An Mtb-Human Protein-Protein Interaction Map Identifies a Switch between Host Antiviral and Antibacterial Responses.

Although macrophages are armed with potent antibacterial functions, Mycobacterium tuberculosis (Mtb) replicates inside these innate immune cells. Determinants of macrophage intrinsic bacterial control, and the Mtb strategies to overcome them, are poorly understood. To further study these processes, we used an affinity tag purification mass spectrometry (AP-MS) approach to identify 187 Mtb-human protein-protein interactions (PPIs) involving 34 secreted Mtb proteins. This interaction map revealed two factors involved in Mtb pathogenesis-the secreted Mtb protein, LpqN, and its binding partner, the human ubiquitin ligase CBL. We discovered that an lpqN Mtb mutant is attenuated in macrophages, but growth is restored when CBL is removed. Conversely, Cbl-/- macrophages are resistant to viral infection, indicating that CBL regulates cell-intrinsic polarization between antibacterial and antiviral immunity. Collectively, these findings illustrate the utility of this Mtb-human PPI map for developing a deeper understanding of the intricate interactions between Mtb and its host.

Mucosal Single-Cell Profiling of Crohn's-Like Disease of the Pouch Reveals Unique Pathogenesis and Therapeutic Targets.

BACKGROUND & AIMS: The pathophysiology of Crohn's-like disease of the pouch (CDP) in patients with a history of ulcerative colitis (UC) is unknown. We examined mucosal cells from patients with and without CDP using single-cell analyses. METHODS: Endoscopic samples were collected from pouch body and prepouch ileum (pouch/ileum) of 50 patients with an ileal pouch-anal anastomosis. Single-cell RNA sequencing was performed on pouch/ileal tissues of patients with normal pouch/ileum and CDP. Mass cytometry was performed on mucosal immune cells from patients with UC with normal pouch/ileum, CDP, pouchitis, and those with familial adenomatous polyposis after pouch formation. Findings were independently validated using immunohistochemistry. RESULTS: The cell populations/states in the pouch body differed from those in the prepouch ileum, likely secondary to increased microbial burden. Compared with the familial adenomatous polyposis pouch, the UC pouch was enriched in colitogenic immune cells even without inflammation. CDP was characterized by increases in T helper 17 cells, inflammatory fibroblasts, inflammatory monocytes, TREM1+ monocytes, clonal expansion of effector T cells, and overexpression of T helper 17 cells-inducing cytokine genes such as IL23, IL1B, and IL6 by mononuclear phagocytes. Ligand-receptor analysis further revealed a stromal-mononuclear phagocytes-lymphocyte circuit in CDP. Integrated analysis showed that up-regulated immune mediators in CDP were similar to those in CD and pouchitis, but not UC. Additionally, CDP pouch/ileum exhibited heightened endoplasmic reticulum stress across all major cell compartments. CONCLUSIONS: CDP likely represents a distinct entity of inflammatory bowel disease with heightened endoplasmic reticulum stress in both immune and nonimmune cells, which may become a novel diagnostic biomarker and therapeutic target for CDP.

Portosinusoidal Vascular Disorder: A Heretofore Unrecognized Manifestation of Sickle Cell Disease?

Portosinusoidal vascular disorder (PSVD) is a recently proposed histopathologic entity that encompasses a spectrum of often subtle hepatic microvascular lesions and related microarchitectural abnormalities. Clinical manifestations may arise years after histologic diagnosis and include extrahepatic portal vein thrombosis and portal hypertension. While the histopathologic features of PSVD have been associated with numerous clinical conditions, most notably prothrombotic/vasculopathic disorders, PSVD has not yet been described in sickle cell disease. This gap is striking given the central role of microvascular dysfunction in sickle cell disease and well-described patterns of hepatic injury and dysfunction in this population. This case series is the first to explore the prevalence and pathogenesis of PSVD in sickle cell disease. Forty-one diagnostically adequate liver biopsies from patients with sickle cell disease were identified across the archives of 5 tertiary medical centers. All biopsies exhibited at least 1 histopathologic feature associated with PSVD (mean 3.8 features/case). Overall, 90.2% of patients met the criteria for a diagnosis of PSVD based on the presence of specific histopathologic and/or clinical findings. Immunohistochemical stains for von Willebrand factor, CD34, and glutamine synthetase were performed on 36 cases (87.8%). Aberrant (centrilobular sinusoidal) CD34 and von Willebrand factor staining was present in 97.2% and 86.1% of cases, respectively. Glutamine synthetase reactivity was at least mildly decreased in zone 3 hepatocytes in 52.8% of cases. We posit that chronic erythrocyte sickling results in dysfunction and remodeling of the portal microvasculature, culminating in regression of zone 3 hepatocytes. The presence of PSVD may explain, at least in part, the hepatic dysfunction observed in this patient population. These patients may also benefit from extended clinical surveillance for portal hypertension and other complications. While subtle and prone to overdiagnosis, the features of PSVD should be carefully considered when interpreting liver biopsies from patients with sickle cell disease.

