<AbstractText>To describe the clinical profiles and risk factors for critical illness in hospitalized children and adolescents with COVID-19.</AbstractText><AbstractText>Children 1 month to 21 years with COVID-19 from a single tertiary care children's hospital between March 15-April 13, 2020 were included. Demographic and clinical data were collected.</AbstractText><AbstractText>67 children tested positive for COVID-19; 21 (31.3%) were managed as outpatients. Of 46 admitted patients, 33 (72%) were admitted to the general pediatric medical unit and 13 (28%) to the pediatric intensive care unit (PICU). Obesity and asthma were highly prevalent but not significantly associated with PICU admission (p=0.99). Admission to the PICU was significantly associated with higher C-reactive protein, procalcitonin, and pro-<em>B</em> type natriuretic peptide levels and platelet counts (p&<em>lt</em>;0.05 for all). Patients in the PICU were more likely to require high-flow nasal cannula (p=0.0001) and were more likely to have received Remdesivir through compassionate release (p&<em>lt</em>;0.05). Severe sepsis and septic shock syndromes were observed in 7 (53.8%) PICU patients. Acute respiratory distress syndrome (ARDS) was observed in 10 (77%) PICU patients, 6 of whom (46.2%) required invasive mechanical ventilation for a median of 9 days. Of the 13 patients in the PICU, 8 (61.5%) were discharged home, and 4 (30.7%) patients remain hospitalized on ventilatory support at day 14. One patient died after withdrawal of life-sustaining therapy because of metastatic cancer.</AbstractText><AbstractText>We describe a higher than previously recognized rate of severe disease requiring PICU admission in pediatric patients admitted to the hospital with COVID-19.</AbstractText>

Even though enterotoxin-producing Escherichia coli (ETEC) is the most important cause of diarrhoea in developing countries and among travellers, no vaccine for use in humans is yet available. New knowledge about virulence factors and protective antigens of ETEC, however, suggests that development of a useful vaccine may soon become possible. Such a vaccine should be given orally and ideally evoke both anticolonization and antitoxic immune responses in the gut. An oral cholera vaccine, containing a component (B subunit) which crossreacts immunologically with the major, heat-labile enterotoxin (LT) of ETEC, has been shown to afford significant protection against diarrhoea caused by LT-producing ETEC. Promising prototype oral ETEC vaccines combining B subunit toxoid with inactivated ETEC bacteria expressing the most prevalent colonization factor antigens (CFAs) have been developed, and work is in progress to find means for adding to this CFA-toxoid vaccine a component that could also provide immunity against heat-stable enterotoxin.

During embryogenesis, the Peyer's patch anlagen are induced by a cell population that produces lymphotoxin (LT) alpha(1)beta(2) following stimulation of IL-7Ralpha. In this study, we show that the LT-producing cell is localized within the IL-7Ralpha(+) and integrin alpha(4)beta(7) (alpha(4)beta(7))(+) population in the embryonic intestine. Lineage commitment to the LT producer phenotype in the fetal liver coincides with expression of alpha(4)beta(7). Before expression of alpha(4)beta(7), the potential of IL-7Ralpha(+) population to generate B cells is lost. However, the progenitors for T cells and LT producer cells reside in the IL-7Ralpha(+)alpha(4)beta(7)(+) cells, but during subsequent differentiation, the potential to give rise to T cells is lost. This IL-7Ralpha(+)alpha(4)beta(7)(+) population migrates to the intestine, where it induces the Peyer's patch anlagen. When stimulated with IL-15 or IL-3 and TNF, the intestinal IL-7Ralpha(+)alpha(4)beta(7)(+) population can differentiate into fully competent NK1.1(+) NK cells or CD11c(+) APCs. Expression of alpha(4)beta(7) is lost during differentiation of both lineages; IL-7Ralpha expression is lost during NK1.1(+) cells differentiation. A newly discovered lineage(-)IL-7Ralpha(+)c-Kit(+)alpha(4)beta(7)(+) population in the fetal liver is committed to T, NK, dendritic, and fetal intestinal LT producer lineage, the latter being an intermediate stage during differentiation of NK and dendritic cells.

BACKGROUND

Ifakara tent traps (ITT) are currently the only sufficiently sensitive, safe, affordable and practical method for routine monitoring host-seeking mosquito densities in Dar es Salaam. However, it is not clear whether ITT catches represent indoors or outdoors biting densities. ITT do not yield samples of resting, fed mosquitoes for blood meal analysis.

METHODS

Outdoors mosquito sampling methods, namely human landing catch (HLC), ITT (Design B) and resting boxes (RB) were conducted in parallel with indoors sampling using HLC, Centers for Disease Control and Prevention miniature light traps (LT) and RB as well as window exit traps (WET) in urban Dar es Salaam, rotating them thirteen times through a 3 × 3 Latin Square experimental design replicated in four blocks of three houses. This study was conducted between 6th May and 2rd July 2008, during the main rainy season when mosquito biting densities reach their annual peak.

