In comparison to secondary lymphoid organs, gut-associated lymphoid tissues such as isolated lymphoid follicles (ILF) and cryptopatches (CP) have been less intensively studied. To gain a better insight into processes regulating organization and function of these structures, which are believed to participate in immune responses and extrathymic T cell development, we characterized the lymphoid structures of the murine small intestine in more detail. The size and cellular composition of small intestinal lymphoid aggregations were analyzed in C57BL/6 and BALB/c wild-type and lymphotoxin (LT)-deficient mice, by flow cytometry, histology and automated multi-color immunofluorescence microscopy evaluating large coherent areas of the intestine. These evaluations demonstrate that aggregated lymphoid structures in the small intestine vary in size and cellular composition, with a majority of structures not matching the current definitions of CP or ILF. Accordingly, significant variations depending on species, age and mouse strain were observed. Furthermore, small bowel transplantation revealed a rapid exchange of B but not T cells between host and grafted tissue. Moreover, LT-deficient animals lack any intestinal lymphoid aggregations yet possess the complete panel of intraepithelial lymphocytes (IEL). In summary, our observations disclose intestinal lymphoid aggregations as dynamic structures with a great deal of inborn plasticity and demonstrate their dispensability for the generation of IEL.

(<em>b</em>)Rationale:</<em>b</em>) One important concern during high-flow nasal cannula (HFNC) therapy in patients with acute hypoxemic respiratory failure is to not delay intu<em>b</em>ation. (<em>b</em>)O<em>b</em>jectives:</<em>b</em>) To validate the diagnostic accuracy of an index (termed ROX and defined as the ratio of oxygen saturation as measured <em>b</em>y pulse oximetry/Fi<su<em>b</em>)O<su<em>b</em>)2</su<em>b</em>)</su<em>b</em>) to respiratory rate) for determining HFNC outcome (need or not for intu<em>b</em>ation). (<em>b</em>)Methods:</<em>b</em>) This was a 2-year mu<em>lt</em>icenter prospective o<em>b</em>servational cohort study including patients with pneumonia treated with HFNC. Identification was through Cox proportional hazards modeling of ROX association with HFNC outcome. The most specific cutoff of the ROX index to predict HFNC failure and success was assessed. (<em>b</em>)Measurements and Main Resu<em>lt</em>s:</<em>b</em>) Among the 191 patients treated with HFNC in the validation cohort, 68 (35.6%) required intu<em>b</em>ation. The prediction accuracy of the ROX index increased over time (area under the receiver operating characteristic curve: 2 h, 0.679; 6 h, 0.703; 12 h, 0.759). ROX greater than or equal to 4.88 measured at 2 (hazard ratio, 0.434; 95% confidence interval, 0.264-0.715; <i>P</i> = 0.001), 6 (hazard ratio, 0.304; 95% confidence interval, 0.182-0.509; <i>P</i> &<em>lt</em>; 0.001), or 12 hours (hazard ratio, 0.291; 95% confidence interval, 0.161-0.524; <i>P</i> &<em>lt</em>; 0.001) after HFNC initiation was consistently associated with a lower risk for intu<em>b</em>ation. A ROX less than 2.85, less than 3.47, and less than 3.85 at 2, 6, and 12 hours of HFNC initiation, respectively, were predictors of HFNC failure. Patients who failed presented a lower increase in the values of the ROX index over the 12 hours. Among components of the index, oxygen saturation as measured <em>b</em>y pulse oximetry/Fi<su<em>b</em>)O<su<em>b</em>)2</su<em>b</em>)</su<em>b</em>) had a greater weight than respiratory rate. (<em>b</em>)Conclusions:</<em>b</em>) In patients with pneumonia with acute respiratory failure treated with HFNC, ROX is an index that can help identify those patients with low and those with high risk for intu<em>b</em>ation. Clinical trial registered with www.clinica<em>lt</em>rials.gov (NCT02845128).