Clinicopathologic Features of Gastrointestinal Tract Langerhans Cell Histiocytosis.

Gastrointestinal (GI) tract involvement by Langerhans cell histiocytosis (LCH) is rare and its clinicopathologic characteristics have only been described in case reports and small series. We reviewed hematoxylin and eosin and CD1a, S100, and Langerin immunohistochemical-stained slides from 47 patients with well-documented demographic and clinical findings. Our cases included 8 children and 39 adults, with a mean follow-up of 63 months. All pediatric patients had concurrent multisystem LCH, presented with GI symptoms, and showed nonpolypoid lesions. Seven (88%) showed multifocal GI disease, including 5 with multiple GI organ involvement. All sampled lesions from children exhibited infiltrative growth. More than half had died of the disease or manifested persistent LCH at last follow-up. Twenty-five of 39 (64%) adults had LCH involving only the GI tract (single system), with the remaining 14 (36%) exhibiting multisystem disease. Adult single-system GI LCH was typically encountered incidentally on screening/surveillance endoscopy (72%). Most exhibited isolated colorectal involvement (88%) as a solitary polyp (92%), with a well-demarcated/noninfiltrative growth pattern (70%), and excellent prognosis (100%). In comparison, adult patients with multisystem LCH more frequently presented with GI symptoms (92%, P < .001), noncolorectal GI site involvement (50%, P = .02), multifocal GI lesions (43%, P = .005), nonpolypoid lesions (71%, P < .001), infiltrative histologic growth pattern (78%, P = .04), and persistent disease (57%, P < .001). Adult patients with multisystem LCH appear to exhibit similar clinicopathologic features to those of pediatric patients. These results demonstrated that adults with single-system LCH involving the GI tract have an excellent prognosis, whereas multisystem LCH occurring at any age carries an unfavorable prognosis. High-risk features of GI LCH include pediatric age, GI symptomatology, noncolorectal GI involvement, multifocal GI disease, nonpolypoid lesions, and infiltrative growth pattern.

Antibody-Mediated Rejection in Liver Transplantation: Immuno-Pathological Characteristics and Long-Term Follow-Up.

The diagnosis of liver antibody-mediated rejection (AMR) is challenging and likely under-recognized. The association of AMR with donor-specific antibodies (DSA), and its clinical course in relation to pathologic findings and treatment are ill defined. We identified cases of liver AMR by following the criteria outlined by the 2016 Banff Working Group. Patient demographics, native liver disease, histopathologic findings, treatment type, clinical outcome, and transaminase levels during AMR diagnosis, treatment, and resolution were determined. Patients (n = 8) with AMR average age was 55.2 years (range: 19-68). Seven of eight cases met the Banff criteria for AMR. Personalized treatment regimens consisted of optimization of immunosuppression, intravenous pulse steroids, plasmapheresis, IVIG, rituximab, and bortezomib. Five patients experienced complete resolution of AMR, return of transaminases to baseline, and decreased DSA at long-term follow-up. One patient developed chronic AMR and two patients required re-transplantation. Follow-up after AMR diagnosis ranged from one to 11 years. Because AMR can present at any time, crossmatch, early biopsy, and routine monitoring of DSA levels should be implemented following transaminase elevation to recognize AMR. Furthermore, treatment should be immediately implemented to reverse AMR and prevent graft failure, chronic damage, re-transplantation, and possibly mortality.

