Localization of copper-zinc superoxide dismutase in the endocrine pancreas

Immunoelectron microscopy and cellular fractionation on sucrose density gradients have been used to examine the intracellular distribution of copper-zinc superoxide dismutase (SOD) in the canine and rat endocrine pancreas. Using rabbit anti-canine copper-zinc SOD as the primary antiserum, immunostaining in canine beta and non-beta islet cells was significantly greater than that in serial sections of the same islet incubated with preimmune serum from the same rabbit. Within the cells, immunostaining was associated with spherical and crystalloid granules as well as with the cytoplasm. Radioimmune assays of cellular fractions or rat islet-cell preparations showed that the granule-rich fraction containing large amounts of insulin was also rich in SOD. This interesting observation of association of SOD with hormone-containing granules suggests that SOD may play a role in protecting these oxidation-sensitive proteins.     

Fetal cerebrohepatorenal (Zellweger) syndrome: dysmorphic, radiologic, biochemical, and pathologic findings in four affected fetuses

Four fetuses with positive family histories for cerebrohepatorenal (Zellweger) syndrome (CHRS) underwent diagnostic amniocentesis or chorionic villus biopsy. Cultured amniocytes or fibroblasts from all of the fetuses displayed abnormal fatty acid ratios, and the parents elected therapeutic abortions. Dysmorphic features in one fetus consisted of micrognathia, proximal implantation of toes, and bilateral talipes equinovarus. Radiologic examination of the fetus confirmed the dysmorphic features and revealed foci of mineralization in the patellae. Biochemical analysis of three of the fetuses demonstrated markedly increased levels of very-long-chain fatty acids, both saturated and monounsaturated, in liver, kidney, adrenal, and brain. Pathologic findings consisted of premature mineralization of patellae; renal cystic tubular dilations; striated cells in adrenal fetal zone and testicular interstitium; dysplastic alterations of inferior olivary nuclei, dentate nuclei, and cerebral cortex; equivocal increases in portal fibrous tissue; and abnormal cytosomes in fetal zone adrenocortical cells, testicular and renal interstitial cells, and brain macrophages. Iron deposition, probably physiologic, was observed only in liver tissue. Distributions of immunoreactive catalase were identical in the fetuses with CHRS and age-matched control subjects. These findings document the accuracy of the prenatal diagnostic test and provide insights into the morphogenesis and pathogenesis of CHRS.     

Application of passive hemagglutination for evaluation of antisperm antibodies and a modified coombs'' test for detecting male autoimmunity to sperm antigens

A rapid, specific, and sensitive assay using passive hemagglutination and Coombs' tests has been established for detecting male autoimmunity and female isoimmunity to sperm antigens. Antisperm antibody titers evaluated by this method were similar in 156 normal females, 400 pregnant females, 25 gonadal dysgenetics, 43 users of Oracon (an oral contraceptive), and 25 normal males. In contrast, in the evaluation of 50 couples with unexplained infertility, 16 males were found to have significantly elevated Coombs' autoantibody titers (primarily IgG) and correspondingly higher antisperm antibody titers in their serum and seminal plasma; another 28 had low autoantibody titers and low serum and seminal plasma antibody titers; and 6 had intermediate titers. The wives of 63% of the males with high autoantibody titers were found to be ‘isoimmune’, as compared with 7% of those with low titers. The isoimmune females had significantly higher antisperm antibody titers than did those with normal titers. In general, comparable titer values were obtained whether sperm extract or seminal plasma was used as the erythrocyte coat for passive hemagglutination. The titers were consistently higher in the seminal plasma than in the serum of males. Very little crossreactivity was apparent between anti-Candida and antisperm antibodies, and antibodies to blood group antigens did not appear to influence the results. When the results obtained by passive hemagglutination were compared with those obtained by immunofluorescence, Franklin-Dukes, and hanging drop interface methods, only those of the passive hemagglutination and Coombs' test correlated well with the results of postcoital tests and seminal analysis.     

