights for development of new treatments.

A Higher Manometric Esophageal Length to Height Ratio in Achalasia Explains the Lower Prevalence of Hiatal Hernia.

Background/Aims: The evidence suggests that a shorter esophageal length (EL) in gastroesophageal reflux disease (GERD) patients is associated with the presence of hiatal hernia (HH). However, there are no reports of this association in patients with achalasia. The aim is to (1) determine the prevalence of hiatal hernia in achalasia patients, (2) compare achalasia EL with GERD patients and healthy volunteers (HV), (3) measure achalasia manometric esophageal length to height (MELH) ratio, and (4) determine if there are differences in symptoms between patients with and without hiatal hernia. Methods: This retrospective and cross-sectional study consist of 87 pre-surgical achalasia patients, 22 GERD patients, and 30 HV. High-resolution manometry (HRM), barium swallow, and upper endoscopy were performed to diagnose HH. The EL and MELH ratio were measured by HRM. Symptoms were assessed with Eckardt, Eating Assessment Tool, and GERD-health-related quality of life questionnaires. Results: The HH in GERD's prevalence was 73% vs 3% in achalasia patients (P < 0.001). Achalasia patients had a longer esophagus and a higher MELH ratio than HV and GERD patients (P < 0.001). GERD patients had a lower MELH ratio than HV (P < 0.05). EAT-10 (P < 0.0001) and Eckardt (P < 0.05) scores were higher in achalasia without HH vs HH. Conclusions: The prevalence of HH in achalasia is significantly lower than in GERD. The longer EL and the higher MELH ratio in achalasia could explain the lower prevalence of HH. Despite the low prevalence of HH in achalasia patients, the surgeon should be encouraged not to rule out HH since the risk of postoperative reflux may increase if this condition is not identified and corrected.

Supplementation with EPA and DHA omega-3 fatty acids improves peripheral immune cell mitochondrial dysfunction and inflammation in subjects with obesity.

Omega-3 fatty acids (w-3 FA) have anti-inflammatory effects and improve mitochondrial function. Nonetheless, little is known about their effect on mitochondrial bioenergetics of peripheral blood mononuclear cells (PBMCs) in individuals with obesity. Thus, this study aimed to determine the mitochondrial bioenergetics status and cell subset composition of PBMCs during obesity, before and after 1 month supplementation with w-3 FA. We performed a case-control study with twelve women with normal BMI (lean group) and 19 with grade 2 obesity (obese group), followed by a before-after prospective study where twelve subjects with obesity received a 1 month intervention with 5.25 g of w-3 FA (3.5 g eicosapentaenoic (EPA) and 1.75 g docosahexaenoic (DHA) acids), and obtained PBMCs from all participants. Mitochondrial bioenergetic markers, including basal and ATP-production associated respiration, proton leak, and nonmitochondrial respiration, were higher in PBMCs from the obese group vs. the lean group. The bioenergetic health index (BHI), a marker of mitochondrial function, was lower in the obese vs. the lean group. In addition, Th1, Th2, Th17, CD4+ Tregs, CD8+ Tregs, and Bregs, M1 monocytes and pDCreg cells were higher in PBMCs from the obese group vs. the lean group. The w-3 FA intervention improved mitochondrial function, mainly by decreasing nonmitochondrial respiration and increasing the reserve respiratory capacity and BHI. The intervention also reduced circulating pro-inflammatory and anti-inflammatory lymphocyte and monocytes subsets in individuals with obesity. The mitochondrial dysfunction of PBMCs and the higher proportion of peripheral pro-inflammatory and anti-inflammatory immune cells in subjects with obesity, improved with 1 month supplementation with EPA and DHA.

Pecans and Its Polyphenols Prevent Obesity, Hepatic Steatosis and Diabetes by Reducing Dysbiosis, Inflammation, and Increasing Energy Expenditure in Mice Fed a High-Fat Diet.