Morphologic and immunophenotypic evaluation of liver allograft biopsies with contemporaneous serum DSA measurements.

BACKGROUND: Acute antibody mediated rejection is increasingly identified in liver allografts as a unique form of alloimmune injury associated with donor specific antibodies (DSA). This manifests pathologically as microvascular injury and C4d uptake. Despite the liver allograft's relative resistance to alloimmune injury, liver allografts are not impervious to cellular and antibody-mediated rejection. METHODS: In this blinded control study, we evaluated CD163 immunohistochemistry and applied the Banff 2016 criteria for diagnosis of acute AMR on a group of indication allograft liver biopsies from DSA positive patients and compared them to indication biopsies from DSA negative controls. RESULTS: Most DSA positive patients were females (75%, p = .027), and underwent transplantation for HCV infection. Significant histopathological predictors of serum DSA positivity were Banff H-score (p = .01), moderate to severe cholestasis (p = .03), and CD163 score > 2 (p = .029). Other morphologic features that showed a trend with DSA positivity include Banff portal C4d-score (p = .06), bile ductular reaction (p = .07), and central perivenulitis (p = .07). The odds of DSA sMFI ≥5000 was 12.5 times higher in those with a C4d score >1 than those with a C4d score ≤ 1 (p = .04). Incidence of definite for aAMR in the DSA positive cohort was 25% (n = 5), and 0% in the DSA negative cohort. A group of 5 DSA positive cases were not classifiable by the current scheme. CONCLUSION: Sinusoidal CD163, Banff H-score, and diffuse C4d are predictors of serum DSA, and facilitate recognition of histopathological features associated with serum DSA and tissue-antibody interaction.

Multicenter Evaluation of the Cepheid Xpert GBS LB XC Test.

Early-onset neonatal sepsis due to Streptococcus agalactiae (group B Streptococcus [GBS]) infection is one of the leading causes of newborn mortality and morbidity. The latest guidelines published in 2019 recommended universal screening of GBS colonization among all pregnant women and intrapartum antibiotic prophylaxis for positive GBS. The updated procedures allow rapid molecular-based GBS screening using nutrient broth-enriched rectovaginal samples. Commercially available molecular assays for GBS diagnosis target mainly the cfb gene, which encodes a hemolysin protein responsible for producing the Christie-Atkins-Munch-Petersen (CAMP) factor. cfb is considered a conserved gene in essentially all GBS isolates. However, false-negative GBS results on Cepheid Xpert GBS and GBS LB tests due to deletions in or near the region that encodes cfb were reported recently. Therefore, the new Xpert GBS LB XC test was developed. This study is a multicenter evaluation of the new test for GBS identification from nutrient broth-enriched rectal/vaginal samples from antepartum women. A total of 621 samples were prospectively enrolled. The samples were tested with the Xpert GBS LB XC test, the composite comparator method, which included the Hologic Panther Fusion GBS test combined with bacterial culture, followed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) identification, and bacterial culture alone, followed by MALDI-TOF MS identification. The respective sensitivity and specificity of the Xpert GBS LB XC test were 99.3% and 98.7% compared to the composite comparator method and 99.1% and 91.8% compared to bacterial culture alone with MALDI-TOF MS identification. Overall, the Xpert GBS LB XC test performed comparatively to the composite comparator method and is equivalent to traditional bacterial culture followed by MALDI-TOF MS.

Electric treatment for hydrophilic ink deinking.

Hydrophilic inks have been widely used due to higher printing speed, competitive cost and being healthy non-organic solvents. However, they cause problems in both product quality and process runnability due to their hydrophilic surface wettability, strong negative surface charge and sub-micron size. Electric treatment was shown to be able to increase the ink sizes from 60 nm to 700 nm through electrocoagulation and electrophoresis. In addition, electric treatment assisted flotation could reduce effective residual ink concentration (ERIC) by 90 ppm, compared with only 20 ppm by traditional flotation. Furthermore, the effect of electric treatment alone on ink separation was investigated by two anode materials, graphite and stainless steel. Both of them could remove hydrophilic inks with less than 1% yield loss via electroflotation and electrophoresis. But graphite is a better material as the anode because graphite reduced ERIC by an additional 100 ppm. The yield loss of flotation following electric treatment was also lower by 17% if graphite was the anode material. The difference between the two electrode materials resulted from electrocoagulation and ink redeposition during electric treatment. An electric pretreatment-flotation-hyperwashing process was conducted to understand the deinking performance in conditions similar to a paper mill, and the ERIC was reduced from 950 ppm to less than 400 ppm.