RESULTS

The mean sensitivities of indoor RB, outdoor RB, WET, LT, ITT (Design B) and HLC placed outdoor relative to HLC placed indoor were 0.01, 0.005, 0.036, 0.052, 0.374, and 1.294 for Anopheles gambiae sensu lato (96% An. gambiae s.s and 4% An. arabiensis), respectively, and 0.017, 0.053, 0.125, 0.423, 0.372 and 1.140 for Culex spp, respectively. The ITT (Design B) catches correlated slightly better to indoor HLC (r(2) = 0.619, P < 0.001, r(2) = 0.231, P = 0.001) than outdoor HLC (r(2) = 0.423, P < 0.001, r(2) = 0.228, P = 0.001) for An. gambiae s.l. and Culex spp respectively but the taxonomic composition of mosquitoes caught by ITT does not match those of the indoor HLC (χ(2) = 607.408, degrees of freedom = 18, P < 0.001). The proportion of An. gambiae caught indoors was unaffected by the use of an LLIN in that house.

CONCLUSIONS

The RB, WET and LT are poor methods for surveillance of malaria vector densities in urban Dar es Salaam compared to ITT and HLC but there is still uncertainty over whether the ITT best reflects indoor or outdoor biting densities. The particular LLIN evaluated here failed to significantly reduce house entry by An. gambiae s.l. suggesting a negligible repellence effect.

A primary obstacle to an HIV-1 cure is long-lived viral reservoirs, which must be eliminated or greatly reduced. Cure strategies have largely focused on monitoring changes in T cell reservoirs in peripheral blood (PB), even though the lymphoid tissues (LT) are primary sites for viral persistence. To track and discriminate viral reservoirs within tissue compartments we developed a specific and sensitive next-generation in situ hybridization approach to detect vRNA, including vRNA+ cells and viral particles ("RNAscope"), vDNA+ cells ("DNAscope") and combined vRNA and vDNA with immunohistochemistry to detect and phenotype active and latently infected cells in the same tissue section. RNAscope is highly sensitive with greater speed of analysis compared to traditional in situ hybridization. The highly sensitive and specific DNAscope detected SIV/HIV vDNA+ cells, including duplexed detection of vDNA and vRNA or immunophenotypic markers in the same section. Analysis of LT samples from macaques prior to and during combination antiretroviral therapy demonstrated that B cell follicles are an important anatomical compartment for both latent and active viral persistence during treatment. These new tools should allow new insights into viral reservoir biology and evaluation of cure strategies.

Thirteen-lined ground squirrels and other circannual hibernators undergo profound physiological changes on an annual basis, transitioning from summer homeothermy [body temperature (T(b)) approximately 37 degrees C] to winter heterothermy (T(b) cycling between 0 degrees C and 37 degrees C). We hypothesize that these physiological changes are reflected in biochemical changes that provide mechanistic insights into, and biomarkers for, hibernation states. Here we report the results of an NMR-based metabolomics analysis of liver extracts from ground squirrels in three distinct physiological states of circannual hibernation: summer active (SA), late torpor (LT), and reentering torpor (Ent) after one of the euthermic arousals. Of the 43 identified and quantified metabolites, 36 differed among these three states and fell into two patterns of variation: 1) SA differed from both of the two winter states; or 2) the two winter states differed from each other, but one of the two was not different from SA. Concentrations of hepatic glucose, lactate, alanine, succinate, beta-hydroxybutyrate, glutamine, and betaine were identified as robust hepatic biomarkers that together distinguish among animals in these three states of the circannual hibernation rhythm. These data are consistent with a proposed two-switch model of hibernation, in which setting the summer-winter switch to winter enables expression of a distinct torpor-arousal switch. The summer-winter switch is characterized by the metabolites associated with the well-known switch from carbohydrate to lipid fuel utilization during hibernation. The torpor-arousal switch is characterized by the accumulation of metabolites of nitrogen (glutamine) and phospholipid (betaine) catabolism in LT with the capacity to act as protective osmolytes.