Pecans (Carya illinoinensis) are considered a functional food due to the high content of polyunsaturated fatty acids, dietary fiber and polyphenols. To determine the effect of whole pecans (WP) or a pecan polyphenol (PP) extract on the development of metabolic abnormalities in mice fed a high-fat (HF) diet, we fed C57BL/6 mice with a Control diet (7% fat), HF diet (23% fat), HF containing 30% WP or an HF diet supplemented with 3.6 or 6 mg/g of PP for 18 weeks. Supplementation of an HF diet with WP or PP reduced fat mass, serum cholesterol, insulin and HOMA-IR by 44, 40, 74 and 91%, respectively, compared to the HF diet. They also enhanced glucose tolerance by 37%, prevented pancreatic islet hypertrophy, and increased oxygen consumption by 27% compared to the HF diet. These beneficial effects were associated with increased thermogenic activity in brown adipose tissue, mitochondrial activity and AMPK activation in skeletal muscle, reduced hypertrophy and macrophage infiltration of subcutaneous and visceral adipocytes, reduced hepatic lipid content and enhanced metabolic signaling. Moreover, the microbial diversity of mice fed WP or PP was higher than those fed HF, and associated with lower circulating lipopolysaccharides (~83-95%). Additionally, a 4-week intervention study with the HF 6PP diet reduced the metabolic abnormalities of obese mice. The present study demonstrates that WP or a PP extract prevented obesity, liver steatosis and diabetes by reducing dysbiosis, inflammation, and increasing mitochondrial content and energy expenditure. Pecan polyphenols were mainly condensed tannin and ellagic acid derivatives including ellagitannins as determined by LC-MS. Herein we also propose a model for the progression of the HF diet-mediated metabolic disorder based on early and late events, and the possible molecular targets of WP and PP extract in preventive and intervention strategies. The body surface area normalization equation gave a conversion equivalent to a daily human intake dose of 2101-3502 mg phenolics that can be obtained from 110-183 g pecan kernels/day (22-38 whole pecans) or 21.6-36 g defatted pecan flour/day for an average person of 60 kg. This work lays the groundwork for future clinical studies.

Vachellia farnesiana Pods or a Polyphenolic Extract Derived from Them Exert Immunomodulatory, Metabolic, Renoprotective, and Prebiotic Effects in Mice Fed a High-Fat Diet.

Obesity causes systemic inflammation, hepatic and renal damage, as well as gut microbiota dysbiosis. Alternative vegetable sources rich in polyphenols are known to prevent or delay the progression of metabolic abnormalities during obesity. Vachellia farnesiana (VF) is a potent source of polyphenols with antioxidant and anti-inflammatory activities with potential anti-obesity effects. We performed an in vivo preventive or an interventional experimental study in mice and in vitro experiments with different cell types. In the preventive study, male C57BL/6 mice were fed with a Control diet, a high-fat diet, or a high-fat diet containing either 0.1% methyl gallate, 10% powdered VFP, or 0.5%, 1%, or 2% of a polyphenolic extract (PE) derived from VFP (Vachellia farnesiana pods) for 14 weeks. In the intervention study, two groups of mice were fed for 14 weeks with a high-fat diet and then one switched to a high-fat diet with 10% powdered VFP for ten additional weeks. In the in vitro studies, we evaluated the effect of a VFPE (Vachellia farnesiana polyphenolic extract) on glucose-stimulated insulin secretion in INS-1E cells or of naringenin or methyl gallate on mitochondrial activity in primary hepatocytes and C2C12 myotubes. VFP or a VFPE increased whole-body energy expenditure and mitochondrial activity in skeletal muscle; prevented insulin resistance, hepatic steatosis, and kidney damage; exerted immunomodulatory effects; and reshaped fecal gut microbiota composition in mice fed a high-fat diet. VFPE decreased insulin secretion in INS-1E cells, and its isolated compounds naringenin and methyl gallate increased mitochondrial activity in primary hepatocytes and C2C12 myotubes. In conclusion VFP or a VFPE prevented systemic inflammation, insulin resistance, and hepatic and renal damage in mice fed a high-fat diet associated with increased energy expenditure, improved mitochondrial function, and reduction in insulin secretion.

Peripheral Immunophenotype in IgG4-Related Disease and Its Association with Clinical Phenotypes and Disease Activity.