Microstickies agglomeration by electric field.

Microstickies deposits on both paper machine and paper products when it agglomerates under step change in ionic strength, pH, temperature and chemical additives. These stickies increase the down time of the paper mill and decrease the quality of paper. The key property of microstickies is its smaller size, which leads to low removal efficiency and difficulties in measurement. Thus the increase of microstickies size help improve both removal efficiency and reduce measurement difficulty. In this paper, a new agglomeration technology based on electric field was investigated. The electric treatment could also increase the size of stickies particles by around 100 times. The synergetic effect between electric field treatment and detacky chemicals/dispersants, including polyvinyl alcohol, poly(diallylmethylammonium chloride) and lignosulfonate, was also studied.

Medically Refractory Lymphocytic Colitis Successfully Treated With Upadacitinib.

Lymphocytic colitis is a microscopic colitis characterized endoscopically by nearly normal-appearing colonic mucosa and histology demonstrating intraepithelial lymphocytosis. Microscopic colitis that is refractory to conventional therapies, including budesonide, is rare but challenging and with scarce evidence. Upadacitinib is a novel Janus kinase 1 selective inhibitor approved by the US Food and Drug Administration for atopic dermatitis, rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, and moderately to severely active ulcerative colitis. We present the first case of lymphocytic colitis refractory to conventional and immunosuppressive therapies, which responded promptly to upadacitinib.

Upregulated expression of cytotoxicity-related genes in IFN-γ knockout mice with Schistosoma japonicum infection.

It is well accepted that IFN-γ is important to the development of acquired resistance against murine schistosomiasis. However, the in vivo role of this immunoregulatory cytokine in helminth infection needs to be further investigated. In this study, parasite burden and host immune response were observed in IFN-γ knockout mice (IFNg KO) infected with Schistosoma japonicum for 6 weeks. The results suggested that deficiency in IFN-γ led to decreased egg burden in mice, with low schistosome-specific IgG antibody response and enhanced activation of T cells during acute infection. Microarray and qRT-PCR data analyses showed significant upregulation of some cytotoxicity-related genes, including those from the granzyme family, tumor necrosis factor, Fas Ligand, and chemokines, in the spleen cells of IFNg KO mice. Furthermore, CD8+ cells instead of NK cells of IFNg KO mice exhibited increased transcription of cytotoxic genes compared with WT mice. Additionally, Schistosoma japonicum-specific egg antigen immunization also could activate CD8+ T cells to upregulate the expression of cytotoxic genes in IFNg KO mice. Our data suggest that IFN-γ is not always a positive regulator of immune responses. In certain situations, the disruption of IFN-γ signaling may up-regulate the cytotoxic T-cell-mediated immune responses to the parasite.

Aberrant Gut Microbiome Contributes to Intestinal Oxidative Stress, Barrier Dysfunction, Inflammation and Systemic Autoimmune Responses in MRL/lpr Mice.