<A<em>b</em>stractText>Short-term treatment for people with type 2 dia<em>b</em>etes using a low dose of the selective endothelin A receptor antagonist atrasentan reduces al<em>b</em>uminuria without causing significant sodium retention. We report the long-term effects of treatment with atrasentan on major renal outcomes.</A<em>b</em>stractText><p><div>(<em>b</em>)METHODS</<em>b</em>)</div>We did this dou<em>b</em>le-<em>b</em>lind, randomised, place<em>b</em>o-controlled trial at 689 sites in 41 countries. We enrolled adu<em>lt</em>s aged 18-85 years with type 2 dia<em>b</em>etes, estimated glomerular fi<em>lt</em>ration rate (eGFR) 25-75 mL/min per 1·73 m² of <em>b</em>ody surface area, and a urine al<em>b</em>umin-to-creatinine ratio (UACR) of 300-5000 mg/g who had received maximum la<em>b</em>elled or tolerated renin-angiotensin system inhi<em>b</em>ition for at least 4 weeks. Participants were given atrasentan 0·75 mg orally daily during an enrichment period <em>b</em>efore random group assignment. Those with a UACR decrease of at least 30% with no su<em>b</em>stantial fluid retention during the enrichment period (responders) were included in the dou<em>b</em>le-<em>b</em>lind treatment period. Responders were randomly assigned to receive either atrasentan 0·75 mg orally daily or place<em>b</em>o. All patients and investigators were masked to treatment assignment. The primary endpoint was a composite of dou<em>b</em>ling of serum creatinine (sustained for ≥30 days) or end-stage kidney disease (eGFR &<em>lt</em>;15 mL/min per 1·73 m² sustained for ≥90 days, chronic dialysis for ≥90 days, kidney transplantation, or death from kidney failure) in the intention-to-treat population of all responders. Safety was assessed in all patients who received at least one dose of their assigned study treatment. The study is registered with ClinicalTrials.gov, num<em>b</em>er NCT01858532.</p><A<em>b</em>stractText>Between May 17, 2013, and July 13, 2017, 11 087 patients were screened; 5117 entered the enrichment period, and 4711 completed the enrichment period. Of these, 2648 patients were responders and were randomly assigned to the atrasentan group (n=1325) or place<em>b</em>o group (n=1323). Median follow-up was 2·2 years (IQR 1·4-2·9). 79 (6·0%) of 1325 patients in the atrasentan group and 105 (7·9%) of 1323 in the place<em>b</em>o group had a primary composite renal endpoint event (hazard ratio [HR] 0·65 [95% CI 0·49-0·88]; p=0·0047). Fluid retention and anaemia adverse events, which have <em>b</em>een previously attri<em>b</em>uted to endothelin receptor antagonists, were more frequent in the atrasentan group than in the place<em>b</em>o group. Hospital admission for heart failure occurred in 47 (3·5%) of 1325 patients in the atrasentan group and 34 (2·6%) of 1323 patients in the place<em>b</em>o group (HR 1·33 [95% CI 0·85-2·07]; p=0·208). 58 (4·4%) patients in the atrasentan group and 52 (3·9%) in the place<em>b</em>o group died (HR 1·09 [95% CI 0·75-1·59]; p=0·65).</A<em>b</em>stractText><A<em>b</em>stractText>Atrasentan reduced the risk of renal events in patients with dia<em>b</em>etes and chronic kidney disease who were selected to optimise efficacy and safety. These data support a potential role for selective endothelin receptor antagonists in protecting renal function in patients with type 2 dia<em>b</em>etes at high risk of developing end-stage kidney disease.</A<em>b</em>stractText><A<em>b</em>stractText>A<em>b</em><em>b</em>Vie.</A<em>b</em>stractText>

The galactose-binding site in cholera toxin and the closely related heat-labile enterotoxin (LT) from Escherichia coli is an attractive target for the rational design of potential anti-cholera drugs. In this paper we analyse the molecular structure of this binding site as seen in several crystal structures, including that of an LT:galactose complex which we report here at 2.2 A resolution. The binding surface on the free toxin contains several tightly associated water molecules and a relatively flexible loop consisting of residues 51-60 of the B subunit. During receptor binding this loop becomes tightly ordered by forming hydrogen bonds jointly to the GM1 pentasaccharide and to a set of water molecules which stabilize the toxin:receptor complex.

Alzheimer's disease (AD) is the most prevalent form of neurodegeneration; however, therapies to prevent or treat AD are inadequate. Amyloid-beta (Abeta) protein accrues in cortical senile plaques, one of the key neuropathological hallmarks of AD, and is elevated in brains of early onset AD patients in a small number of families that bear certain genetic mutations, further implicating its role in this devastating neurological disease. In addition, soluble Abeta oligomers have been shown to be detrimental to neuronal function. Therapeutic strategies aimed at lowering cerebral Abeta levels are currently under development. One strategy is to immunize AD patients with Abeta peptides so that they will generate antibodies that bind to Abeta protein and enhance its clearance. As of 1999, Abeta immunotherapy, either through active immunization with Abeta peptides or through passive transfer of Abeta-specific antibodies, has been shown to reduce cerebral Abeta levels and improve cognitive deficits in AD mouse models and lower plaque load in nonhuman primates. However, a Phase II clinical trial of active immunization using full-length human Abetabeta peptide as a self-protein may have induced an adverse autoimmune response in these patients. Although only approximately 20% of immunized patients generated anti-Abeta titers, responders showed some general slowing of cognitive decline. Focal cortical regions devoid of Abeta plaques were observed in brain tissues of several immunized patients who have since come to autopsy. In order to avoid a deleterious immune response, passive Abeta immunotherapy is under investigation by administering monthly intravenous injections of humanized Abeta monoclonal antibodies to AD patients. However, a safe and effective active Abeta vaccine would be more cost-effective and more readily available to a larger AD population. We have developed several novel short Abeta immunogens that target the Abeta N-terminus containing a strong B cell epitope while avoiding the Abeta mid-region and C-terminus containing T cell epitopes. These immunogens include dendrimeric Abetabetabetabetabetabetabeta fragment immunogens and a mucosal adjuvant, mutant Escherichia coli heat-labile enterotoxin LT(R192G), resulted in reduced cerebral Abeta levels, plaque deposition, and gliosis, as well as increased plasma Abeta levels and improved cognition in a transgenic mouse model of AD. Preclinical trials in nonhuman primates, and human clinical trials using similar Abeta immunogens, are now underway. Abeta immunotherapy looks promising but must be made safer and more effective at generating antibody titers in the elderly. It is hoped that these novel immunogens will enhance Abeta antibody generation across a broad population and avoid the adverse events seen in the earlier clinical trial.