Diverse immune cell subsets have been described in IgG4-related disease (IgG4-RD). If there is a different immunophenotype according to clinical phenotype and activity status is not known. Levels of IL-4-, IL-13-, IL-5-, and IL-21-producing CD4+ T cells (Th2 subsets), CD4+ cytotoxic T lymphocytes (CD4+CTLs), T helper 9 cells, T follicular helper cells (Tfh; Tfh1/Tfh2/Tfh17/Tf regulatory [Tfr]), Foxp3+ regulatory T cells, Type 1 regulatory T cells (Tr1), T helper 3 regulatory cells (Th3), IL-10-producing regulatory B cells (Bregs), IL-10-expressing regulatory plasmacytoid dendritic (pDC IL-10+) cells, and M1 and M2 monocytes were determined by flow cytometry in 43 IgG4-RD patients and 12 controls. All immune subsets were higher in patients vs. controls. CD4+/IL-4+, CD4+/IL-5+, CD4+CTLs, Tfh2, Tfh17, Tfr, and M1 monocyte cell number was different among IgG4-RD clinical phenotypes. The pancreato-hepato-biliary phenotype was characterized by a higher CD4+CTLs, Tfh17, Tfh2, and Tfr and lower M1 cell number. An increased CD4+CTLs and Th3 cell number distinguished the head and neck-limited phenotype, while the retroperitoneal/aortic and Mikulicz/systemic phenotypes were characterized by increased Th2 subsets. Tfh17, Tr1, Th3, pDC, M1, and M2 monocytes were augmented in active patients. In summary, the clinical heterogeneity of IgG4-RD might be driven by the participation of different immunophenotypes and, consequently, by a different fibroinflammatory process.

Is the Sars-CoV-2 virus a possible trigger agent for the development of achalasia?

BACKGROUND: Achalasia is an autoimmune disease whose probable causal agent is a neurotropic virus that chronically infects the myenteric plexus of the esophagus and induces the disease in a genetically susceptible host. The association between achalasia and coronaviruses has not been reported. AIMS: To evaluate the presence of the SARS-CoV-2 virus, the ACE2 expression, the tissue architecture, and immune response in the lower esophageal sphincter muscle (LESm) of achalasia patients who posteriorly had SARS-CoV-2 (achalasia-COVID-19) infection before laparoscopic Heller myotomy (LHM) and compare the findings with type II achalasia patients and transplant donors (controls) without COVID-19. METHODS: The LESm of 7 achalasia-COVID-19 patients (diagnosed by PCR), ten achalasia patients, and ten controls without COVID-19 were included. The presence of the virus was evaluated by in situ PCR and immunohistochemistry. ACE2 receptor expression and effector CD4 T cell and regulatory subsets were determined by immunohistochemistry. KEY RESULTS: Coronavirus was detected in 6/7 patients-COVID-19. The SARS-CoV-2 was undetectable in the LESm of the achalasia patients and controls. ACE2 receptor was expressed in all the patients and controls. One patient developed achalasia type II post-COVID-19. The percentage of Th22/Th17/Th1/pDCreg was higher in achalasia and achalasia-COVID-19 pre-HLM vs. controls. The Th2/Treg/Breg cell percentages were higher only in achalasia vs. controls. CONCLUSION & INFERENCES: SARS-CoV2 and its receptor expression in the LESm of achalasia patients who posteriorly had COVID-19 but not in the controls suggests that it could affect the myenteric plexus. Unlike achalasia, patients-COVID-19 have an imbalance between effector CD4 T cells and the regulatory mechanisms.

Down-Regulation-Resistant STAT4 Risk Haplotype Contributes to Lupus Nephritis Through CD4+ T Cell Interferon-γ Production.

OBJECTIVE: Variants in STAT4 are associated with systemic lupus erythematosus (SLE) and other autoimmune diseases. We undertook this study to investigate how disease-associated variants affect STAT4 expression, in particular in CD4+ T cells where STAT4 plays an essential role. METHODS: We compared Th1 differentiation between naive CD4+ T cells from healthy donors homozygous for the risk (R/R) or nonrisk (NR/NR) alleles. We analyzed epigenetic marks in STAT4 and evaluated the relevance of its third intron, assessed the consequences of Stat4 overexpression in vivo in mice, and analyzed the effects of the STAT4 genotype in patients with lupus nephritis. RESULTS: Naive CD4+ T cells from NR/NR healthy donors down-regulated STAT4 in response to interleukin-12 (IL-12). In contrast, cells from R/R healthy donors maintained high levels. R/R cells exhibited a higher abundance of transcriptionally active STAT4 and increased interferon-γ production. Accordingly, R/R healthy donors exhibited a stronger induction of local active enhancer marks. Genetic editing confirmed the presence of a negative regulatory region in the STAT4 third intron, where most of the SLE-associated STAT4 single-nucleotide polymorphisms (SNPs) are located. In vivo forced expression demonstrated that increases in Stat4 levels in T cells enhanced glomerulonephritis in mice. Accordingly, the R/R genotype was associated with suboptimal response to treatment and with worse clinical outcomes in patients with proliferative lupus nephritis. CONCLUSION: The SLE-associated STAT4 haplotype correlates with an abnormal IL-12-mediated STAT4 transcriptional regulation. Carriers of the risk variant exhibit exaggerated CD4+ proinflammatory capacities that, in the context of SLE, contribute to more severe disease. R/R patients may benefit from blockade of the IL-12/STAT4 pathway.

