Alveolar macrophages (AM) are very important for pulmonary innate immune responses against invading inhaled pathogens because they directly kill the organisms and initiate a cascade of innate and adaptive immune responses. Although several factors contribute to inhalational anthrax, we hypothesized that unimpeded infection of Bacillus anthracis is directly linked to disabling the innate immune functions contributed by AM. Here, we investigated the effects of lethal toxin (LT), one of the binary complex virulence factors produced by B. anthracis, on freshly isolated nonhuman primate AM. Exposure of AM to doses of LT that killed susceptible macrophages had no effect on the viability of AM, despite complete MEK1 cleavage. Intoxicated AM remained fully capable of B. anthracis spore phagocytosis. However, pretreatment of AM with LT resulted in a significant decrease in the clearance of both the Sterne strain and the fully virulent Ames strain of B. anthracis, which may have been a result of impaired AM secretion of proinflammatory cytokines. Our data imply that cytolysis does not correlate with MEK1 cleavage, and this is the first report of LT-mediated impairment of nonhuman primate AM bactericidal activity against B. anthracis.

OBJECTIVE

To describe clinicopathologic features and to identify prognostic factors in a large series of primary cutaneous diffuse large B-cell lymphoma, leg type (PCLBCL LT), as defined in the recent World Health Organization-European Organization for Research and Treatment of Cancer classification of cutaneous lymphomas.

METHODS

Retrospective multicenter study from the French Study Group on Cutaneous Lymphomas.

METHODS

Nineteen departments of dermatology in 10 regions of France.

METHODS

Sixty patients with a PCLBCL LT included in the registry of the French Study Group on Cutaneous Lymphomas.

METHODS

Age, sex, outcome, therapy, B symptoms, cutaneous extent, number of lesions, location (leg vs nonleg), serum lactate dehydrogenase level, and MUM-1 and Bcl-2 expression were recorded. Disease-specific survival was used as the main end point. Prognostic factors were identified using a Cox proportional hazards model.

RESULTS

Primary cutaneous diffuse large B-cell lymphoma, leg type is characterized by a predilection for the leg (72%), a high proportion of Bcl-2 expression (85%), an advanced age at onset (mean age, 76 years), and frequent relapses and extracutaneous dissemination. The overall 5-year disease-specific survival rate was 41%. Location on the leg and multiple skin lesions were predictive of death in multivariate analysis. Although no variable related to therapy was significantly associated with survival, patients recently treated with combinations of anthracycline-containing chemotherapies and rituximab had a more favorable short-term outcome.

CONCLUSIONS

Primary cutaneous diffuse large B-cell lymphoma, leg type is a distinct entity with a poor prognosis, particularly in patients with multiple tumors on the legs. Despite the advanced age of many patients, the prognosis could be improved with combinations of anthracycline-containing chemotherapies and rituximab.

Leukotriene B(4) (LTB(4)) biosynthesis by polymorphonuclear leukocytes (PMNs) is an important factor of inflammatory responses. PMNs also release LTA(4), an unstable intermediate that can be taken up by neighboring cells and metabolized into LTC(4). Most studies of LT synthesis have been carried out using human PMNs, but very little information is available about mouse PMNs. Mouse bone marrow PMNs were found to synthesize eicosanoids upon stimulation with A23187, fMLP, or zymosan. The major eicosanoids produced are LTB(4) and 5-hydroxyeicosatetraenoic acid, with some nonenzymatic products of LTA(4) hydrolysis. No cysteinyl leukotrienes were produced, in contrast to what was observed with human blood neutrophil preparations. Human megakaryoblast-like MEG-01 cells synthesized thromboxane B(2) and prostaglandin E(2) in response to A23187 but produced no 5-lipoxygenase (5-LO)-derived eicosanoids. When mouse bone marrow cells (mBMCs) and MEG-01 cells were stimulated during coincubation, LTC(4) and LTD(4) were produced. Mouse peritoneal macrophages from 5-LO-deficient mice were able to synthesize LTC(4) when incubated with mBMCs from wild-type mice, demonstrating transcellular exchange of LTA(4) from mBMCs into murine peritoneal macrophages. These data demonstrate that murine bone marrow PMNs are a valid model for the study of LT biosynthesis, which now offers the possibility to investigate specific biochemical pathways through the use of transgenic mice.

Purified human peripheral blood monocytes were stimulated with aggregated human myeloma proteins of different classes or the calcium ionophore A23187 and the release of leukotrienes C4 and <em>B</em>4 (<em>LT</em>C4, <em>LT</em><em>B</em>4), and prostaglandin E2 (PGE2) into the supernatant was determined. The ionophore induced release of 10 +/- 5 ng <em>LT</em>C4/10(6) cells and 25 +/- 8 ng <em>LT</em><em>B</em>4/10(6) cells. Aggregated IgG, IgA, and IgE, but not IgM or monomeric immunoglobulins (Ig), induced release of <em>LT</em>C4 and <em>LT</em><em>B</em>4 that was approximately 10 to 20% of that induced by ionophore. In addition, IgG, IgA, and IgE, but not IgM, induced release of PGE2 (range 0.015 to 0.22 ng/10(6) cells). Aggregated Ig induced <em>LT</em>C4, <em>LT</em><em>B</em>4, and PGE2 release in a dose-dependent manner; maximal leukotriene (<em>LT</em>) release was observed by 30 min, in contrast to PG release, which continued to increase up to 2.5 hr. <em>B</em>oth ionophore- and Ig-induced <em>LT</em>C4 and <em>LT</em><em>B</em>4 release were completely inhibited by removal of calcium from the media and by preincubation of cells with nordihydroguaiaretic acid. Indomethacin inhibited Ig-induced PGE2 release by 80%. Phagocytosis of the Ig aggregates was not required for <em>LT</em> or PGE2 release, since release was not inhibited by cytochalasin <em>B</em>. Release of <em>LT</em>C4, <em>LT</em><em>B</em>4, and PGE2 induced by IgG, IgA, and IgE, but not IgM, correlated with the presence or absence of monocyte Fc receptors (FcR) as determined by rosette assays. The data suggest that IgG, IgA, and IgE immune complexes mostly likely induce monocyte arachidonic acid metabolism via cross-linking of FcR. The ability of monocytes to release eicosanoids in the absence of phagocytosis suggests that interaction of monocytes with immobilized immune complexes, such as those deposited in blood vessel walls or glomerular basement membranes, could initiate metabolism of arachidonic acid by monocytes. Such a mechanism could contribute to inflammatory reactions characterized by mononuclear cell infiltrates.