Previous studies suggest that the core protein of hepatitis C virus (HCV) has a pleiotropic function in the replication cycle of the virus. To understand the role of this protein in HCV pathogenesis, we used a yeast two-hybrid protein interaction cloning system to search for cellular proteins physically interacting with the HCV core protein. One such cellular gene was isolated and characterized as the gene encoding the lymphotoxin-beta receptor (LT-betaR). In vitro binding analysis demonstrated that the HCV core protein binds to the C-terminal 98 amino acids within the intracellular domain of the LT-betaR that is involved in signal transduction, although the binding affinity of the full-length HCV core protein was weaker than that of its C-terminally truncated form. Our results also indicated that the N-terminal 40-amino-acid segment of the HCV core protein was sufficient for interaction with LT-betaR and that the core protein could form complexes with the oligomeric form of the intracellular domain of LT-betaR, which is a prerequisite for downstream signaling of this receptor. Similar to other members of the tumor necrosis factor (TNF) receptor superfamily, LT-betaR is involved in the cytotoxic effect of the signaling pathway, and thus we have elucidated the biological consequence of interaction between the HCV core protein and LT-betaR. Our results indicated that in the presence of the synergizing agent gamma interferon, the HCV core protein enhances the cytotoxic effects of recombinant forms of LT-betaR ligand in HeLa cells but not in hepatoma cells. Furthermore, this enhancement of the cytolytic activity was cytokine specific, since in the presence of cycloheximide, the expression of the HCV core protein did not elicit an increase in the cytolytic activity of TNF in both HeLa and hepatoma cells. In summary, the HCV core protein can associate with LT-betaR, and this protein-protein interaction has a modulatory effect on the signaling pathway of LT-betaR in certain cell types. Given the known roles of LT-betaR/LT-alpha1,betaLT-betaR, resulting in pathogenesis in HCV-infected cells.

The 5' terminus of the gene that codes for the heat-stable enterotoxin of Escherichia coli (ST) was genetically fused to the 3' terminus of the gene that codes for the binding subunit of the heat-labile enterotoxin of E. coli (LT-B). The ST-encoding gene used for these studies was constructed synthetically with appropriate restriction sites to permit in-frame, downstream insertion of the oligomer. For this construction, maximum expression of ST antigenicity was obtained when a seven-amino-acid, proline-containing linker was included between the LT-B and ST moieties. The LT-B-ST fusion peptide was purified by affinity chromatography and consisted of a single polypeptide chain with an apparent molecular weight of 18,000 when examined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. There was no evidence of multimer formation and no change in the mobility of the fusion peptide when it was boiled in SDS or in SDS with dithiothreitol. The LT-B-ST fusion peptide was nontoxic, and immunologic determinants of both LT and ST were recognized by antibodies to the native toxins. More importantly, the LT-B-ST fusion peptide was immunogenic. Animals immunized with crude or purified preparations containing the hybrid molecule produced antibodies that were able to recognize native toxin in vitro. Significantly, these antibodies were able to neutralize the biological activity of native ST.

Lymphotoxin (LT) plays an important role in inflammation and lymphoid organ development, though the mechanisms by which it promotes these processes are poorly understood. Toward this end, the biologic activities of a recently generated recombinant murine (m) LT alpha preparation were evaluated. This cytokine preparation was effective at inducing cytotoxicity of WEHI target cells with 50% maximal killing observed with 1.2 ng/ml. mLT alpha also induced the expression of inflammatory mediators in the murine endothelial cell line bEnd.3. rmLT alpha induced expression of the adhesion molecules VCAM, ICAM, E-selectin, and the mucosal addressin cellular adhesion molecule, MAdCAM-1. When mLT alpha, human (h) LT alpha, and mTNF-alpha were compared, mLT alpha was the most potent inducer of MAdCAM-1. None of these cytokines induced the peripheral node addressin, PNAd. mLT alpha also induced expression of the chemokines RANTES, IFN-inducible protein 10 (IP-10), and monocyte chemotactic protein 1 (MCP-1). mRNA levels peaked 4 h following treatment with mLT alpha and declined through the 24-h treatment period. LT alpha also induced chemokine protein within 8 h of treatment, which increased through the 24-h treatment period. These data demonstrate that the proinflammatory effects of LT alpha3 may be mediated in part through the induction of adhesion molecule and chemokine expression. Further, LT alpha3 may promote development of lymphoid tissue through induction of chemokines and the mucosal addressin MAdCAM-1. These data confirm previous observations in transgenic and knockout mice that LT alpha3 in the absence of LT beta carries out unique biologic activities.

Rheumatoid arthritis (RA) is characterised by breach of self-tolerance towards citrullinated antigens with generation of anti-citrullinated peptide/proteins antibodies (ACPA). Currently, the nature and source of citrullinated antigens driving the humoral autoimmune response within synovial ectopic lymphoid structures (ELS) is a crucial unknown aspect of RA pathogenesis. Here we characterised the autoreactive B-cell response of lesional B cells isolated from ELS+RA synovium.

Single synovial tissue CD19+cells were Fluorescence Activated Cell Sorting (FACS)-sorted and VH/VL Ig genes cloned to generate recombinant monoclonal antibodies (rmAbs) from patients with ELS+/ACPA+RA.