Dendritic cells drive profibrotic inflammation and aberrant T cell polarization in systemic sclerosis.

OBJECTIVES: SSc is a devastating autoimmune disease characterized by fibrosis and obliterative vasculopathy affecting the skin and visceral organs. While the processes mediating excessive extracellular matrix deposition and fibroblast proliferation are clear, the exact link between autoimmunity and fibrosis remains elusive. Th17 cells have been proposed as critical drivers of profibrotic inflammation during SSc, but little is known about the immune components supporting their pathogenic role. Our aim was to determine cytokine responses of stimulated monocyte-derived dendritic cells (Mo-DCs) and to determine how they influence T-cell cytokine production in SSc. MATERIAL AND METHODS: Dendritic cells (DCs) activate and shape T cell differentiation by producing polarizing cytokines. Hence, we investigated the cytokine responses of monocyte-derived DCs (Mo-DCs) from patients with limited cutaneous SSc (lcSSc), diffuse cutaneous SSc (dcSSc) and healthy controls (HCs) after stimulation with toll-like receptor (TLR) agonists. Also, using co-culture assays, we analysed T cell subpopulations after contact with autologous TLR-activated Mo-DCs. RESULTS: In general, we observed an increased production of Th17-related cytokines like IL-1ß, IL-17F, IL-21 and IL-22 by SSc compared with HC Mo-DCs, with variations between lcSSc vs dcSSc and early- vs late-stage subgroups. Noticeably, we found a significant increment in IL-33 production by Mo-DCs in all SSc cases regardless of their clinical phenotype. Strikingly, T cells displayed Th2, Th17 and dual Th2-Th17 phenotypes after exposure to autologous TLR-stimulated Mo-DCs from SSc patients but not HCs. These changes were pronounced in individuals with early-stage dcSSc and less significant in the late-stage lcSSc subgroup. CONCLUSIONS: Our findings suggest that functional alterations of DCs promote immune mechanisms favouring the aberrant T cell polarization and profibrotic inflammation behind clinical SSc heterogeneity.

Predictive factors associated with the persistence of chest pain in post-laparoscopic myotomy and fundoplication in patients with achalasia.

Background: Episodic angina-like retrosternal pain is a prevalent symptom for achalasia patients pre- and post-treatment. The cause of postoperative chest pain remains poorly understood. Moreover, there are no reports on their predictive value for chest pain in the long-term post-treatment. The effect of laparoscopic Heller myotomy (LHM) and fundoplication techniques (Dor vs. Toupet) is unclear. Methods: We analyzed a cohort of 129 achalasia cases treated with LHM and randomly assigned fundoplication technique. All the patients were diagnosed with achalasia by high-resolution manometry (HRM). Patients were followed up at 1-, 6-, 12-, and 24-month post-treatment. We implemented unadjusted and adjusted logistic regression analyses to evaluate the predictive significance of pre- and post-operative clinical factors. Results: Preoperative chest pain with every meal was associated with an increased risk of occasional postoperative chest pain [unadjusted model: odds ratio (OR) = 12, 95% CI: 2.2-63.9, P = 0.006; adjusted model: OR = 26, 95% CI: 2.6-259.1, P = 0.005]. In type II achalasia, hypercontraction was also associated with an increased risk of chest pain (unadjusted model: OR = 2.6 e9 in all the patients). No significant differences were associated with age, type of achalasia, dysphagia, esophageal shape, and integrated relaxation pressure (IRP) with an increased risk of occasional postoperative chest pain. Also, there was no significant difference between fundoplication techniques or surgical approaches (e.g., length of myotomy). Conclusion: Preoperative chest pain with every meal was associated with a higher risk of occasionally postoperative chest pain.

Autoantigen characterization in the lower esophageal sphincter muscle of patients with achalasia.