A first-generation oral inactivated whole-cell enterotoxigenic Escherichia coli (ETEC) vaccine, comprising formalin-killed ETEC bacteria expressing different colonization factor (CF) antigens combined with cholera toxin B subunit (CTB), when tested in phase III studies did not significantly reduce overall (generally mild) ETEC diarrhea in travelers or children although it reduced more severe ETEC diarrhea in travelers by almost 80%. We have now developed a novel more immunogenic ETEC vaccine based on recombinant non-toxigenic E. coli strains engineered to express increased amounts of CF antigens, including CS6 as well as an ETEC-based B subunit protein (LCTBA), and the optional combination with a nontoxic double-mutant heat-labile toxin (LT) molecule (dmLT) as an adjuvant. Two test vaccines were prepared under GMP: (1) A prototype E. coli CFA/I-only formalin-killed whole-cell+LCTBA vaccine, and (2) A "complete" inactivated multivalent ETEC-CF (CFA/I, CS3, CS5 and CS6 antigens) whole-cell+LCTBA vaccine. These vaccines, when given intragastrically alone or together with dmLT in mice, were well tolerated and induced strong intestinal-mucosal IgA antibody responses as well as serum IgG and IgA responses to each of the vaccine CF antigens as well as to LT B subunit (LTB). Both mucosal and serum responses were further enhanced (adjuvanted) when the vaccines were co-administered with dmLT. We conclude that the new multivalent oral ETEC vaccine, both alone and especially in combination with the dmLT adjuvant, shows great promise for further testing in humans.

T-cell responses may be shaped by sterile "danger signals" that are constituted by damage-associated molecular patterns (DAMP). However, whether and what type of adaptive immune responses are triggered in vivo by DAMPs induced by tumor progression are not well characterized. In this study, we report that the production of HMGB1, an established DAMP released by dying cells, was critical for tumor progression in an established mouse model of prostate cancer. HMGB1 was required for the activation and intratumoral accumulation of T cells that expressed cytokine lymphotoxinα(1)β(2) (LT) on their surface. Intriguingly, these tumor-activated T cells recruited macrophages to the lesion and were essential to promote the preneoplasia to invasive carcinoma in an LTβ receptor (LTβR)-dependent manner. Taken together, our findings suggest that the release of HMGB1 as an endogenous danger signal is important for priming an adaptive immune response that promotes malignant progression, with implications for cancer prevention and therapy.

1. The alpha5 subunit participates to the formation of native neuronal nicotinic receptors, particularly in autonomic ganglia. Like the related betabeta subunits, but its effect on the properties of the resulting alphabetaalpha5 receptor depends on the alpha and beta subunits chosen and on the expression system. We used a reporter mutation approach to test whether alpha5, like betabetabetabetabetabetaLT)betaLT)betabetaLT)alpha5 vs. alpha3betaLT)). The much greater effect observed in alpha3betaLT)alpha5 receptors accords with the hypothesis that alpha5 takes the place of a beta subunit in the receptor. 3. Introducing a 9'T mutation in alpha5 had no effect on the agonist sensitivity of alpha3betabetabetabeta and therefore only one copy of alpha5.

LT-IIa and LT-IIb, the type II heat-labile enterotoxins of Escherichia coli, are closely related in structure and function to cholera toxin and LT-I, the type I heat-labile enterotoxins of Vibrio cholerae and E. coli, respectively. Recent studies from our group demonstrated that LT-IIa and LT-IIb are potent systemic and mucosal adjuvants. To determine whether binding of LT-IIa and LT-IIb to their specific ganglioside receptors is essential for adjuvant activity, LT-IIa and LT-IIb enterotoxins were compared with their respective single-point substitution mutants which have no detectable binding activity for their major ganglioside receptors [e.g., LT-IIa(T34I) and LT-IIb(T13I)]. Both mutant enterotoxins exhibited an extremely low capacity for intoxicating mouse Y1 adrenal cells and for inducing production of cyclic AMP in a macrophage cell line. BALB/c female mice were immunized by the intranasal route with the surface adhesin protein AgI/II of Streptococcus mutans alone or in combination with LT-IIa, LT-IIa(T34I), LT-IIb, or LT-IIb(T13I). Both LT-IIa and LT-IIb potentiated strong mucosal and systemic immune responses against AgI/II. Of the two mutant enterotoxins, only LT-IIb(T13I) had the capacity to strongly potentiate mucosal anti-AgI/II and systemic anti-AgI/II antibody responses. Upon boosting with AgI/II, however, both LT-IIa(T34I) and LT-IIb(T13I) enhanced humoral memory responses to AgI/II. Flow cytometry demonstrated that LT-IIa(T34I) had no affinity for cervical lymph node lymphocytes. In contrast, LT-IIb(T13I) retained binding activity for T cells, B cells, and macrophages, indicating that this immunostimulatory mutant enterotoxin interacts with one or more unknown lymphoid cell receptors.