Microbiome composition and function have been implicated as contributing factors in the pathogenesis of autoimmune diseases (ADs), including systemic lupus erythematosus (SLE), rheumatoid arthritis and autoimmune hepatitis (AIH). Furthermore, dysbiosis of gut microbiome is associated with impaired barrier function and mucosal immune dysregulation. However, mechanisms by which gut microbiome contributes to the ADs and whether antioxidant treatment can restore gut homeostasis and ameliorate the disease outcome are not known. This study was, therefore, focused on examining the involvement of gut microbiome and host responses in the pathogenesis of SLE using unique female mouse models (C57BL/6, MRL+/+ and MRL/lpr) of 6 and 18 weeks with varying degrees of disease progression. Fecal microbiome diversity and composition, gut oxidative stress (OS), barrier function and inflammation, as well as systemic autoimmunity were determined. Interestingly, each mouse strain had distinct bacterial community as revealed by ß-diversity. A lower Firmicutes/Bacteroidetes ratio in 6-week-old MRL/lpr mice was observed, evidenced by decrease in Peptostreptococcaceae under Firmicutes phylum along with enrichment of Rikenellaceae under Bacteroidetes phylum. Additionally, we observed increases in colonic OS [4-hydroxynonenal (HNE)-adducts and HNE-specific immune complexes], permeability changes (lower tight junction protein ZO-2; increased fecal albumin and IgA levels) and inflammatory responses (increased phos-NF-κB, IL-6 and IgG levels) in 18-week-old MRL/lpr mice. These changes were associated with markedly elevated AD markers (antinuclear and anti-smooth muscle antibodies) along with hepatic portal inflammation and severe glomerulonephritis. Notably, antioxidant N-acetylcysteine treatment influenced the microbial composition (decreased Rikenellaceae; increased Akkeransiaceae, Erysipelotrichaceae and Muribaculaceae) and attenuated the systemic autoimmunity in MRL/lpr mice. Our data thus show that gut microbiome dysbiosis is associated with increased colonic OS, barrier dysfunction, inflammatory responses and systemic autoimmunity markers. These findings apart from delineating a role for gut microbiome dysbiosis, also support the contribution of gut OS, permeability changes and inflammatory responses in the pathogenesis of ADs.

Iron Overload in an HFE Heterozygous Carrier: A Case Report and Literature Review.

Hereditary hemochromatosis (HH) is an autosomal recessive disorder of iron metabolism characterized by increased iron absorption and tissue deposition. Three loss-of-function mutations in the hemochromatosis gene (HFE), namely, C282Y (c.845G>A), H63D (c.187C>G), and S65C (c.193A>T), account for the vast majority of HH cases. These mutations cause alterations in HFE membrane expression, structure, and/or activity, leading to dysregulation of iron absorption. It is well established that the phenotypic expression of HFE mutations varies markedly. Herein, we describe a 64-year-old Caucasian woman with a reported history of hemochromatosis. The father of the patient had died of complications due to iron overload. Testing of HFE codon C282Y, H63D, and S65C mutations showed heterozygous C282Y. The patient had significantly elevated transferrin saturation (TS) and serum ferritin (SF) levels. Her liver function test results showed elevated alanine transaminase (ALT) and aspartate aminotransferase (AST) levels. The patient has been treated with regular phlebotomy to prevent the clinical manifestations of hemochromatosis.

Redox regulation of hepatic NLRP3 inflammasome activation and immune dysregulation in trichloroethene-mediated autoimmunity.

Trichloroethene (TCE) exposure is associated with the development of various autoimmune diseases (ADs), including autoimmune hepatitis (AIH) and systemic lupus erythematosus (SLE), potentially through the generation of excessive reactive oxygen and nitrogen species (RONS; oxidative stress). However, the mechanisms by which oxidative stress contributes to these TCE-mediated ADs are not fully understood, and are the focus of current investigation. Female MRL+/+ mice were treated with TCE along with or without antioxidant N-acetylcysteine (NAC) for 6 weeks (TCE, 10 mmol/kg, i. p., every 4th day; NAC, 250 mg/kg/day via drinking water). TCE-treated mice had elevated antinuclear antibodies (ANA) and 4-hydroxynonenal (HNE)-specific circulating immune complexes, suggesting the association of TCE-induced oxidative stress with autoimmune response. In addition, TCE exposure led to prominent lobular inflammation with sinusoid dilation, increased sinusoidal cellularity and increased staining for proliferating cell nuclear antigen (PCNA), confirming inflammatory and hepatocellular cell proliferation. Importantly, TCE exposure resulted in the activation of hepatic inflammasome (NLRP3 and caspase-1) and up-regulation of pro-inflammatory cytokine IL-1ß, and these changes were attenuated by NAC supplementation. TCE treatment also led to dysregulation of hepatic immune response as evident from markedly increased hepatic lymphocyte infiltration (especially B cells) and imbalance between Tregs (decreased) and Th17 cells (increased). Interestingly, TCE-mediated dysregulation of various hepatic and splenic immune cells was also effectively attenuated by NAC. Taken together, our findings provide evidence for TCE-mediated inflammasome activation, infiltration of various immune cells, and skewed balance of Treg and Th17 cells in the liver. The attenuation of TCE-mediated hepatic inflammasome activation and immune responses by NAC further supports a critical role of oxidative stress in TCE-mediated inflammation and autoimmunity. These novel findings could help in designing therapeutic strategies for such ADs.