BACKGROUND: Serum anti-myenteric autoantibodies define autoimmune achalasia and tissue MMP-9 activity may locally process autoantigenic proteins in the muscle of the lower esophageal sphincter (LES) of achalasia patients. METHODS: Biopsies of the LES muscle from 36 achalasia patients, 6 esophagogastric junction outflow obstruction (EGJOO) patients, and 16 transplant donors (TD) were compared in a blind cross-sectional study. Histological characteristics such as inflammation, fibrosis, presence of ganglion cells, cells of Cajal, GAD65, PNMA2, S-100, P substance, and MMP-9 proteoforms in tissue were assessed by H&E and Picrosirius Red staining and immunohistochemistry analysis. Anti-neuronal antibodies, onconeural antigens, recoverin, SOX-1, titin, zic4, GAD65, and Tr were evaluated by immunoblot/line assay. KEY RESULTS: Tissue of achalasia patients had heterogeneous inflammatory infiltrates with fibrosis and contrasting higher levels of activated MMP-9, as compared with EGJOO and TD. Moreover, lower ganglion cell percentages and cell of Cajal percentages were determined in esophageal tissues of achalasia patients versus TD. The tissues of achalasia versus EGJOO patients had higher GAD65 and PNMA2 protein expression. Unexpectedly, these proteins were absent in TD tissue. S-100 and P substance had similar expression levels in tissues of achalasia patients versus TD and EGJOO. Most of the achalasia sera had anti-GAD65 (83%) and anti-PNMA2 (90%) autoantibodies versus EGJOO (17% and 33%, respectively) and healthy volunteers (10% and 0%, respectively). CONCLUSIONS AND INFERENCES: Tissue-specific ectopic expression of GAD65 and PNMA/Ta2 and active MMP-9, associated with the presence of specific autoantibodies directed against these proteins, might participate in the pathophysiology of achalasia triggering and/or perpetuating autoimmune disease.

Differential Th follicular cell subsets in minor salivary glands of patients with primary Sjögren's syndrome and systemic lupus erythematosus associated with Sjögren's syndrome.

OBJECTIVES: T follicular helper cells (Tfh) have been recognised in minor salivary glands (MSG) of patients with primary Sjögren's syndrome (pSS). Nevertheless, if the Tfh1, Tfh2, Tfh17, Tfr phenotype is different when comparing pSS and associated SS in systemic lupus erythematosus (SLE) is unknown. METHODS: We included MSG biopsies from 8 pSS, 8 SLE/SS patients, 7 SLE patients, and 2 non-SS sicca patients. To determine the subpopulation of Tfh, a double-staining procedure for transcription factor B cell lymphoma 6 (Bcl-6)+/IL-17A+, Bcl6+/IL-4+, Bcl6+/IFN-γ+, and Bcl6+/Foxp3+ cells was performed. We estimated the mean percentage of positively staining cells in four fields per sample. RESULTS: Tfh1, Tfh2, and Tfh17 cells were highly expressed in pSS compared with the rest of the groups; conversely, in patients with SLE/SS predominated, the Tfh17 and in SLE patients the Tfh1 cells. Regulatory Tfh cells (Tfr) were similar in pSS and the rest of the patients. However, the lowest frequency was found in the SLE group. A positive correlation was observed between anti-Ro/SSA autoantibody and Tfh17 subset (r=0.726, p=0.0001); and with the (Tfh2+Tfh17)/Tfh1 ratio (r=0.844, p<0.0001) in the MSG of patients with pSS. CONCLUSIONS: We showed a differential Tfh profile in primary SS and SLE with associated SS. Whether this Tfh differential profile participates in the increased risk of lymphoproliferative disease in pSS compared with associated SS, or other outcomes, is yet to be determined in future studies.

AKAP12/Gravin is over-expressed in patients with ulcerative colitis.