<A<em>b</em>stractText>The effects of sodium-glucose co-transporter-2 (SGLT2) inhi<em>b</em>itors on kidney failure, particularly the need for dialysis or transplantation or death due to kidney disease, is uncertain. Additionally, previous studies have <em>b</em>een underpowered to ro<em>b</em>ustly assess heterogeneity of effects on kidney outcomes <em>b</em>y different levels of estimated glomerular fi<em>lt</em>ration rate (eGFR) and al<em>b</em>uminuria. We aimed to do a systematic review and meta-analysis to assess the effects of SGLT2 inhi<em>b</em>itors on major kidney outcomes in patients with type 2 dia<em>b</em>etes and to determine the consistency of effect size across trials and different levels of eGFR and al<em>b</em>uminuria.</A<em>b</em>stractText><A<em>b</em>stractText>We did a systematic review and meta-analysis of randomised, controlled, cardiovascular or kidney outcome trials of SGLT2 inhi<em>b</em>itors that reported effects on major kidney outcomes in people with type 2 dia<em>b</em>etes. We searched MEDLINE and Em<em>b</em>ase from data<em>b</em>ase inception to June 14, 2019, to identify eligi<em>b</em>le trials. The primary outcome was a composite of dialysis, transplantation, or death due to kidney disease. We used random-effects models to o<em>b</em>tain summary relative risks (RRs) with 95% CIs and random-effects meta-regression to explore effect modification <em>b</em>y su<em>b</em>groups of <em>b</em>aseline eGFR, al<em>b</em>uminuria, and use of renin-angiotensin system (RAS) <em>b</em>lockade. This review is registered with PROSPERO (CRD42019131774).</A<em>b</em>stractText><p><div>(<em>b</em>)FINDINGS</<em>b</em>)</div>From 2085 records identified, four studies met our inclusion criteria, assessing three SGLT2 inhi<em>b</em>itors: empagliflozin (EMPA-REG OUTCOME), canagliflozin (CANVAS Program and CREDENCE), and dapagliflozin (DECLARE-TIMI 58). From a total of 38 723 participants, 252 required dialysis or transplantation or died of kidney disease, 335 developed end-stage kidney disease, and 943 had acute kidney injury. SGLT2 inhi<em>b</em>itors su<em>b</em>stantially reduced the risk of dialysis, transplantation, or death due to kidney disease (RR 0·67, 95% CI 0·52-0·86, p=0·0019), an effect consistent across studies (I²=0%, p<su<em>b</em>)heterogeneity</su<em>b</em>)=0·53). SGLT2 inhi<em>b</em>itors also reduced end-stage kidney disease (0·65, 0·53-0·81, p&<em>lt</em>;0·0001), and acute kidney injury (0·75, 0·66-0·85, p&<em>lt</em>;0·0001), with consistent <em>b</em>enefits across studies. A<em>lt</em>hough we identified some evidence that the proportional effect of SGLT2 inhi<em>b</em>itors might attenuate with declining kidney function (p<su<em>b</em>)trend</su<em>b</em>)=0·073), there was clear, separate evidence of <em>b</em>enefit for all eGFR su<em>b</em>groups, including for participants with a <em>b</em>aseline eGFR 30-45 mL/min per 1·73 m² (RR 0·70, 95% CI 0·54-0·91, p=0·0080). Renoprotection was also consistent across studies irrespective of <em>b</em>aseline al<em>b</em>uminuria (p<su<em>b</em>)trend</su<em>b</em>)=0·66) and use of RAS <em>b</em>lockade (p<su<em>b</em>)heterogeneity</su<em>b</em>)=0·31).</p><A<em>b</em>stractText>SGLT2 inhi<em>b</em>itors reduced the risk of dialysis, transplantation, or death due to kidney disease in individuals with type 2 dia<em>b</em>etes and provided protection against acute kidney injury. These data provide su<em>b</em>stantive evidence supporting the use of SGLT2 inhi<em>b</em>itors to prevent major kidney outcomes in people with type 2 dia<em>b</em>etes.</A<em>b</em>stractText><A<em>b</em>stractText>None.</A<em>b</em>stractText>