Autoantibodies and immunoglobulin allotypes in healthy North American Blacks of different age groups

A population of 291 healthy North American Black subjects of different ages was studied for immunoglobulin (Ig) allotypes and the prevalence of autoantibodies, to determine possible associations between Ig allotypes and age, autoantibodies and age, and Ig allotypes and autoantibodies. Indirect immunofluorescence was used to detect anti-gastric parietal cell, anti-smooth muscle, anti-thyroid microsomal, anti-nuclear, and anti-mitochondrial antibodies. The sera were typed for the Ig allotypes Gm(1, 2, 3, 5, 6, 13, 14, 17, and 21) and Km(1) with a hemagglutination-inhibition assay. A significant association between advanced age and an increased prevalence of anti-nuclear antibodies was observed in females. There was no significant association between Ig allotypes and the autoantibodies tested. The results suggest that Ig allotypes are not involved in the development of autoantibodies in healthy Blacks.     

Bacteroides ovatus Promotes IL-22 Production and Reduces Trinitrobenzene Sulfonic Acid–Driven Colonic Inflammation

The intestinal microbiota influences the development and function of the mucosal immune system. However, the exact mechanisms by which commensal microbes modulate immunity is not clear. We previously demonstrated that commensal Bacteroides ovatus ATCC 8384 reduces mucosal inflammation. Herein, we aimed to identify immunomodulatory pathways employed by B. ovatus. In germ-free mice, mono-association with B. ovatus shifted the CD11b⁺/CD11c⁺ and CD103⁺/CD11c⁺ dendritic cell populations. Because indole compounds are known to modulate dendritic cells, B. ovatus cell-free supernatant was screened for tryptophan metabolites by liquid chromatography–tandem mass spectrometry and larger quantities of indole-3-acetic acid were detected. Analysis of cecal and fecal samples from germ-free and B. ovatus mono-associated mice confirmed that B. ovatus could elevate indole-3-acetic acid concentrations in vivo. Indole metabolites have previously been shown to stimulate immune cells to secrete the reparative cytokine IL-22. Addition of B. ovatus cell-free supernatant to immature bone marrow–derived dendritic cells stimulated IL-22 secretion. The ability of IL-22 to drive repair in the intestinal epithelium was confirmed using a physiologically relevant human intestinal enteroid model. Finally, B. ovatus shifted the immune cell populations in trinitrobenzene sulfonic acid–treated mice and up-regulated colonic IL-22 expression, effects that correlated with decreased inflammation. Our data suggest that B. ovatus–produced indole-3-acetic acid promotes IL-22 production by immune cells, yielding beneficial effects on colitis.

Crohn disease and ulcerative colitis are chronic, relapsing and remitting inflammatory bowel diseases (IBDs) that impact over three million Americans.¹ Current treatments include aminosalicylates, systemic steroids, immunomodulators such as 6-mercaptopurine and methotrexate, and biologic medications such as anti–tumor necrosis factor agents. These medications can cause significant adverse effects, including bone marrow suppression and malignancy,² highlighting a critical need for novel and safe adjuvant treatment strategies for IBD.The complex yet undefined etiology of IBD involves an intricate interplay of genetic predisposition, environmental factors, the host immune system, and the intestinal microbiota. Decreased fecal and mucosal Bacteroides³ are strongly associated with the development of IBD.⁴ Bacteroides species comprise nearly 40% of the average healthy child''s gut microbiome, with their abundance increasing with age.⁵ In general, Bacteroides beneficially influence immunoregulatory, physiological, and metabolic homeostasis in the host.⁶ The beneficial effects of Bacteroides species have been demonstrated in several colitis models. Bacteroides thetaiotaomicron ameliorates colitis in both dextran sulfate sodium (DSS) and IL-10 knockout colitis models.⁷ Bacteroides fragilis treatment decreases inflammation in trinitrobenzene sulfonic acid (TNBS) and DSS colitis models.^8,9 Similarly, monocolonization with Bacteroides ovatus in immunodeficient mice decrease mortality associated with DSS-induced colitis.¹⁰Testing three Bacteroides species (Bacteroides vulgatus, B. thetaiotaomicron, and B. ovatus) in a DSS colitis model¹¹ showed that B. ovatus ATCC 8483 monotherapy was most effective in reducing intestinal inflammation, enhancing epithelial recovery, and prolonging host survival.¹¹ Although commensal Bacteroides species are typically characterized as beneficial microbes, several Bacteroides species possess virulence factors and cause human infection.¹² More importantly, these virulence factors appear to be absent in B. ovatus.¹³ Despite these findings, B. ovatus has not been traditionally considered as a probiotic and its therapeutic potential in the setting of colitis has not been thoroughly investigated. We sought to further characterize the anti-inflammatory properties of B. ovatus in a gnotobiotic mouse model and TNBS model of murine colitis.     