Leukotriene A(4) hydrolase (LTA(4)H) catalyzes production of the proinflammatory lipid mediator, leukotriene (LT) B(4), which is implicated in a number of inflammatory diseases. We have identified a potent and selective inhibitor of both the epoxide hydrolase and aminopeptidase activities of recombinant human LTA(4)H (IC(50), approximately 10 nM). In a murine model of arachidonic acid-induced ear inflammation, the LTA(4)H inhibitor, JNJ-26993135 (1-[4-(benzothiazol-2-yloxy)-benzyl]-piperidine-4-carboxylic acid), dose-dependently inhibited ex vivo LTB(4) production in blood, in parallel with dose-dependent inhibition of neutrophil influx (ED(50), 1-3 mg/kg) and ear edema. In murine whole blood and in zymosan-induced peritonitis, JNJ-26993135 selectively inhibited LTB(4) production, without affecting cysteinyl leukotriene production, while maintaining or increasing production of the anti-inflammatory mediator, lipoxin (LX) A(4). The 5-lipoxygenase (5-LO) inhibitor zileuton showed inhibition of LTB(4), LTC(4), and LXA(4) production. Although zileuton inhibited LTB(4) production in the peritonitis model more effectively than the LTA(4)H inhibitor, the influx of neutrophils into the peritoneum after 1 and 2 h was significantly higher in zileuton- versus JNJ-26993135-treated animals. This difference may have been mediated by the increased LXA(4) levels in the presence of the LTA(4)H inhibitor. The selective inhibition of LTB(4) production by JNJ-26993135, while increasing levels of the anti-inflammatory mediator, LXA(4), may translate to superior therapeutic efficacy versus 5-LO or 5-LO-activating protein inhibitors in LTB(4)-mediated inflammatory diseases.

Intraperitoneal administration of zymosan to mice resulted in marked biosynthesis of eicosanoids and influx of neutrophils with distinct time course profiles. 6-Keto-prostaglandin-F1 alpha (6-KPA) increased between 30 and 60 min and rapidly decreased thereafter. Leukotriene (LT)C4 levels showed similar patterns, but were sustained for several hours. LTB4 increased in a biphasic manner with peak increases between 2 to 3 hr. Repeated injections with zymosan suggested that incoming neutrophils generate most of the LTB4. Myeloperoxidase (MPO), an enzyme marker for neutrophils, continued to increase throughout the time course. Mast cells regulate LTB4 biosynthesis and neutrophil trafficking, whereas resident macrophages contribute to 6-KPA and LTC4 biosynthesis. The complement fragment C5a has a minimal role in zymosan-induced inflammation. Selective 5-lipoxygenase (5-LO) inhibitors, zileuton [N-(1-benzo[b]thienyl-2yl-ethyl)-N-hydroxyurea], TZI-41127 [2-(4-hydroxy-3,5-dimethylphenyl)-5-methoxy-3-methylindole] and cyclooxygenase (CO) inhibitors selectively modulated eicosanoid biosynthesis. Both 5-LO and CO inhibitors attenuated influx of neutrophils to varying degrees. A LTB4 receptor antagonist, SC-41930 [7-(3-(4-acetyl-3-methoxy-2-propylphenoxy)-propoxy]-3,4-dihydro-8- propyl-2H-1-benzopyran-2-carboxylic acid) and an LTD4 receptor antagonist, LY-171883 [1-(2-hydroxy-3-propyl-4-(4-1H-tetrazol-5-yl)butoxy-phenyl) ethanone)] (i.v.) attenuated influx of neutrophils and associated LTB4 biosynthesis. These results suggest that both 5-LO and CO metabolites regulate neutrophil influx in this model. Marked eicosanoid biosynthesis and cellular influx in response to zymosan provides an attractive experimental paradigm to evaluate anti-inflammatory effects of inhibitors of arachidonate CO or 5-LO pathways.

Heat-labile enterotoxin (LT) produced by enterotoxigenic Escherichia coli (ETEC) and cholera toxin (CT) produced by Vibrio cholerae have been shown to function as potent mucosal adjuvants. A number of studies have examined the effects of different mutations at either the active site or the protease site of LT and CT and the influence of those mutations on toxicity and adjuvanticity. However, different observations reported by various groups using a variety of animal models with different antigens or different routes of immunization have provided contradictory findings and evoked many questions regarding the underlying mechanisms of mucosal adjuvanticity of LT and CT. In this study, the role of cAMP in mucosal adjuvanticity was examined by comparing three LT active site mutants (S61F, A69G, E112K), a protease site mutant (R192G) and recombinant LT-B for toxicity, cAMP activity and mucosal adjuvanticity using tetanus toxoid (TT) as a model antigen. While all mutants examined showed reduced toxicity, the effects of each mutation on its ability to function as an adjuvant varied. Following intranasal immunization, native LT as well as protease and active site mutants of LT induced serum anti-TT IgG and their responses were virtually indistinguishable from one another. In addition, LT-B was also able to enhance production of serum anti-TT IgG, though at a level significantly lower than that achieved by native LT and mutants. Following oral immunization, the best serum anti-TT IgG responses were obtained with native LT and mutants that retained the ability to induce accumulation of cAMP. Despite the nearly identical serum anti-TT IgG responses following intranasal immunization, there was a strong correlation between the ability to induce accumulation of cAMP in cultured Caco-2 cells and the ability to elicit production of antigen-specific Th1 or Th2 cytokines.