RA-rmAbs immunoreactivity analysis provided the following key findings: (1) in a chIP-based array containing 300 autoantigens and in a 'citrullinome' multiplex assay, a strong reactivity against citrullinated histones H2A/H2B (citH2A/H2B) was observed in ∼40% of RA-rmAbs, followed by cit-fibrinogen and cit-vimentin; (2) anti-citH2A/H2B-reactive RA-rmAbs (but not anti-citH2A/H2B negative) selectively recognised neutrophil extracellular traps (NETs) from peripheral blood and/or RA joint neutrophils; (3) anti-citH2A/citH2B and anti-NET immunobinding was dependent on affinity maturation and was completely abrogated following reversion of hypermutated IgVH/VL genes to germline sequences; (4) ELS+ (not ELS-) RA synovial tissues engrafted into Severe Combined ImmunoDeficiency (SCID) mice released human anti-citH2A/citH2B and anti-NET antibodies in association with the intra-graft expression of CXCL13 and lymphotoxin (LT)-β, two master regulators of ELS.

We provided novel evidence that B cells differentiated within synovial ELS in the RA joints frequent target deiminated proteins which could be generated during NETosis of RA synovial neutrophils including histones. Thus, NETs could represent a source of citrullinated antigens fuelling the ACPA autoimmune response within the RA synovium.

<A<em>b</em>stractText><em>B</em>-cell anomalies play a role in the pathogenesis of mem<em>b</em>ranous nephropathy. <em>B</em>-cell depletion with rituxima<em>b</em> may therefore <em>b</em>e noninferior to treatment with cyclosporine for inducing and maintaining a complete or partial remission of proteinuria in patients with this condition.</A<em>b</em>stractText><p><div>(<em>b</em>)METHODS</<em>b</em>)</div>We randomly assigned patients who had mem<em>b</em>ranous nephropathy, proteinuria of at least 5 g per 24 hours, and a quantified creatinine clearance of at least 40 ml per minute per 1.73 m² of <em>b</em>ody-surface area and had <em>b</em>een receiving angiotensin-system <em>b</em>lockade for at least 3 months to receive intravenous rituxima<em>b</em> (two infusions, 1000 mg each, administered 14 days apart; repeated at 6 months in case of partial response) or oral cyclosporine (starting at a dose of 3.5 mg per kilogram of <em>b</em>ody weight per day for 12 months). Patients were followed for 24 months. The primary outcome was a composite of complete or partial remission of proteinuria at 24 months. La<em>b</em>oratory varia<em>b</em>les and safety were also assessed.</p><p><div>(<em>b</em>)RESULTS</<em>b</em>)</div>A total of 130 patients underwent randomization. At 12 months, 39 of 65 patients (60%) in the rituxima<em>b</em> group and 34 of 65 (52%) in the cyclosporine group had a complete or partial remission (risk difference, 8 percentage points; 95% confidence interval [CI], -9 to 25; P = 0.004 for noninferiority). At 24 months, 39 patients (60%) in the rituxima<em>b</em> group and 13 (20%) in the cyclosporine group had a complete or partial remission (risk difference, 40 percentage points; 95% CI, 25 to 55; P&<em>lt</em>;0.001 for <em>b</em>oth noninferiority and superiority). Among patients in remission who tested positive for anti-phospholipase A<su<em>b</em>)2</su<em>b</em>) receptor (PLA2R) anti<em>b</em>odies, the decline in autoanti<em>b</em>odies to anti-PLA2R was faster and of greater magnitude and duration in the rituxima<em>b</em> group than in the cyclosporine group. Serious adverse events occurred in 11 patients (17%) in the rituxima<em>b</em> group and in 20 (31%) in the cyclosporine group (P = 0.06).</p><A<em>b</em>stractText>Rituxima<em>b</em> was noninferior to cyclosporine in inducing complete or partial remission of proteinuria at 12 months and was superior in maintaining proteinuria remission up to 24 months. (Funded <em>b</em>y Genentech and the Fulk Family Foundation; MENTOR ClinicalTrials.gov num<em>b</em>er, NCT01180036.).</A<em>b</em>stractText>