Interaction of Peptide Aptamers with Prion Protein Central Domain Promotes α-Cleavage of PrPC.

Prion diseases are infectious and fatal neurodegenerative diseases affecting humans and animals. Transmission is possible within and between species with zoonotic potential. Currently, no prophylaxis or treatment exists. Prions are composed of the misfolded isoform PrPSc of the cellular prion protein PrPC. Expression of PrPC is a prerequisite for prion infection, and conformational conversion of PrPC is induced upon its direct interaction with PrPSc. Inhibition of this interaction can abrogate prion propagation, and we have previously established peptide aptamers (PAs) binding to PrPC as new anti-prion compounds. Here, we mapped the interaction site of PA8 in PrP and modeled the complex in silico to design targeted mutations in PA8 which presumably enhance binding properties. Using these PA8 variants, we could improve PA-mediated inhibition of PrPSc replication and de novo infection of neuronal cells. Furthermore, we demonstrate that binding of PA8 and its variants increases PrPC α-cleavage and interferes with its internalization. This gives rise to high levels of the membrane-anchored PrP-C1 fragment, a transdominant negative inhibitor of prion replication. PA8 and its variants interact with PrPC at its central and most highly conserved domain, a region which is crucial for prion conversion and facilitates toxic signaling of Aß oligomers characteristic for Alzheimer's disease. Our strategy allows for the first time to induce α-cleavage, which occurs within this central domain, independent of targeting the responsible protease. Therefore, interaction of PAs with PrPC and enhancement of α-cleavage represent mechanisms that can be beneficial for the treatment of prion and other neurodegenerative diseases.

Noble metal catalyzed aqueous phase hydrogenation and hydrodeoxygenation of lignin-derived pyrolysis oil and related model compounds.

Aqueous phase hydrodeoxygenation of lignin pyrolysis oil and related model compounds were investigated using four noble metals supported on activated carbon. The hydrodeoxygenation of guaiacol has three major reaction pathways and the demethylation reaction, mainly catalyzed by Pd, Pt and Rh, produces catechol as the products. The presence of catechol and guaiacol in the reaction is responsible for the coke formation and the catalysts deactivation. As expected, there was a significant decrease in the specific surface area of Pd, Pt and Rh catalysts during the catalytic reaction because of the coke deposition. In contrast, no catechol was produced from guaiacol when Ru was used so a completely hydrogenation was accomplished. The lignin pyrolysis oil upgrading with Pt and Ru catalysts further validated the reaction mechanism deduced from model compounds. Fully hydrogenated bio-oil was produced with Ru catalyst.

IFN-inducible p47 GTPases display differential responses to Schistosoma japonicum acute infection.

Interferon gamma induced GTPase (IGTP) (also named Irgm3) and interferon gamma inducible protein 47 (IRG-47) (also named Irgd) are interferon (IFN)-inducible p47 GTPases that have been shown to regulate host resistance to intracellular pathogens. Little knowledge has been known about the role of p47 GTPases in host responses against extracellular pathogens. To investigate possible roles of IGTP and IRG-47 in the course of Schistosoma japonicum infection, IGTP and IRG-47 knockout and wild-type (WT) mice were challenged with cercariae of S. japonicum, and host responses were analyzed. At the acute stage of S. japonicum infection, mice that lacked IGTP displayed similar parasite burden and pathological damage to WT mice. Importantly, S. japonicum-infected IRG-47-deficient mice, in contrast to IGTP-deficient mice and WT mice, showed significantly reduced worms and lower egg-burden, but intense granulomatous reaction evoked by schistosome eggs in peripheral parts of liver lobes. In addition, upregulation of inflammation-related gene expression was observed in the spleen of IRG-47-deficient mice using oligonucleotide microarrays, in which multiple pathways of cytokine-cytokine receptor interaction, T-cell receptor signaling, complement, coagulation cascades and cell adhesion molecules were highlighted. Taken together, these data suggest that IGTP and IRG-47 might have distinct features that were differentially required for resistance to S. japonicum.