Memory T helper cells (Th cells) play an important role in host defense against pathogens but also contribute to the pathogenesis of inflammatory disorders. We found that a soluble decoy lymphotoxin β receptor (LT-βR)-Fc, which can block tumor necrosis factor (TNF)-related ligands LIGHT (TNFSF14) and LT-αβ binding to the herpesvirus entry mediator (HVEM) and the LT-βR, inhibited the accumulation of memory Th2 cells after antigen encounter and correspondingly reduced inflammatory responses in vivo. Showing that this was a function of the receptor for LIGHT, antigen-specific memory CD4 T cells deficient in HVEM were also unable to persist, despite having a normal immediate response to recall antigen. HVEM(-/-) memory Th2 cells displayed reduced activity of PKB (protein kinase B; Akt), and constitutively active Akt rescued their survival and restored strong inflammation after antigen rechallenge. This was not restricted to Th2 memory cells as HVEM-deficient Th1 memory cells were also impaired in surviving after encounter with recall antigen. Furthermore, the absence of LIGHT on T cells recapitulated the defect seen with the absence of HVEM, suggesting that activated T cells communicate through LIGHT-HVEM interactions. Collectively, our results demonstrate a critical role of HVEM signals in the persistence of large pools of memory CD4 T cells.

A total of 1,226 Escherichia coli strains isolated from 1979 to 1989 from pigs with diarrhea were examined for serogroup and fimbrial antigen F4 (K88) production. Four main patterns of isolation of the various serogroups were observed, depending on the ages of the pigs from which isolates were obtained and the production of F4. In pattern I, serogroups O8:K"S16", O9:K35, O9/O101:K30, O9/O101:K103, O9 (group), O20:K101, and O64:K"V142" were predominant in pigs aged 0 to 6 days (41.9% of isolates) and were less frequent in pigs aged 7 to 27 days (24.6% of isolates) but were rarely found in pigs aged 28 to 60 days (4.0% of isolates). In pattern II, the F4-associated serogroups O8:K"4627", O157:K"V17", O149:K91, and O147:K89 were predominant in pigs aged 7 to 27 days (29.8% of isolates) and in pigs aged 28 to 60 days (35.0% of isolates). In pattern III, serogroups O8 (group), O115:K"V165", and O147:K89 were rarely isolated from pigs aged 0 to 6 days but were equally distributed in pigs aged 7 to 27 days (10.1% of isolates) and in pigs aged 28 to 60 days (10.9% of isolates). In pattern IV, serogroups O138:K81, O139:K82, O141:K85ac, O45:K"E65", and O26:K60 were most frequently isolated in pigs aged 28 to 60 days (19.3% isolates). Over the period from 1979 to 1989, the proportion of isolates belonging to serogroups of pattern II and the proportion of F4 isolates within the serogroup O157:K"V17" declined, whereas the proportion of isolates of serogroups O147:K89, O8:K"S16", and O9:K35 increased. For 228 isolates selected from the most important serogroups, good agreement was observed between the results of gene probes and immunofluorescence for the detection of fimbrial antigens F4 (K88), F5 (K99), F6 (987P), and F41 and between the results of gene probes and biological assays for the detection of heat-labile enterotoxin (LT) and heat-stable enterotoxins a and b (STa and STb). The STa gene was mostly associated with isolates of pattern I serogroups, which had the F5, F6, and F41 genes alone or in various combinations. The LT and/or STb genes, with the F4 gene, mostly were observed in isolates of pattern II serogroups. The STb gene alone was observed mostly in isolates of pattern III serogroups, although isolates were negative for all fimbrial antigen genes. Similarly, isolates of pattern IV serogroups were negative for all fimbrial antigen genes and rarely positive for the enterotoxin genes. However, verotoxin production was associated with isolates of serogroups O138:K81 and O139:K82. The most important pathotypes among enterotoxigenic isolates in this study were F4:LT:STb, F5:STa, STb, F5:F41:STa, F4:STb, F6, STa, and LT.

Human mesenchymal stem cell (MSC) extracellular vesicles (EV) can reduce the severity of bacterial pneumonia, but little is known about the mechanisms underlying their antimicrobial activity. In the current study, we found that bacterial clearance induced by MSC EV in <i>Escherichia coli</i> pneumonia in C57<em>B</em>L/6 mice was associated with high levels of leukotriene (<em>LT</em>) <em>B</em>₄ in the injured alveolus. More importantly, the antimicrobial effect of MSC EV was abrogated by cotreatment with a <em>LT</em><em>B</em>₄ <em>B</em><em>LT</em>1 antagonist. To determine the role of MSC EV on <em>LT</em> metabolism, we measured the effect of MSC EV on a known ATP-binding cassette transporter, multidrug resistance-associated protein 1 (MRP1), and found that MSC EV suppressed MRP1 mRNA, protein, and pump function in LPS-stimulated Raw264.7 cells in vitro. The synthesis of <em>LT</em><em>B</em>₄ and <em>LT</em>C₄ from <em>LT</em>A₄ are competitive, and MRP1 is the efflux pump for <em>LT</em>C₄ Inhibition of MRP1 will increase <em>LT</em><em>B</em>₄ production. In addition, administration of a nonspecific MRP1 inhibitor (MK-571) reduced <em>LT</em>C₄ and subsequently increased <em>LT</em><em>B</em>₄ levels in C57<em>B</em>L/6 mice with acute lung injury, increasing overall antimicrobial activity. We previously found that the biological effects of MSC EV were through the transfer of its content, such as mRNA, microRNA, and proteins, to target cells. In the current study, miR-145 knockdown abolished the effect of MSC EV on the inhibition of MRP1 in vitro and the antimicrobial effect in vivo. In summary, MSC EV suppressed MRP1 activity through transfer of miR-145, thereby resulting in enhanced <em>LT</em><em>B</em>₄ production and antimicrobial activity through <em>LT</em><em>B</em>₄/<em>B</em><em>LT</em>1 signaling.