The gene of A-kinase anchor protein 12 (AKAP12) regulates cell cycle progression, cell motility, and morphology through its multiple scaffolding domains. However, the role of AKAP12 expression in ulcerative colitis (UC) patients has not been yet described. The aim of the study was to describe the gene and protein of AKAP12 expression in patients with UC and its association regarding the disease severity. We included a total of 40 patients with confirmed diagnosis of UC and 25 controls without endoscopic evidence of colitis or neoplasia. The relative quantification of the gene expression was performed by real-time PCR for AKAP12. Kruskal-Wallis was used to test differences among groups, and Spearman correlation to assess the relationship between AKAP12 gene and clinical outcomes. The extent of disease was evaluated using total colonoscopy, and biopsies were taken from rectum segments. The AKAP12 gene expression was increased in colonic mucosa from patients with active UC when compared with UC remission and control group. The overexpression of AKAP12 in patients with UC was associated with the presence of extensive colitis (p = 0.04, RM = 12, IC = 1.29-186.37). AKAP12/CD16 double positive cells were higher in submucosa (p = 0.04), muscular (p < 0.001), and cells from serosa (p < 0.001) in patients affected by UC in comparison to controls. The overexpression of AKAP12 was associated with the extent of disease. This is the first report about the role of AKAP12 in patients with UC suggesting that this gene and its protein could be involved in the modulation of the disease.

Intestinal production of secreted protein acidic and rich in cysteine (SPARC) in patients with ulcerative colitis.

BACKGROUND: Ulcerative colitis (UC) is an inflammatory disease of the intestine. The genetics factors play an important role in the pathogenesis of UC. SPARC exacerbates colonic inflammatory symptoms in dextran sodium sulphate-induced murine colitis. The aim of the study was to measure the gene expression and intestinal production of SPARC in patients with UC and controls as well as, to determine its correlation with histological activity. METHODS: We included 40 patients with confirmed diagnosis of UC, and 20 controls without endoscopic evidence of any type of colitis or neoplasia. The relative quantification of the gene expression was performed by real time PCR. GAPDH was used as housekeeping gene for normalization purposes and quality controls. Protein expression was determined by immunohistochemistry. RESULTS: The gene expression of SPARC was increased in patients with active UC vs in remission UC and vs. controls (P = 0.005). There was no significant difference between patients with remission UC and controls. The overexpression of SPARC in patients with active UC correlated significantly with mild histological activity (P = 0.06, OR = 7.77, IC = 0.77-77.9) moderate (P = 0.06, OR = 8.1, IC 95%=0.79-82.73), and severe (P = 0.03, OR = 6.5, IC 95%=1.09-38.6). Double positive SPARC+/CD16+ cells were localized mainly in submucosa, muscular layer, and adventitia, and in perivascular inflammatory infiltrates in patients with active UC. CONCLUSION: The gene and protein expression of SPARC is increased in active UC. SPARC could be a marker of intestinal inflammation and its expression correlates with histological activity.

Does laparoscopic reoperation yield symptomatic improvements similar to those of primary laparoscopic Heller myotomy in achalasia patients?

BACKGROUND: Laparoscopic Heller myotomy fails in approximately 3.5% to 15% of patients. Evidence of successful laparoscopic reoperation is limited to a few studies. METHODS: This case-control study was conducted in patients who underwent laparoscopic Heller myotomy reoperation (LHM-R) from 2008 to 2016. The operative outcomes, preoperative and last follow-up manometric parameters, and symptom questionnaire results, including the Eckardt, Gastroesophageal Reflux Disease-Health Related Quality of Life (GERD-HRQL) and eating assessment tool (EAT-10) scores, were obtained. The data were compared with those of patients who underwent primary laparoscopic Heller myotomy (LHM-1). RESULTS: Thirty-five patients who underwent LHM-R and 35 patients who underwent LHM-1 were included. The reasons for failure in the LHM-R patient group included incomplete myotomy (71.4%), myotomy fibrosis (25.7%) and structural alterations in fundoplication (2.9%). The follow-up duration was 34 months for the LHM-R group and 24 months for the LHM-1 group (p = 0.557). The procedure was performed by laparoscopy in 100% of the patients in the two groups. No differences were found regarding surgical morbidity (11.4% LHM-R vs. 2.9% LHM-1, p = 0.164). The symptomatic outcomes were equivalent between groups (Eckardt p = 0.063, EAT-10 p = 0.166, GERD-HRQL p = 0.075). An IRP < 15 mmHg was achieved in 100% of the LHM-R and LHM-1 patients. At the last follow-up, 82.1% of the LHM-R patients and 91.4% of the LHM-1 patients were in symptomatic remission (p = 0.271). CONCLUSION: The results achieved with LHM-R are similar to those achieved with LHM-1. Laparoscopic reoperation should be considered an effective and safe treatment after a failed Heller myotomy.