<A<em>b</em>stractText>We examined whether plasma <em>β</em>-amyloid (A<em>β</em>)42/A<em>β</em>40, as measured <em>b</em>y a high-precision assay, accurately diagnosed <em>b</em>rain amyloidosis using amyloid PET or CSF p-tau181/A<em>β</em>42 as reference standards.</A<em>b</em>stractText><A<em>b</em>stractText>Using an immunoprecipitation and liquid chromatography-mass spectrometry assay, we measured A<em>β</em>42/A<em>β</em>40 in plasma and CSF samples from 158 mostly cognitively normal individuals that were collected within 18 months of an amyloid PET scan.</A<em>b</em>stractText><p><div>(<em>b</em>)RESULTS</<em>b</em>)</div>Plasma A<em>β</em>42/A<em>β</em>40 had a high correspondence with amyloid PET status (receiver operating characteristic area under the curve [AUC] 0.88, 95% confidence interval [CI] 0.82-0.93) and CSF p-tau181/A<em>β</em>42 (AUC 0.85, 95% CI 0.79-0.92). The com<em>b</em>ination of plasma A<em>β</em>42/A<em>β</em>40, age, and <i>APOE</i> ε4 status had a very high correspondence with amyloid PET (AUC 0.94, 95% CI 0.90-0.97). Individuals with a negative amyloid PET scan at <em>b</em>aseline and a positive plasma A<em>β</em>42/A<em>β</em>40 (&<em>lt</em>;0.1218) had a 15-fold greater risk of conversion to amyloid PET-positive compared to individuals with a negative plasma A<em>β</em>42/A<em>β</em>40 (<i>p</i> = 0.01).</p><p><div>(<em>b</em>)CONCLUSIONS</<em>b</em>)</div>Plasma A<em>β</em>42/A<em>β</em>40, especially when com<em>b</em>ined with age and <i>APOE</i> ε4 status, accurately diagnoses <em>b</em>rain amyloidosis and can <em>b</em>e used to screen cognitively normal individuals for <em>b</em>rain amyloidosis. Individuals with a negative amyloid PET scan and positive plasma A<em>β</em>42/A<em>β</em>40 are at increased risk for converting to amyloid PET-positive. Plasma A<em>β</em>42/A<em>β</em>40 could <em>b</em>e used in prevention trials to screen for individuals likely to <em>b</em>e amyloid PET-positive and at risk for Alzheimer disease dementia.</p><A<em>b</em>stractText>This study provides Class II evidence that plasma A<em>β</em>42/A<em>β</em>40 levels accurately determine amyloid PET status in cognitively normal research participants.</A<em>b</em>stractText>

(1) Background: The novel coronavirus disease 2019 (COVID-19) is a global public health emergency that has caused worldwide concern. Vast resources have been allocated to control the pandemic and treat patients. However, little attention has been paid to the adverse impact on mental health or effective mitigation strategies to improve mental health. (2) Purpose: The aim of this study was to assess the adverse impact of the COVID-19 outbreak on Chinese college students' mental health, understand the underlying mechanisms, and explore feasible mitigation strategies. (3) Methods: During the peak time of the COVID-19 outbreak in China, we conducted longitudinal surveys of sixty-six college students. Structured questionnaires collected information on demographics, physical activity, negative emotions, sleep quality, and aggressiveness level. A mixed-effect model was used to evaluate associations between variables, and the mediating effect of sleep quality was further explored. A generalized additive model was used to determine the dose-response relationships between the COVID-19 death count, physical activity, and negative emotions. (4) Results: The COVID-19 death count showed a direct negative impact on general sleep quality (β = 1.37, 95% confidence interval [95% CI]: 0.55, 2.19) and reduced aggressiveness (β = -6.57, 95% CI: -12.78, -0.36). In contrast, the COVID-19 death count imposed not a direct but an indirect impact on general negative emotions (indirect effect (IE) = 0.81, p = 0.012), stress (IE = 0.40, p &lt; 0.001), and anxiety (IE = 0.27, p = 0.004) with sleep quality as a mediator. Moreover, physical activity directly alleviated general negative emotions (β = -0.12, 95% CI: -0.22, -0.01), and the maximal mitigation effect occurred when weekly physical activity was about 2500 METs. (5) Conclusions: (a) The severity of the COVID-19 outbreak has an indirect effect on negative emotions by affecting sleep quality. (b) A possible mitigation strategy for improving mental health includes taking suitable amounts of daily physical activity and sleeping well.

Keywords: coronavirus; mental health; mitigation strategies; physical activity; sleep quality.

Background: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an emerging infection causing a widely spread pandemic of Coronavirus disease 2019 (COVID-19). The current COVID-2019 pandemic is prompting fear of falling sick, dying, helplessness and stigma, urgent and timely understanding of mental health status is needed to help the community. Our investigation designed to survey the general population in Saudi Arabia to assess the degree of psychological impact during the pandemic.