<A<em>b</em>stractText>In late Fe<em>b</em>ruary 2020, due to the spread of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the Italian Government closed down all educational and sport activities. In March, it introduced further measures to stop the spread of coronavirus disease (COVID-19), placing the country in a state of almost complete lockdown. We report the impact of these restrictions on glucose control among people with type 1 dia<em>b</em>etes (T1D).</A<em>b</em>stractText><A<em>b</em>stractText>Data were collected on 33 individuals with T1D who were monitoring their glucose levels using a flash glucose monitoring device and remotely connected to the dia<em>b</em>etes clinic on a cloud platform. We retrieved information on average glucose, standard deviation and percentage time in hypoglycaemia (&<em>lt</em>; 70 mg/dl), glucose range (70-180 mg/dl) and hyperglycaemia (> 180 mg/dl). We compared glycaemic measures collected during lockdown to those collected <em>b</em>efore the SARS-CoV-2 epidemic and to the periods immediately <em>b</em>efore lockdown.</A<em>b</em>stractText><p><div>(<em>b</em>)Resu<em>lt</em>s</<em>b</em>)</div>In 20 patients who had stopped working and were at home as a resu<em>lt</em> of the lockdown, overall glycaemic control improved during the first 7 days of the lockdown as compared to the weeks <em>b</em>efore the spread of SARS-CoV-2. Average glucose declined from 177 ± 45 mg/dl (week <em>b</em>efore lockdown) to 160 ± 40 mg/dl (lockdown; <i>p</i> = 0.005) and the standard deviation improved significantly. Time in range increased from 54.4 to 65.2% (<i>p</i> = 0.010), and time in hyperglycaemia decreased from 42.3 to 31.6% (<i>p</i> = 0.016). The num<em>b</em>er of scans per day remained unchanged. In 13 patients who continued working, none of the measures of glycaemic control changed during lockdown.</p><A<em>b</em>stractText>Despite the limited possi<em>b</em>ility to exercise and the incum<em>b</em>ent psychologic stress, glycaemic control improved in patients with T1D who stopped working during the lockdown, suggesting that slowing down routine daily activities can have <em>b</em>eneficial effects on T1D management, at least in the short term.</A<em>b</em>stractText>

Dorsal horn neurons in the young rat spinal cord-hindlimb preparation were physiologically classified as wide dynamic range (WDR), nociceptive specific (NS) or low threshold (LT) according to their excitatory responses to low and high intensity mechanical stimuli applied to the hindlimb skin. Two additional types were classified: neurons displaying only sub-threshold excitations (SUB) and neurons displaying inhibitory events (INH), such as inhibitory post-synaptic potentials or interruption of spontaneous spiking following cutaneous stimulation. Direct intracellular current injection revealed four different patterns of spiking behaviour: group A neurons were characterized by tonic firing in response to depolarizing current pulses; group B neurons were strongly phasic, producing only one spike at the beginning of the pulse; group A-B neurons generated an early unsustained (< 300 ms) burst of spikes; and group C neurons exhibited anomalous rectification in response to hyperpolarizing current which was followed by a voltage-dependent rebound excitation. A statistically significant (P < or = 0.01) association existed between a neuron's physiological classification and its electrophysiological profile. The majority of WDR neurons responded with tonic firing and were assigned to group A, while NS neurons were strongly represented in group A-B. All INH neurons were assigned to group C. LT neurons were distributed between groups A and A-B, and SUB neurons were distributed between groups A and B. These data indicate, firstly, that dorsal horn neurons possess heterogeneous membrane properties and, secondly, that a relationship exists between a neuron's biophysical profile and its excitatory or inhibitory response to peripheral cutaneous afferent stimulation. The implications for dorsal horn somatosensory processing are discussed.

Background Inability to tolerate statins because of muscle symptoms contributes to uncontrolled cholesterol levels and insufficient cardiovascular risk reduction. Bempedoic acid, a prodrug that is activated by a hepatic enzyme not present in skeletal muscle, inhibits ATP -citrate lyase, an enzyme upstream of β-hydroxy β-methylglutaryl-coenzyme A reductase in the cholesterol biosynthesis pathway. Methods and Results The phase 3, double-blind, placebo-controlled CLEAR (Cholesterol Lowering via Bempedoic acid, an ACL-Inhibiting Regimen) Serenity study randomized 345 patients with hypercholesterolemia and a history of intolerance to at least 2 statins (1 at the lowest available dose) 2:1 to bempedoic acid 180 mg or placebo once daily for 24 weeks. The primary end point was mean percent change from baseline to week 12 in low-density lipoprotein cholesterol. The mean age was 65.2 years, mean baseline low-density lipoprotein cholesterol was 157.6 mg/dL, and 93% of patients reported a history of statin-associated muscle symptoms. Bempedoic acid treatment significantly reduced low-density lipoprotein cholesterol from baseline to week 12 (placebo-corrected difference, -21.4% [95% CI, -25.1% to -17.7%]; P&lt;0.001). Significant reductions with bempedoic acid versus placebo were also observed in non-high-density lipoprotein cholesterol (-17.9%), total cholesterol (-14.8%), apolipoprotein B (-15.0%), and high-sensitivity C-reactive protein (-24.3%; P&lt;0.001 for all comparisons). Bempedoic acid was safe and well tolerated. The most common muscle-related adverse event, myalgia, occurred in 4.7% and 7.2% of patients who received bempedoic acid or placebo, respectively. Conclusions Bempedoic acid offers a safe and effective oral therapeutic option for lipid lowering in patients who cannot tolerate statins. Clinical Trial Registration URL : https://www.clinicaltrials.gov . Unique identifier: NCT 02988115.