<A<em>b</em>stractText>Patients with transplantation-ineligi<em>b</em>le relapsed/refractory (R/R) diffuse large <em>B</em>-cell lymphoma (DL<em>B</em>CL) fare poorly, with limited treatment options. The anti<em>b</em>ody-drug conjugate polatuzuma<em>b</em> vedotin targets CD79<em>b</em>, a <em>B</em>-cell receptor component.</A<em>b</em>stractText><A<em>b</em>stractText>Safety and efficacy of polatuzuma<em>b</em> vedotin with <em>b</em>endamustine and o<em>b</em>inutuzuma<em>b</em> (pola-<em>B</em>G) was evaluated in a single-arm cohort. Polatuzuma<em>b</em> vedotin com<em>b</em>ined with <em>b</em>endamustine and rituxima<em>b</em> (pola-<em>B</em>R) was compared with <em>b</em>endamustine and rituxima<em>b</em> (<em>B</em>R) in a randomly assigned cohort of patients with transplantation-ineligi<em>b</em>le R/R DL<em>B</em>CL (primary end point: independent review committee [IRC] assessed complete response [CR] rate at the end of treatment). Duration of response, progression-free survival (PFS), and overall survival (OS) were analyzed using Kaplan-Meier and Cox regression methods.</A<em>b</em>stractText><p><div>(<em>b</em>)RESULTS</<em>b</em>)</div>Pola-<em>B</em>G and pola-<em>B</em>R had a tolera<em>b</em>le safety profile. The phase I<em>b</em>/II pola-<em>B</em>G cohort (n = 27) had a CR rate of 29.6% and a median OS of 10.8 months (median follow-up, 27.0 months). In the randomly assigned cohort (n = 80; 40 per arm), pola-<em>B</em>R patients had a significantly higher IRC-assessed CR rate (40.0% <i>v</i> 17.5%; <i>P</i> = .026) and longer IRC-assessed PFS (median, 9.5 <i>v</i> 3.7 months; hazard ratio [HR], 0.36, 95% CI, 0.21 to 0.63; <i>P</i> &<em>lt</em>; .001) and OS (median, 12.4 <i>v</i> 4.7 months; HR, 0.42; 95% CI, 0.24 to 0.75; <i>P</i> = .002; median follow-up, 22.3 months). Pola-<em>B</em>R patients had higher rates of grade 3-4 neutropenia (46.2% <i>v</i> 33.3%), anemia (28.2% <i>v</i> 17.9%), and throm<em>b</em>ocytopenia (41% <i>v</i> 23.1%), <em>b</em>ut similar grade 3-4 infections (23.1% <i>v</i> 20.5%), versus the <em>B</em>R group. Peripheral neuropathy associated with polatuzuma<em>b</em> vedotin (43.6% of patients) was grade 1-2 and resolved in most patients.</p><A<em>b</em>stractText>Polatuzuma<em>b</em> vedotin com<em>b</em>ined with <em>B</em>R resu<em>lt</em>ed in a significantly higher CR rate and reduced the risk of death <em>b</em>y 58% compared with <em>B</em>R in patients with transplantation-ineligi<em>b</em>le R/R DL<em>B</em>CL.</A<em>b</em>stractText>

Cholera toxin and heat-labile enterotoxin (LT) are structurally similar oligomeric proteins which are capable of being efficiently secreted from Vibrio cholerae. Here we report that these proteins transiently enter the periplasm of V. cholerae as they traverse the cell envelope to reach the extracellular milieu. Pulse-chase experiments on V. cholerae TRH7000 harboring an LT-encoding plasmid revealed that radiolabeled LT A and B subunits entered the periplasm rapidly, followed by their slow efflux (half-time, 13 min) into the medium. LT B-subunit efflux from the periplasm was calculated to be at a rate of ca. 170 monomers per min per cell (which is equivalent to 34 assembled LT holotoxin molecules per min per cell). These values were estimated to be sufficient to account for the increase in extracellular enterotoxin concentration during exponential cell growth. Thus, all enterotoxin subunits which are secreted into the medium can be assumed to be channelled via the periplasm. These findings led to an improved model of the pathway of toxin secretion by V. cholerae.

Escherichia coli enterotoxin (LT) and the homologous cholera toxin (CT) are A-B toxins that cause travelers' diarrhea and cholera, respectively. So far, experimental live and killed vaccines against these diseases have been developed using only the nontoxic B portion of these toxins. The enzymatically active A subunit has not been used because it is responsible for the toxicity and it is reported to induce a negligible titer of toxin neutralizing antibodies. We used site-directed mutagenesis to inactivate the ADP-ribosyltransferase activity of the A subunit and obtained nontoxic derivatives of LT that elicited a good titer of neutralizing antibodies recognizing the A subunit. These LT mutants and equivalent mutants of CT may be used to improve live and killed vaccines against cholera and enterotoxinogenic E. coli.

<A<em>b</em>stractText>CALGB/SWOG 80405 was a randomized phase III trial that found no statistically significant difference in overall survival (OS) in patients with first-line metastatic colorectal cancer treated with chemotherapy plus either <em>b</em>evacizuma<em>b</em> or cetuxima<em>b</em>. Primary tumor DNA from 843 patients has <em>b</em>een used to discover genetic markers of OS.</A<em>b</em>stractText><A<em>b</em>stractText>Gene mutations were determined <em>b</em>y polymerase chain reaction. Microsatellite status was determined <em>b</em>y genotyping of microsatellites. Tumor mutational <em>b</em>urden (TMB) was determined <em>b</em>y next-generation sequencing. Cox proportional hazard models were used, with adjusting factors. Interaction of molecular a<em>lt</em>erations with either the <em>b</em>evacizuma<em>b</em> or the cetuxima<em>b</em> arms was tested.</A<em>b</em>stractText><p><div>(<em>b</em>)RESULTS</<em>b</em>)</div>Patients with high TMB in their tumors had longer OS than did patients with low TMB (hazard ratio [HR], 0.73 [95% CI, 0.57 to 0.95]; <i>P</i> = .02). In patients with microsatellite insta<em>b</em>ility-high (MSI-H) tumors, longer OS was o<em>b</em>served in the <em>b</em>evacizuma<em>b</em> arm than in the cetuxima<em>b</em> arm (HR, 0.13 [95% CI, 0.06 to 0.30]; interaction <i>P</i> &<em>lt</em>; .001 for interaction <em>b</em>etween microsatellite status and the two arms). Patients with <i>BRAF</i> mutant tumors had shorter OS than did patients with wild-type (WT) tumors (HR, 2.01 [95% CI, 1.49 to 2.71]; <i>P</i> &<em>lt</em>; .001). Patients with extended <i>RAS</i> mutant tumors had shorter OS than did patients with WT tumors (HR, 1.52 [95% CI, 1.26 to 1.84]; <i>P</i> &<em>lt</em>; .001). Patients with triple-negative tumors (WT for <i>NRAS</i>/<i>KRAS</i>/<i>BRAF</i>) had a median OS of 35.9 months (95% CI, 33.0 to 38.8 months) versus 22.2 months (95% CI, 19.6 to 24.4 months ) in patients with at least one mutated gene in their tumors (<i>P</i> &<em>lt</em>; .001).</p><p><div>(<em>b</em>)CONCLUSION</<em>b</em>)</div>In patients with metastatic colorectal cancer treated in first line, low TMB, and <i>BRAF</i> and <i>RAS</i> mutations are negative prognostic factors. Patients with MSI-H tumors <em>b</em>enefited more from <em>b</em>evacizuma<em>b</em> than from cetuxima<em>b</em>, and studies to confirm this effect of MSI-H are warranted.</p>