Lactobacillus acidophilus Induces a Strain-specific and Toll-Like Receptor 2–Dependent Enhancement of Intestinal Epithelial Tight Junction Barrier and Protection Against Intestinal Inflammation

Defective intestinal tight junction (TJ) barrier is an important pathogenic factor of inflammatory bowel disease. To date, no effective therapies that specifically target the intestinal TJ barrier are available. The purpose of this study was to identify probiotic bacterial species or strains that induce a rapid and sustained enhancement of intestinal TJ barrier and protect against the development of intestinal inflammation by targeting the TJ barrier. After high-throughput screening of >20 Lactobacillus and other probiotic bacterial species or strains, a specific strain of Lactobacillus acidophilus, referred to as LA1, uniquely produced a marked enhancement of the intestinal TJ barrier. LA1 attached to the apical membrane surface of intestinal epithelial cells in a Toll-like receptor (TLR)-2–dependent manner and caused a rapid increase in enterocyte TLR-2 membrane expression and TLR-2/TLR-1 and TLR-2/TLR-6 hetero-complex–dependent enhancement in intestinal TJ barrier function. Oral administration of LA1 caused a rapid enhancement in mouse intestinal TJ barrier, protected against a dextran sodium sulfate (DSS) increase in intestinal permeability, and prevented the DSS-induced colitis in a TLR-2– and intestinal TJ barrier–dependent manner. In conclusion, we report for the first time that a specific strain of LA causes a strain-specific enhancement of intestinal TJ barrier through a novel mechanism that involves the TLR-2 receptor complex and protects against the DSS-induced colitis by targeting the intestinal TJ barrier.