<A<em>b</em>stractText>Socioeconomic status is associated with differences in risk factors for cardiovascular disease incidence and outcomes, including mortality. However, it is unclear whether the associations <em>b</em>etween cardiovascular disease and common measures of socioeconomic status-wea<em>lt</em>h and education-differ among high-income, middle-income, and low-income countries, and, if so, why these differences exist. We explored the association <em>b</em>etween education and household wea<em>lt</em>h and cardiovascular disease and mortality to assess which marker is the stronger predictor of outcomes, and examined whether any differences in cardiovascular disease <em>b</em>y socioeconomic status parallel differences in risk factor levels or differences in management.</A<em>b</em>stractText><A<em>b</em>stractText>In this large-scale prospective cohort study, we recruited adu<em>lt</em>s aged <em>b</em>etween 35 years and 70 years from 367 ur<em>b</em>an and 302 rural communities in 20 countries. We collected data on families and households in two questionnaires, and data on cardiovascular risk factors in a third questionnaire, which was supplemented with physical examination. We assessed socioeconomic status using education and a household wea<em>lt</em>h index. Education was categorised as no or primary school education only, secondary school education, or higher education, defined as completion of trade school, college, or university. Household wea<em>lt</em>h, calculated at the household level and with household data, was defined <em>b</em>y an index on the <em>b</em>asis of ownership of assets and housing characteristics. Primary outcomes were major cardiovascular disease (a composite of cardiovascular deaths, strokes, myocardial infarction, and heart failure), cardiovascular mortality, and all-cause mortality. Information on specific events was o<em>b</em>tained from participants or their family.</A<em>b</em>stractText><p><div>(<em>b</em>)FINDINGS</<em>b</em>)</div>Recruitment to the study <em>b</em>egan on Jan 12, 2001, with most participants enrolled <em>b</em>etween Jan 6, 2005, and Dec 4, 2014. 160 299 (87·9%) of 182 375 participants with <em>b</em>aseline data had availa<em>b</em>le follow-up event data and were eligi<em>b</em>le for inclusion. After exclusion of 6130 (3·8%) participants without complete <em>b</em>aseline or follow-up data, 154 169 individuals remained for analysis, from five low-income, 11 middle-income, and four high-income countries. Participants were followed-up for a mean of 7·5 years. Major cardiovascular events were more common among those with low levels of education in all types of country studied, <em>b</em>ut much more so in low-income countries. After adjustment for wea<em>lt</em>h and other factors, the HR (low level of education vs high level of education) was 1·23 (95% CI 0·96-1·58) for high-income countries, 1·59 (1·42-1·78) in middle-income countries, and 2·23 (1·79-2·77) in low-income countries (p<su<em>b</em>)interaction</su<em>b</em>)&<em>lt</em>;0·0001). We o<em>b</em>served similar resu<em>lt</em>s for all-cause mortality, with HRs of 1·50 (1·14-1·98) for high-income countries, 1·80 (1·58-2·06) in middle-income countries, and 2·76 (2·29-3·31) in low-income countries (p<su<em>b</em>)interaction</su<em>b</em>)&<em>lt</em>;0·0001). By contrast, we found no or weak associations <em>b</em>etween wea<em>lt</em>h and these two outcomes. Differences in outcomes <em>b</em>etween educational groups were not explained <em>b</em>y differences in risk factors, which decreased as the level of education increased in high-income countries, <em>b</em>ut increased as the level of education increased in low-income countries (p<su<em>b</em>)interaction</su<em>b</em>)&<em>lt</em>;0·0001). Medical care (eg, management of hypertension, dia<em>b</em>etes, and secondary prevention) seemed to play an important part in adverse cardiovascular disease outcomes <em>b</em>ecause such care is likely to <em>b</em>e poorer in people with the lowest levels of education compared to those with higher levels of education in low-income countries; however, we o<em>b</em>served less marked differences in care <em>b</em>ased on level of education in middle-income countries and no or minor differences in high-income countries.</p><A<em>b</em>stractText>A<em>lt</em>hough people with a lower level of education in low-income and middle-income countries have higher incidence of and mortality from cardiovascular disease, they have <em>b</em>etter overall risk factor profiles. However, these individuals have markedly poorer hea<em>lt</em>h care. Policies to reduce hea<em>lt</em>h inequities glo<em>b</em>ally must include strategies to overcome <em>b</em>arriers to care, especially for those with lower levels of education.</A<em>b</em>stractText><A<em>b</em>stractText>Full funding sources are listed at the end of the paper (see Acknowledgments).</A<em>b</em>stractText>