Methods: During the early stage of the outbreak, we conducted an online-based survey using a snowballing sample technique. The surveys collected data about several aspects of participant sociodemographic, knowledge, concerns, psychological impact, and mental health status. We assessed the psychological impact and mental health status using the Impact of Event Scale-Revised (IES-R), and the Depression, Anxiety, and Stress Scale (DASS-21).

Results: Our survey recruited 1160 respondents of the general public of Saudi Arabia. Of them, 23.6% reported moderate or severe psychological impact of the outbreak, 28.3%,24%, and 22.3% reported moderate to severe depressive, anxiety, and stress symptoms, respectively. Females reported IES-R (B: 5.46, 95% CI: 3.61 to 7.31) and DASS subscales B coefficient ranged from 1.65 to 2.63, along with high-school students, working in the medical field, and poor self-reported health status was significantly associated with a high level of IES-R and DASS scales (p &lt; .05). Experiencing breathing difficulty and dizziness showed a stronger association with higher IES-R and DASS subscales than other somatic symptoms (e.g., headache and fever);(p &lt; .001). Respondents who practiced specific preventative measures (e.g., hand washing, social distancing) demonstrated a protective effect against stress, anxiety, and depression symptoms. Social distancing appeared to be protective on stress and anxiety subscales (B: -1.49, 95% CI: -2.79 to -0.19),(B: -1.53, 95% CI: -2.50 to -0.57),respectively; and hand hygiene on depression subscale (B: -2.43, 95% CI: -4.44 to -0.42).

Conclusion: Throughout the early stage of the COVID-19 outbreak in Saudi Arabia, the results showed that nearly one-fourth of the sampled general population experienced moderate to severe psychological impact. Following specific precautionary measures appeared to have a protective effect on the individual's mental health. Our findings can be used to construct psychological interventions directed toward vulnerable populations and to implement public mental health strategies in the early stages of the outbreak.

Keywords: Anxiety; Coronavirus; Depression; IES; Knowledge; Pandemic; Precaution; Psychological impact; Saudi Arabia; Stress.

Supernatants of peripheral blood mononuclear leukocytes (PBMC) treated with Sendai virus were found to exert significant cytotoxic effects mediated by leukocyte-produced proteins distinct from interferon. Fractionation of the PBMC into adherent and nonadherent cells indicated that these virus-induced cytotoxins (CTX) were produced primarily in the mononuclear phagocytes. Cells of the monocyte-like U937 line pretreated with 4 beta-phorbol-12-myristate-13-acetate could also be induced with Sendai virus to produce CTX. The nonadherent mononuclear cells of the peripheral blood responded poorly to the virus with regard to CTX production, even though they could be induced to produce CTX with phytohemagglutinin (PHA). With the use of monospecific antibodies to tumor necrosis factor (TNF) and to lymphotoxin (LT), it was found that TNF is the major CTX produced by PBMC and by the U937 cells after 24 hr stimulation by the virus, whereas LT is not induced under these conditions to any measurable extent. TNF was also found to be produced in significant amounts together with LT upon stimulation of the nonadherent fraction of the PBMC by PHA. These findings indicate that besides bacterial lipopolysaccharides, other biological agents including viruses can be effective inducers of tumor necrosis factor, suggesting implications regarding the physiologic role of this protein.

Ectopic or tertiary lymphoid tissues (TLTs) are often induced at sites of chronic inflammation. They typically contain various hematopoietic cell types, high endothelial venules, and follicular dendritic cells; and are organized in lymph node-like structures. Although fibroblastic stromal cells may play a role in TLT induction and persistence, they have remained poorly defined. Herein, we report that TLTs arising during inflammation in mice and humans in a variety of tissues (eg, pancreas, kidney, liver, and salivary gland) contain stromal cell networks consisting of podoplanin(+) T-zone fibroblastic reticular cells (TRCs), distinct from follicular dendritic cells. Similar to lymph nodes, TRCs were present throughout T-cell-rich areas and had dendritic cells associated with them. They expressed lymphotoxin (LT) β receptor (LTβR), produced CCL21, and formed a functional conduit system. In rat insulin promoter-CXCL13-transgenic pancreas, the maintenance of TRC networks and conduits was partially dependent on LTβR and on lymphoid tissue inducer cells expressing LTβR ligands. In conclusion, TRCs and conduits are hallmarks of secondary lymphoid organs and of well-developed TLTs, in both mice and humans, and are likely to act as important scaffold and organizer cells of the T-cell-rich zone.

