Benzylpenicillin preparations can evoke a systemic anaphylactic reaction in guinea pigs

All of the five commercially available benzylpenicillin preparations obtained from different sources and a PcG preparation prepared by filtration of a commercial PcG on Sephadex G10 elicited the systemic anaphylactic reactions in guinea pigs which had been immunized with benzylpenicilloyl (BPO)-Ascaris extract conjugate (BPO-As) mixed with aluminum hydroxide gel. These preparations could evoke no such reactions in guinea pigs immunized with BPO-bovine gamma globulin conjugate (BPO-BGG) emulsified with complete Freund's adjuvant. The severity of the systemic anaphylactic reactions correlated significantly with the titers of either 8-day passive cutaneous anaphylactic (8-day PCA) reactions or 4-hr PCA reactions evoked with the same benzylpenicillin preparations. In vitro benzylpenicillin preparation contracted the tracheas of the guinea pigs immunized with BPO-As. These results indicated that the commercially available benzylpenicillin preparations have enough antigenicity to evoke systemic anaphylactic reactions in guinea pigs immunized with BPO-As mixed with aluminum hydroxide gel. Such guinea pigs represent an animal model for investigation of penicillin allergy.     

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.     

Dual β-Catenin and γ-Catenin Loss in Hepatocytes Impacts Their Polarity through Altered Transforming Growth Factor-β and Hepatocyte Nuclear Factor 4α Signaling

Hepatocytes are highly polarized epithelia. Loss of hepatocyte polarity is associated with various liver diseases, including cholestasis. However, the molecular underpinnings of hepatocyte polarization remain poorly understood. Loss of β-catenin at adherens junctions is compensated by γ-catenin and dual loss of both catenins in double knockouts (DKOs) in mice liver leads to progressive intrahepatic cholestasis. However, the clinical relevance of this observation, and further phenotypic characterization of the phenotype, is important. Herein, simultaneous loss of β-catenin and γ-catenin was identified in a subset of liver samples from patients of progressive familial intrahepatic cholestasis and primary sclerosing cholangitis. Hepatocytes in DKO mice exhibited defects in apical-basolateral localization of polarity proteins, impaired bile canaliculi formation, and loss of microvilli. Loss of polarity in DKO livers manifested as epithelial-mesenchymal transition, increased hepatocyte proliferation, and suppression of hepatocyte differentiation, which was associated with up-regulation of transforming growth factor-β signaling and repression of hepatocyte nuclear factor 4α expression and activity. In conclusion, concomitant loss of the two catenins in the liver may play a pathogenic role in subsets of cholangiopathies. The findings also support a previously unknown role of β-catenin and γ-catenin in the maintenance of hepatocyte polarity. Improved understanding of the regulation of hepatocyte polarization processes by β-catenin and γ-catenin may potentially benefit development of new therapies for cholestasis.

A hallmark of epithelial cells is polarization, which is achieved by the orchestration of external cues, such as cellular contact, extracellular matrix, signal transduction, growth factors, and spatial organization.¹ Hepatocytes in the liver show a unique polarity by forming several apical and basolateral poles within a cell.² The apical poles of adjacent hepatocytes form a continuous network of bile canaliculi into which bile is secreted, whereas the basolateral membrane domain forms the sinusoidal pole, which secretes various components, such as proteins or drugs, into the blood circulation.³ Loss of hepatic polarity has been associated with several cholestatic and developmental disorders, including progressive familial intrahepatic cholestasis (PFIC) and primary sclerosing cholangitis (PSC).^4,5 Although the molecular mechanisms governing hepatocyte polarity have been extensively studied in the in vitro systems, there is still a significant gap in our understanding of how polarity is established within the context of tissue during development or maintained during homeostasis.^6,7 Similarly, the molecular pathways contributing to hepatic polarity are not entirely understood, and a better comprehension of hepatic polarity regulation is thus warranted.Previous studies have confirmed the role of hepatocellular junctions, such as tight and gap junctions, in the maintenance of hepatocyte polarity.^8,9 Studies done in vitro and in vivo have shown that loss of junctional proteins, such as zonula occludens protein (ZO)-1, junctional adhesion molecule-A, and claudins, lead to impairment of polarity and distorted bile canaliculi formation.10, 11, 12, 13 In addition, proteins involved in tight junction assembly, such as liver kinase B1, are also involved in polarity maintenance.¹⁴ Among adherens junction proteins, various in vitro cell culture models have confirmed the role of E-cadherin in the regulation of hepatocyte polarity, possibly through its interaction with β-catenin.^15,16 However, there is a lack of an in vivo model to study the role of adherens junction proteins in hepatocyte polarity and their misexpression contributing to various liver diseases.β-Catenin plays diverse functions in the liver during development, regeneration, zonation, and tumorigenesis.17, 18, 19 The relative contribution of β-catenin as part of the adherens junction is challenging to study because like in other tissues, γ-catenin compensates for the β-catenin loss in the liver.^20,21 To address this redundancy, we previously reported a hepatocyte-specific β-catenin and γ-catenin double-knockout (DKO) mouse model was reported.²² Simultaneous deletion of β-catenin and γ-catenin in mice livers led to cholestasis, partially through the breach of cell-cell junctions. However, more comprehensive understanding of the molecular underpinnings of the phenotype is needed.In the current study, prior preclinical findings of dual β-catenin and γ-catenin loss were extended to a subset of PFIC and PSC patients. In vivo studies using the murine model with hepatocyte-specific dual loss of β-catenin and γ-catenin showed complete loss of hepatocyte polarity compared to the wild-type controls (CONs). Loss of polarity in DKO liver was accompanied by epithelial-mesenchymal transition (EMT), activation of transforming growth factor (TGF)-β signaling, and reduced expression of hepatocyte nuclear factor 4α (HNF4α). Our findings suggest that β-catenin and γ-catenin and in turn adherens junction integrity, are critical for the maintenance of hepatocyte polarity, and any perturbations in this process can contribute to the pathogenesis of cholestatic liver disease.     