<A<em>b</em>stractText>Genetic factors associated with non-alcoholic fatty liver disease (NAFLD) remain incompletely understood. To date, most GWAS studies have adopted radiologically assessed hepatic triglyceride content as reference phenotype and so cannot address steatohepatitis or fi<em>b</em>rosis. We descri<em>b</em>e a genome-wide association study (GWAS) encompassing the full spectrum of histologically characterized NAFLD.</A<em>b</em>stractText><A<em>b</em>stractText>The GWAS involved 1483 European NAFLD cases and 17781 genetically-matched population controls. A replication cohort of 559 NAFLD cases and 945 controls was genotyped to confirm signals showing genome-wide or close to genome-wide significance.</A<em>b</em>stractText><p><div>(<em>b</em>)RESULTS</<em>b</em>)</div>Case-control analysis identified signals showing p-values ≤ 5 x 10^-8 at four locations (chromosome (chr) 2 GCKR/C2ORF16; chr4 HSD17B13; chr19 TM6SF2; chr22 PNPLA3) together with two other signals with p&<em>lt</em>;1 x10^-7 (chr1 near LEPR and chr8 near IDO2/TC1). Case-only analysis of quantitative traits steatosis, disease activity score, NAS and fi<em>b</em>rosis showed that the PNPLA3 signal (rs738409) was genome-wide significantly associated with steatosis, fi<em>b</em>rosis and NAS score and identified a new signal (PYGO1 rs62021874) with close to genome-wide significance for steatosis (p=8.2 x 10^-8). Su<em>b</em>group case-control analysis for NASH confirmed the PNPLA3 signal. The chr1 LEPR SNP also showed genome-wide significance for this phenotype. Considering the su<em>b</em>group with advanced fi<em>b</em>rosis (≥F3), the signals on chromosomes 2, 19 and 22 remained genome-wide significant. With the exception of GCKR/C2ORF16, the genome-wide significant signals replicated.</p><A<em>b</em>stractText>This study confirms PNPLA3 as a risk factor for the full histological spectrum of NAFLD at genome-wide significance levels, with important contri<em>b</em>utions from TM6SF2 and HSD17B13. PYGO1 is a novel steatosis modifier, suggesting relevance of Wnt signalling pathways in NAFLD pathogenesis.</A<em>b</em>stractText>

(<em>b</em>)Introduction:</<em>b</em>) COVID-19 Ag Respi-Strip, an immunochromatographic (ICT) assay for the rapid detection of SARS-CoV-2 antigen on nasopharyngeal specimen, has <em>b</em>een developed to identify positive COVID-19 patients allowing prompt clinical and quarantine decisions. In this original research article, we descri<em>b</em>e the conception, the analytical and clinical performances as well as the risk management of implementing the COVID-19 Ag Respi-Strip in a diagnostic decision algorithm. (<em>b</em>)Materials and Methods:</<em>b</em>) Development of the COVID-19 Ag Respi-Strip resu<em>lt</em>ed in a ready-to-use ICT assay <em>b</em>ased on a mem<em>b</em>rane technology with colloidal gold nanoparticles using monoclonal anti<em>b</em>odies directed against the SARS-CoV and SARS-CoV-2 highly conserved nucleoprotein antigen. Four hundred o<em>b</em>servations were recorded for the analytical performance study and thirty tests were analyzed for the cross-reactivity study. The clinical performance study was performed in a retrospective mu<em>lt</em>i-centric evaluation on aliquots of 328 nasopharyngeal samples. COVID-19 Ag Respi-Strip resu<em>lt</em>s were compared with qRT-PCR as golden standard for COVID-19 diagnostics. (<em>b</em>)Resu<em>lt</em>s:</<em>b</em>) In the analytical performance study, the reproduci<em>b</em>ility showed a <em>b</em>etween-o<em>b</em>server disagreement of 1.7%, a ro<em>b</em>ustness of 98%, an overall satisfying user friendliness and no cross-reactivity with other virus-infected nasopharyngeal samples. In the clinical performance study performed in three different clinical la<em>b</em>oratories during the ascendant phase of the epidemiological curve, we found an overall sensitivity and specificity of 57.6 and 99.5%, respectively with an accuracy of 82.6%. The cut-off of the ICT was found at CT &<em>lt</em>;22. User-friendliness analysis and risk management assessment through Ishikawa diagram demonstrate that COVID-19 Ag Respi-Strip may <em>b</em>e implemented in clinical la<em>b</em>oratories according to <em>b</em>iosafety recommendations. (<em>b</em>)Conclusion:</<em>b</em>) The COVID-19 Ag Respi-Strip represents a promising rapid SARS-CoV-2 antigen assay for the first-line diagnosis of COVID-19 in 15 min at the peak of the pandemic. Its role in the proposed diagnostic algorithm is complementary to the currently-used molecular techniques.

Keywords: COVID-19; SARS-CoV-2; antigen; diagnostic; immunochromatographic test.