Lymphotoxin-α (LTα), lymphotoxin-β (LTβ), and tumor necrosis factor-α (TNFα) are inflammatory mediators that play crucial roles in lymphoid organ development. We demonstrate here that LTα also contributes to the function of lymphatic vessels and to lymphangiogenesis during inflammation. LTα(-/-) mice exhibited reduced lymph flow velocities and increased interstitial fluid pressure. Airways of LTβ(-/-) mice infected with Mycoplasma pulmonis had significantly more lymphangiogenesis than wild type (WT) or LTα(-/-) mice, as did the skin draining immunization sites of LTβ(-/-) mice. Macrophages, B cells, and T cells, known sources of LT and TNFα, were apparent in the skin surrounding the immunization sites as were LTα, LTβ, and TNFα mRNAs. Ectopic expression of LTα led to the development of LYVE-1 and Prox1-positive lymphatic vessels within tertiary lymphoid organs (TLOs). Quantification of pancreatic lymphatic vessel density in RIPLTαLTβ(-/-) and WT mice revealed that LTα was sufficient for inducing lymphangiogenesis and that LTβ was not required for this process. Kidneys of inducible LTα transgenic mice developed lymphatic vessels before the appearance of obvious TLOs. These data indicate that LTα plays a significant role in lymphatic vessel function and in inflammation-associated lymphangiogenesis.

Cholera and enterotoxigenic Escherichia coli (ETEC) are among the most common causes of acute infantile gastroenteritis globally. We previously developed a rice-based vaccine that expressed cholera toxin B subunit (MucoRice-CTB) and had the advantages of being cold chain-free and providing protection against cholera toxin (CT)-induced diarrhea. To advance the development of MucoRice-CTB for human clinical application, we investigated whether the CTB-specific secretory IgA (SIgA) induced by MucoRice-CTB gives longstanding protection against diarrhea induced by Vibrio cholerae and heat-labile enterotoxin (LT)-producing ETEC (LT-ETEC) in mice. Oral immunization with MucoRice-CTB stored at room temperature for more than 3 y provided effective SIgA-mediated protection against CT- or LT-induced diarrhea, but the protection was impaired in polymeric Ig receptor-deficient mice lacking SIgA. The vaccine gave longstanding protection against CT- or LT-induced diarrhea (for>> or = 6 months after primary immunization), and a single booster immunization extended the duration of protective immunity by at least 4 months. Furthermore, MucoRice-CTB vaccination prevented diarrhea in the event of V. cholerae and LT-ETEC challenges. Thus, MucoRice-CTB is an effective long-term cold chain-free oral vaccine that induces CTB-specific SIgA-mediated longstanding protection against V. cholerae- or LT-ETEC-induced diarrhea.

BACKGROUND

Cholera toxin from Vibrio cholerae and the type I heat-labile enterotoxins (LT-Is) from Escherichia coli are oligomeric proteins with ABLT-IIs) from E. coli are structurally similar to, but antigenically distinct from, the type I enterotoxins. The A subunits of type I and type II enterotoxins are homologous and activate adenylate cyclase by ADP-ribosylation of a G protein subunit, G8 alpha. However, the B subunits of type I and type II enterotoxins differ dramatically in amino acid sequence and ganglioside-binding specificity. The structure of LT-IIb was determined both as a prototype for other LT-IIs and to provide additional insights into structure/function relationships among members of the heat-labile enterotoxin family and the superfamily of ADP-ribosylating protein toxins.

RESULTS

The 2.25 A crystal structure of the LT-IIb holotoxin has been determined. The structure reveals striking similarities with LT-I in both the catalytic A subunit and the ganglioside-binding B subunits. The latter form a pentamer which has a central pore with a diameter of 10-18 A. Despite their similarities, the relative orientation between the A polypeptide and the B pentamer differs by 24 degrees in LT-I and LT-IIb. A common hydrophobic ring was observed at the A-BBLT-I and cholera toxin, possibly involved in assembly, is also present in LT-IIb. The ganglioside receptor binding sites are localized, as suggested by mutagenesis, and are in a position roughly similar to the sites where LT-I binds its receptor.