Mechanisms of respiration and phosphorylation in Ascaris muscle mitochondria

In Ascaris muscle mitochondria the major respiratory chain-linked phosphorylation activity is accomplished by a NADH-linked reduction of fumarate to succinate. Oxygen can also be employed as a terminal electron acceptor via a cyanide- and salicyl-hydroxamate-resistant terminal oxidase. As in fumarate-dependent electron transport this process appears to be coupled to energy conservation at phosphorylation site I. The branchpoint from which electrons are taken from the main respiratory chain to either the alternative oxidase or fumarate reductase is likely to be on the oxygen side of the NADH dehydrogenase segment.Malate and succinate are the only substrates which appreciably support respiration in the mitochondrion of the nematode. Regardless of the presence or absence of oxygen malate is utilized by an oxidation-reduction reaction resulting in the formation of pyruvate, acetate, succinate, propionate and CO₂. In addition, aerobically, hydrogen peroxide is formed as the product of oxygen reduction. Succinate accumulation was found to be significantly higher in the anaerobic as compared to the aerobic incubation mixtures. This effect was accompanied by an increase in anaerobic malate consumption. ATP generation and the formation of pyruvate, acetate and propionate were found to be similar in the presence and absence of oxygen.In malate-supported respiration of intact Ascaris mitochondria reducing equivalents (NADH) are produced exclusively through pyruvate and acetate formation. These enzymatic reactions are functionally coupled to the electron transport-linked reductions of fumarate to succinate and oxygen to hydrogen peroxide, respectively. In accordance with the position of the redox potentials of the fumarate/succinate and O₂/H₂O₂ couples, anaerobic and aerobic respiration was found to be associated with relatively low energy conservation efficiencies. Thus one molecule of ATP was conserved per 2e^? transferred to fumarate or oxygen, respectively. No evidence could be obtained for a significant activity of energy conservation sites II and III and electron transfer through the alternative oxidase pathway was shown not to be coupled to phosphorylation.     

Double minutes and other chromosomal aberrations in two malignant cell lines of the German cockroach Blattella germanica

Structural anomalies of mitotic chromosomes from two tumorigenic cell lines of the German cockroach (Blattella germanica) is described. Aberrations, such as unpairable marker chromosomes, double minutes, di- and tricentrics, ring chromosomes, tri- and quadriradials, and chromosome and chromatid gaps and breaks, were observed in varying proportions. This study reports that the double minute chromosomes (DMs) are associated with insect tumor cells, similar to the findings in both murine and human tumor cells.     

Engulfment of mast cell secretory granules on skin inflammation boosts dendritic cell migration and priming efficiency

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Spontaneous age-associated amyloidosis in senescence-accelerated mouse (SAM)

Morphological studies on spontaneous systemic amyloidosis were conducted on 222 senescence-accelerated mice (SAM) (P) and on 150 mice in the senescence-resistant series (R).Among the pathologic findings, amyloidosis showed the highest incidence in both SAM (79.7%) and R (32.7%) Although an extensive deposition of amyloid was evident in some aged mice in the R series, a more severe amyloidosis occurred with a higher incidence in the P series. There was a statistical significance between the incidence of amyloidosis and age, in both the P and R series. There were no differences in organ distribution and mode of amyloid deposition between the P and R series or between the sexes. In about 60% of the amyloid-positive cases in the 28 killed SAM and 7 mice in the R series, there were no signs of inflammation or neoplasm.The morphological features in SAM more closely resembled those seen in cases of murine spontaneous senile amyloidosis than the features seen in cases of experimentally induced amyloidosis. This model is expected to be a valuable tool with which to assess the relationship between amyloid deposition and the aging process or senescence, perhaps even cases of human senile amyloidosis.     

The effect of 2-amino-4-phosphonobutyrate (APB) on acetylcholine release from the rabbit retina: evidence for on-channel input to cholinergic amacrine cells

The light-evoked release of acetylcholine (ACh) from the rabbit retina was taken as a measure of cholinergic amacrine cell activity. The glutamate analogue DL-(+/-)-2-amino-4-phosphonobutyric acid (APB) prevented the light-evoked release of ACh and also selectively abolished the ON-responses of ganglion cells and the ERG b-wave. It is concluded that the input to cholinergic amacrine cells involves mainly the depolarizing bipolar cells, which subserve ON-channels. L-(+)-stereoisomer of APB was 15 times more potent than the D-(-)-isomer in suppressing ACh release and the b-wave, suggesting that the mechanism of action of APB does not involve antagonism of excitatory amino acids.