(<em>b</em>)Purpose:</<em>b</em>) Studies on long-term sustaina<em>b</em>ility of low-car<em>b</em>ohydrate approaches to treat dia<em>b</em>etes are limited. We previously reported the effectiveness of a novel digitally-monitored continuous care intervention (CCI) including nutritional ketosis in improving weight, glycemic outcomes, lipid, and liver marker changes at 1 year. Here, we assess the effects of the CCI at 2 years. (<em>b</em>)Materials and methods:</<em>b</em>) An open la<em>b</em>el, non-randomized, controlled study with 262 and 87 participants with T2D were enrolled in the CCI and usual care (UC) groups, respectively. Primary outcomes were retention, glycemic control, and weight changes at 2 years. Secondary outcomes included changes in <em>b</em>ody composition, liver, cardiovascular, kidney, thyroid and inflammatory markers, dia<em>b</em>etes medication use and disease status. (<em>b</em>)Resu<em>lt</em>s:</<em>b</em>) Reductions from <em>b</em>aseline to 2 years in the CCI group resu<em>lt</em>ing from intent-to-treat analyses included: H<em>b</em>A1c, fasting glucose, fasting insulin, weight, systolic <em>b</em>lood pressure, diastolic <em>b</em>lood pressure, triglycerides, and liver alanine transaminase, and HDL-C increased. Spine <em>b</em>one mineral density in the CCI group was unchanged. Use of any glycemic control medication (excluding metformin) among CCI participants declined (from 55.7 to 26.8%) including insulin (-62%) and sulfonylureas (-100%). The UC group had no changes in these parameters (except uric acid and anion gap) or dia<em>b</em>etes medication use. There was also resolution of dia<em>b</em>etes (reversal, 53.5%; remission, 17.6%) in the CCI group <em>b</em>ut not in UC. All the reported improvements had <i>p</i> &<em>lt</em>; 0.00012. (<em>b</em>)Conclusion:</<em>b</em>) The CCI group sustained long-term <em>b</em>eneficial effects on mu<em>lt</em>iple clinical markers of dia<em>b</em>etes and cardiometa<em>b</em>olic hea<em>lt</em>h at 2 years while utilizing less medication. The intervention was also effective in the resolution of dia<em>b</em>etes and visceral o<em>b</em>esity with no adverse effect on <em>b</em>one hea<em>lt</em>h. (<em>b</em>)Clinical Trial Registration:</<em>b</em>) Clinica<em>lt</em>rials.gov NCT02519309.

<A<em>b</em>stractText>The presence of tumor-infi<em>lt</em>rating lymphocytes (TILs) is associated with improved survival in head and neck squamous cell carcinoma. However, the prognostic value of TILs remains unclear in oral squamous cell carcinoma (OSCC).</A<em>b</em>stractText><p><div>(<em>b</em>)METHODS</<em>b</em>)</div>We evaluated the associations <em>b</em>etween tumor-infi<em>lt</em>rating CD8⁺ T-cell density and survival in five distinct compartments in 139 OSCC cases.</p><p><div>(<em>b</em>)RESULTS</<em>b</em>)</div>There was a significant association <em>b</em>etween increased tumor-infi<em>lt</em>rating CD8⁺ T cells and their distri<em>b</em>ution. High parenchymal CD8⁺ T-cell density at the invading tumor edge was associated with improved overall survival (OS) and disease-specific survival (DSS; P &<em>lt</em>; 0.01 and P &<em>lt</em>; 0.01, respectively). High stromal CD8⁺ T-cell density at the tumor periphery was also associated with improved recurrence-free survival (RFS; P &<em>lt</em>; 0.01). Cox regression analysis revealed that high stromal CD8⁺ T-cell density at the tumor periphery and high parenchymal CD8⁺ T-cell density at the invading edge were independent prognostic makers (hazard ratio: 0.38 and 0.19, 95% confidence interval, 0.18-0.80 and 0.05-0.72, P = 0.01 and 0.01, respectively) for RFS and OS, respectively.</p><p><div>(<em>b</em>)CONCLUSIONS</<em>b</em>)</div>Assessment of CD8⁺ T cells at the parenchyma of the invading edge and peripheral stroma provides an indicator of tumor recurrence and prognosis.</p>

Background: Ofatumumab, a subcutaneous anti-CD20 monoclonal antibody, selectively depletes B cells. Teriflunomide, an oral inhibitor of pyrimidine synthesis, reduces T-cell and B-cell activation. The relative effects of these two drugs in patients with multiple sclerosis are not known.

Methods: In two double-blind, double-dummy, phase 3 trials, we randomly assigned patients with relapsing multiple sclerosis to receive subcutaneous ofatumumab (20 mg every 4 weeks after 20-mg loading doses at days 1, 7, and 14) or oral teriflunomide (14 mg daily) for up to 30 months. The primary end point was the annualized relapse rate. Secondary end points included disability worsening confirmed at 3 months or 6 months, disability improvement confirmed at 6 months, the number of gadolinium-enhancing lesions per T1-weighted magnetic resonance imaging (MRI) scan, the annualized rate of new or enlarging lesions on T2-weighted MRI, serum neurofilament light chain levels at month 3, and change in brain volume.

Results: Overall, 946 patients were assigned to receive ofatumumab and 936 to receive teriflunomide; the median follow-up was 1.6 years. The annualized relapse rates in the ofatumumab and teriflunomide groups were 0.11 and 0.22, respectively, in trial 1 (difference, -0.11; 95% confidence interval [CI], -0.16 to -0.06; P<0.001) and 0.10 and 0.25 in trial 2 (difference, -0.15; 95% CI, -0.20 to -0.09; P<0.001). In the pooled trials, the percentage of patients with disability worsening confirmed at 3 months was 10.9% with ofatumumab and 15.0% with teriflunomide (hazard ratio, 0.66; P = 0.002); the percentage with disability worsening confirmed at 6 months was 8.1% and 12.0%, respectively (hazard ratio, 0.68; P = 0.01); and the percentage with disability improvement confirmed at 6 months was 11.0% and 8.1% (hazard ratio, 1.35; P = 0.09). The number of gadolinium-enhancing lesions per T1-weighted MRI scan, the annualized rate of lesions on T2-weighted MRI, and serum neurofilament light chain levels, but not the change in brain volume, were in the same direction as the primary end point. Injection-related reactions occurred in 20.2% in the ofatumumab group and in 15.0% in the teriflunomide group (placebo injections). Serious infections occurred in 2.5% and 1.8% of the patients in the respective groups.

Conclusions: Among patients with multiple sclerosis, ofatumumab was associated with lower annualized relapse rates than teriflunomide. (Funded by Novartis; ASCLEPIOS I and II ClinicalTrials.gov numbers, NCT02792218 and NCT02792231.).