Intestinal epithelial tight junctions (TJs) are the apical-most junctional complexes and act as a functional and structural barrier against the paracellular permeation of harmful luminal antigens, which promote intestinal inflammation.¹ The increased intestinal permeability caused by defective intestinal epithelial TJ barrier or a leaky gut is an important pathogenic factor that contributes to the development of intestinal inflammation in inflammatory bowel disease (IBD) and other inflammatory conditions of the gut, including necrotizing enterocolitis and celiac disease.^2,3 Clinical studies in patients with IBD have found that a persistent increase in intestinal permeability after clinical remission is predictive of poor clinical outcome and early recurrence of the disease, whereas normalization of intestinal permeability correlates with a sustained long-term clinical remission.4, 5, 6 Accumulating evidence has found that a defective intestinal TJ barrier plays an important role in exacerbation and prolongation of intestinal inflammation in IBD. Currently, no effective therapies that specifically target the tightening of the intestinal TJ barrier are available.Intestinal microbiota play an important role in modulating the immune system and in the pathogenesis of intestinal inflammation.⁷ Patients with IBD have bacterial dysbiosis in the gut, characterized by a decrease in bacterial diversity and an aberrant increase in some commensal bacteria, which are an important factor in the pathogenesis of intestinal inflammation.^8,9 Normal microbial flora of the gastrointestinal tract consists both of bacteria that are known to have beneficial effects (probiotic bacteria) on intestinal homeostasis and bacteria that could potentially have detrimental effects on gut health (pathogenic bacteria).¹⁰ The modulation of intestinal microflora affects the physiologic and pathologic states in humans and animals. For example, fecal transplantation from healthy, unaffected individuals to patients with refractory Clostridium difficile colitis is curative in up to 94% of the treated patients, and transfer of stool microbiome from obese mice induces obesity in previous lean mice, whereas transfer of microbiome from lean mice preserves the lean phenotype.11, 12, 13 The beneficial effects of gut microbiota are host and bacterial species-specific.¹⁴ Although multiple studies indicate that some commensal bacteria play a beneficial role in gut homeostasis by preserving or promoting the intestinal barrier function, because of conflicting reports, it remains unclear which probiotic species cause a persistent predictable enhancement in the TJ barrier and could be used to treat intestinal inflammation by targeting the TJ barrier. For example, some studies suggest that Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus plantarum, or Lactobacillus rhamnosus cause a modest enhancement in the intestinal epithelial TJ barrier, whereas others have found minimal or no effect of these probiotic species on the intestinal TJ barrier.15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 The major aim the current study was to perform a high-throughput screening of Lactobacillus and other bacterial species to identify probiotic species that induce a rapid, predictable, and marked increase in the intestinal epithelial TJ barrier and protect against the development of intestinal inflammation by preserving the intestinal TJ barrier.In the studies described herein, most of the probiotic species tested (>20 species or strains) had a modest or minimal effect on intestinal TJ barrier function. L. acidophilus uniquely caused a rapid and marked increase in intestinal TJ barrier function. Further analysis indicated that the effect of L. acidophilus was strain-specific, limited to a specific strain of L. acidophilus, and did not extend to other L. acidophilus strains. The L. acidophilus enhancement of the intestinal TJ barrier was mediated by live bacterial-enterocyte interaction that involved Toll-like receptor (TLR)-2 heterodimeric complexes on the apical membrane surface of intestinal epithelial cells. Our animal studies also found that L. acidophilus causes a marked enhancement in mouse intestinal barrier function and protects against the dextran sodium sulfate (DSS)–induced colitis by preserving and augmenting the mouse intestinal barrier function in a strain-specific manner.     

Cytochemistry of type II pneumocytes in Chediak-Higashi syndrome of mice

The type II pneumocytes of beige mice with the Chediak-Higashi syndrome (CHS) have been compared ultrastructurally and histochemically with those of normal black mice in both newborn and adult animals. In normal black mice the inner face of the limiting membrane of lamellar bodies near the cell surface revealed continuous dialyzed iron (DI) staining as did the luminal surface of the cell. The periodate-thiocarbohydrazide-silver proteinate (PA-TCH-SP) method stained selectively the luminal surface of the granular pneumocyte together with multivesicular bodies and the inner face of the limiting membrane of lamellar bodies, in normal black mice thus supporting the role of multivesicular bodies as precursors of lamellar bodies.The beige mice lacked the layer of continuous DI staining lining the superficial lamellar bodies. Moreover, the PA-TCH-SP staining was negligible on the limiting membrane of large lamellar bodies in beige mice. Immunostaining for lysozyme appeared weaker in the large lamellar bodies of type II pneumocytes of the beige mice. Peroxisomes were normal in size but somewhat increased in number in type II pneumocytes of beige mice.The multivesicular bodies as well as the lamellar bodies increased in size and decreased in number in the beige mice and, in some cell profiles, multivesicular bodies or lamellar bodies appeared to be fusing. Since the multivesicular bodies are thought to be precursors of lamellar bodies, these findings indicate that atypical fusion of the lysosomes in type II pneumocytes occurs at early to late stages of maturation. Newborn beige mice disclosed a greater and more consistent increase in size of the lamellar bodies than did adult beige mice. Compared with newborn black mice the newborn beige mice revealed fewer secreted lamellar bodies in the alveolar space. The cytochemical and morphological changes in lamellar bodies and their precusor multivesicular bodies in type II pneumocytes of beige mice are interpreted as resulting from increased fusion of the precursor bodies and lamellar bodies with one another. Impaired secretion of lamellar bodies possibly also contributes to increased size of lamellar bodies and content of lamellar body material in granular pneumocytes of beige mice.     