Bacillus anthracis, the causative agent of anthrax disease, is lethal owing to the actions of two exotoxins: anthrax lethal toxin (LT) and oedema toxin (ET). The key tissue targets responsible for the lethal effects of these toxins are unknown. Here we generated cell-type-specific anthrax toxin receptor capillary morphogenesis protein-2 (CMG2)-null mice and cell-type-specific CMG2-expressing mice and challenged them with the toxins. Our results show that lethality induced by LT and ET occurs through damage to distinct cell types; whereas targeting cardiomyocytes and vascular smooth muscle cells is required for LT-induced mortality, ET-induced lethality occurs mainly through its action in hepatocytes. Notably, and in contradiction to what has been previously postulated, targeting of endothelial cells by either toxin does not seem to contribute significantly to lethality. Our findings demonstrate that B. anthracis has evolved to use LT and ET to induce host lethality by coordinately damaging two distinct vital systems.

Schuknecht proposed a discrete form of presbycusis in which hearing loss results principally from degeneration of cochlear stria vascularis and decline of the endocochlear potential (EP). This form was asserted to be genetically linked, and to arise independently from age-related pathology of either the organ of Corti or cochlear neurons. Although extensive strial degeneration in humans coincides with hearing loss, EPs have never been measured in humans, and age-related EP reduction has never been verified. No human genes that promote strial presbycusis have been identified, nor is its pathophysiology well understood. Effective application of animal models to this issue requires models demonstrating EP decline, and preferably, genetically distinct strains that vary in patterns of EP decline and its cellular correlates. Until recently, only two models, Mongolian gerbils and Tyrp1(B-lt) mice, were known to undergo age-associated EP reduction. Detailed studies of seven inbred mouse strains have now revealed three strains (C57BL/6J, BBA/J) showing essentially no EP decline with age, and four strains ranging from modest to severe EP reduction (C57BL/6-Tyr(c-2J), BALB/cJ, CBA/CaJ, NOD.NON-H2(nbl)/LtJ). Collectively, animal models support five basic principles regarding a strial form of presbycusis: 1) Progressive EP decline from initially normal levels as a defining characteristic; 2) Non-universality, not all age-associated hearing loss involves EP decline; 3) A clear genetic basis; 4) Modulation by environment or stochastic events; and 5) Independent strial, organ of Corti, and neural pathology. Shared features between human strial presbycusis, gerbils, and BALB/cJ and C57BL/6-Tyr(c-2J) mice further suggest this condition frequently begins with strial marginal cell dysfunction and loss. By contrast, NOD.NON-H2(nbl) mice may model a sequence more closely associated with strial microvascular disease. Additional studies of these and other inbred mouse and rat models should reveal candidate processes and genes that promote EP decline in humans.

The lymphotoxin (LT)/tumor necrosis factor (TNF) family has been implicated in the neurologic inflammatory diseases multiple sclerosis (MS) and experimental allergic encephalomyelitis (EAE). To determine the role of individual family members in EAE, C57BL/6 mice, LT-alpha-deficient (LT-alpha-/- mice), or LT-beta-deficient (LT-beta-/- mice), and their wild-type (WT) littermates were immunized with rat myelin oligodendrocyte glycoprotein (MOG) peptide 35-55. C57BL/6 and WT mice developed chronic, sustained paralytic disease with average maximum clinical scores of 3.5 and disease indices (a measure of day of onset and sustained disease scores) ranging from 367 to 663 with central nervous system (CNS) inflammation and demyelination. LT-alpha-/- mice were primed so that their splenic lymphocytes proliferated in response to MOG 35-55 and the mice produced anti-MOG antibody. However, LT-alpha-/- mice were quite resistant to EAE with low average clinical scores (<1), an average disease index of 61, and the negligible CNS inflammation and demyelination. WT T cells transferred EAE to LT-alpha-/- recipients. LT-beta-/- mice were susceptible to EAE, though less than WT, with an average maximum clinical score of 1.9 and disease index of 312. These data implicate T cell production of LT-alpha in MOG EAE and support a major role for LT-alpha3, a minor role for the LT-alpha/beta complex, and by inference, no role for TNF-alpha.