CONCLUSIONS

The structure of <em>LT</em>-IIb provides insight into the sequence diversity and structural similarity of the A<em>B</em>5 toxin family. New knowledge has been gained regarding the assembly of A<em>B</em>5 toxins and their active-site architecture.

Epoxy- and dihydroxy-eicosatrienoic acids (EETs and DHETs) are vasoactive cytochrome P450 metabolites of arachidonic acid. Interestingly, however, the mechanism(s) by which EETs/DHETs mediate smooth muscle relaxation remains unclear. In contrast to previous reports, where dilation was purportedly large-conductance Ca(2+)-activated K(+) (BK(Ca)) and/or transient receptor potential cation channel, subfamily V, member 4 (TRPV4) channel-mediated, 14,15-EET-induced vasodilation [reversal of contractile tone established with the thromboxane receptor (TP) agonist 15-hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic acid (U-46619)] was unaltered in BK(Ca) and TRPV4 knockout mouse isolated aortae compared with wild-type controls, indicating a significant BK(Ca)/TRPV4-resistant mechanism. Whereas all EET and DHET regioisomers reversed U-46619 contraction in rat aortae and mouse mesenteric resistance arteries, these eicosanoids failed to alter phenylephrine-induced contraction, suggesting that they mediated dilation via a "TP-selective" mechanism. Competitive TP antagonism was also observed in nonvascular tissue, including rat fundus and tertiary bronchus, indicating that the effect is not specific to blood vessels. Such effects were TP-selective because 14,15-EET failed to inhibit "non-TP" prostanoid receptor-mediated function in multiple cell/tissue-based assays (K(b)>> 10 microM). In accordance, 14,15-EET inhibited specific [(3)H]7-(3-((2-((phenylamino)carbonyl)hydrazino)-methyl)-7-oxabicyclo(2.2.1)hept-2-yl)-5-heptenoic acid (SQ-29548) binding to human recombinant TP receptor, with a K(i) value of 3.2 microM, and it showed weaker affinity for non-TP prostanoid receptors, including DP, FP, EP(1-4), and IP receptors (K(i) values of 6.1, 5.3, 42.6, 19.7, 13.2, 20.2, and >25 microM, respectively) and no appreciable affinity (K(i) values >10 microM) for a diverse array of pharmacologically distinct receptors, including the leukotriene receptors Cys-LT(1/2) and BLT(1). As such, EETs/DHETs represent a unique class of "endogenous" G protein-coupled receptor competitive antagonists, inducing vasodilation via direct TP inhibition. Thus, EETs/DHETs represent novel autoregulatory agents, directly modulating the actions of cyclooxygenase-derived eicosanoids following arachidonic acid mobilization.

Aging is associated with an inability to mount protective antibody responses to vaccines and infectious agents. This decline is associated with acquisition of defects in multiple cellular compartments, including B cells. While peripheral B-cell numbers do not decline with aging, the composition of the compartment appears to change, with loss of naïve follicular B cells, accumulation of antigen-experienced cells, and alteration of the antibody repertoire. The underlying cause of this change is unknown. We tested the hypothesis that aging-associated repertoire changes can be attributed directly to decreased B lymphopoiesis. Using an Ig transgenic model to report changes in the B-cell repertoire, we show that the reduced B-cell generative capacity of "aged" long-term reconstituting hematopoietic stem cells (LT-HSCs) alters the representation of antigen specificities in the peripheral B-cell repertoire. Further, we show that reconstitution using suboptimal numbers of fully functional LT-HSCs results in the generation of a similarly altered B-cell repertoire. This may be an important factor to consider when deciding the number of bone marrow cells to transplant in the clinical setting. In conclusion, when B lymphopoiesis is limited peripheral B-cell homeostasis is altered. This is reflected in reduced diversity of the B-cell repertoire, which likely reduces the protective quality of the immune response.

We examined changes in muscle buffer capacity (beta m(in vitro)), VO2peak and the lactate threshold (LT) after 5 weeks of high-intensity interval training (INT) above the LT or moderate-intensity continuous training (CON) just below the LT. Prior to and immediately after training, 16 female subjects performed a graded exercise test to determine VO2peak and the LT, followed 2 days later by a resting muscle biopsy from the vastus lateralis muscle to determine beta m(in vitro). Following baseline testing, the subjects were randomly placed into the INT (n=8) or CON training group (n=8). Subjects then performed 5 weeks of cycle training (3 days per week), performing either high-intensity INT (6-10x2 min at 120-140% LT with 1 min rest) or moderate-intensity CON (80-95% LT) training. Total training volume was matched between the two groups. After the training period, both groups had significant improvements in VO2peak (12-14%; P<0.05) and the LT (7-10%; P<0.05), with no significant differences between groups. The INT group, however, had significantly greater improvements in beta m(in vitro) (25%; 123+/-5-153+/-7 micromol H+ x g muscle dm(-1) x pH(-1); P<0.05) than the CON group (2%; 130+/-12-133+/-7 micromol H+ x g muscle dm(-1) x pH(-1), P>0.05). Our results show that when matched for training volume, high-intensity interval training above the LT results in similar improvements in VO2peak and the LT, but greater improvements in beta m(in vitro) than moderate-intensity continuous training below the LT. This suggests that training intensity is an important determinant of changes to beta m(in vitro).