MicroRNA155 Plays a Critical Role in the Pathogenesis of Cutaneous Leishmania major Infection by Promoting a Th2 Response and Attenuating Dendritic Cell Activity

Interferon (IFN)-γ is indispensable in the resolution of cutaneous leishmaniasis (CL), while the Th2 cytokines IL-4, IL-10, and IL-13 mediate susceptibility. A recent study found that miR155, which promotes CD4⁺ Th1 response and IFN-γ production, is dispensable in the control of Leishmania donovani infection. Here, the role of miR155 in CL caused by L. major was investigated using miR155-deficient (miR155^−/−) mice. Infection was controlled significantly quicker in the miR155^−/− mice than in their wild-type (WT) counterparts, indicating that miR155 contributes to the pathogenesis of CL. Faster resolution of infection in miR155^−/− mice was associated with increased levels of Th1-associated IL-12 and IFN-γ and reduced production of Th2- associated IL-4, IL-10, and IL-13. Concentrations of IFN-γ⁺CD8⁺ T cells and natural killer cells in draining lymph nodes were significantly higher in the L. major−infected miR155^−/− mice than in the infected WT mice, as indicated by flow-cytometry. After in vitro IFN-γ stimulation, nitric oxide and IL-12 production were increased, IL-10 production was decreased, and parasite clearance was enhanced in L. major−infected miR155^−/− DCs compared to those in WT DCs. Furthermore, IFN-γ production from activated miR155^−/− T cells was significantly enhanced in L. major−infected miR155^−/− DCs. Together, these findings demonstrate that miR155 promotes susceptibility to CL caused by L. major by promoting Th2 response and inhibiting DC function.

Leishmania are obligate intracellular protozoans that infect phagocytes and cause a spectrum of clinical diseases such as cutaneous leishmaniasis (CL) and visceral leishmaniasis. Common in the tropical and subtropical regions, leishmaniasis affects over 1 billion people worldwide, with an incidence of up to 1 million cases per year.¹ CL is the most common type of Leishmania infection, manifesting as localized skin lesions that can become chronic, leading to significant tissue destruction and disfigurement.^2,3 It is well documented that the induction of a Th1 response and interferon (IFN)-γ are indispensable in the resolution of CL caused by Leishmania major,⁴ whereas disease progression is associated with the induction of a Th2 response and the production of cytokines such as IL-4 and IL-10.⁵ Establishing a disease-resolving response in the host is largely dependent on the ability to mount an appropriate Th1 immune response.⁴ Crucial in this response is the stimulation and activation of DCs that direct T-cell proliferation and differentiation toward IFN-γ–producing Th1 cells.^6,7 In addition to activating of phagocytic cells, IFN-γ induces the production of reactive nitrogen species, specifically nitric oxide (NO), leading to enhanced parasite clearance.⁴miR155 is a recognized regulator of immune cell function and immune response. miR155 enhances macrophage and DC activation and induces inflammatory response,^8,9 and up-regulation of miR155 in CD4⁺ T cells promotes preferential Th1 differentiation and IFN-γ production¹⁰ by suppressing the expression of suppressor of cytokine signaling (SOCS)-1.11, 12, 13, 14 Conversely, miR155 gene–deficient mice exhibit diminished levels of Th1/Th17 cells, macrophages, and DCs.¹⁵ miR155 has also been shown to play a role in regulating effector Th2 response.16, 17, 18 Collectively, these findings suggest that miR155 regulates both Th1 and Th2 responses, which control the outcome of CL caused by L. major. Therefore, the role of miR155 in immunity to L. major using miR155^−/− mice was investigated in the present study. The findings show that miR155 is not required for the induction of a Th1 response and IFN-γ in L. major infection. Rather, miR155 plays a disease-exacerbating role in CL by attenuating DC function and Th1 response and promoting Th2 response.