<A<em>b</em>stractText>Cystic fi<em>b</em>rosis transmem<em>b</em>rane conductance regulator (CFTR) modulators correct the <em>b</em>asic defect caused <em>b</em>y CFTR mutations. Improvements in hea<em>lt</em>h outcomes have <em>b</em>een achieved with the com<em>b</em>ination of a CFTR corrector and potentiator in people with cystic fi<em>b</em>rosis homozygous for the F508del mutation. The addition of elexacaftor (VX-445), a next-generation CFTR corrector, to tezacaftor plus ivacaftor further improved F508del-CFTR function and clinical outcomes in a phase 2 study in people with cystic fi<em>b</em>rosis homozygous for the F508del mutation.</A<em>b</em>stractText><p><div>(<em>b</em>)METHODS</<em>b</em>)</div>This phase 3, mu<em>lt</em>icentre, randomised, dou<em>b</em>le-<em>b</em>lind, active-controlled trial of elexacaftor in com<em>b</em>ination with tezacaftor plus ivacaftor was done at 44 sites in four countries. Eligi<em>b</em>le participants were those with cystic fi<em>b</em>rosis homozygous for the F508del mutation, aged 12 years or older with sta<em>b</em>le disease, and with a percentage predicted forced expiratory volume in 1 s (ppFEV<su<em>b</em>)1</su<em>b</em>)) of 40-90%, inclusive. After a 4-week tezacaftor plus ivacaftor run-in period, participants were randomly assigned (1:1) to 4 weeks of elexacaftor 200 mg orally once daily plus tezacaftor 100 mg orally once daily plus ivacaftor 150 mg orally every 12 h versus tezacaftor 100 mg orally once daily plus ivacaftor 150 mg orally every 12 h alone. The primary outcome was the a<em>b</em>solute change from <em>b</em>aseline (measured at the end of the tezacaftor plus ivacaftor run-in) in ppFEV<su<em>b</em>)1</su<em>b</em>) at week 4. Key secondary outcomes were a<em>b</em>solute change in sweat chloride and Cystic Fi<em>b</em>rosis Questionnaire-Revised respiratory domain (CFQ-R RD) score. This study is registered with ClinicalTrials.gov, NCT03525548.</p><p><div>(<em>b</em>)FINDINGS</<em>b</em>)</div>Between Aug 3 and Dec 28, 2018, 113 participants were enrolled. Following the run-in, 107 participants were randomly assigned (55 in the elexacaftor plus tezacaftor plus ivacaftor group and 52 in the tezacaftor plus ivacaftor group) and completed the 4-week treatment period. The elexacaftor plus tezacaftor plus ivacaftor group had improvements in the primary outcome of ppFEV<su<em>b</em>)1</su<em>b</em>) (least squares mean [LSM] treatment difference of 10·0 percentage points [95% CI 7·4 to 12·6], p&<em>lt</em>;0·0001) and the key secondary outcomes of sweat chloride concentration (LSM treatment difference -45·1 mmol/L [95% CI -50·1 to -40·1], p&<em>lt</em>;0·0001), and CFQ-R RD score (LSM treatment difference 17·4 points [95% CI 11·8 to 23·0], p&<em>lt</em>;0·0001) compared with the tezacaftor plus ivacaftor group. The triple com<em>b</em>ination regimen was well tolerated, with no discontinuations. Most adverse events were mild or moderate; serious adverse events occurred in two (4%) participants receiving elexacaftor plus tezacaftor plus ivacaftor and in one (2%) receiving tezacaftor plus ivacaftor.</p><A<em>b</em>stractText>Elexacaftor plus tezacaftor plus ivacaftor provided clinically ro<em>b</em>ust <em>b</em>enefit compared with tezacaftor plus ivacaftor alone, with a favoura<em>b</em>le safety profile, and shows the potential to lead to transformative improvements in the lives of people with cystic fi<em>b</em>rosis who are homozygous for the F508del mutation.</A<em>b</em>stractText><A<em>b</em>stractText>Vertex Pharmaceuticals.</A<em>b</em>stractText>

Eosinophils are multifunctional leukocytes that reside in the gastrointestinal (GI) lamina propria, where their basal function remains largely unexplored. In this study, by examining mice with a selective deficiency of systemic eosinophils (by lineage ablation) or GI eosinophils (eotaxin-1/2 double deficient or CC chemokine receptor 3 deficient), we show that eosinophils support immunoglobulin A (IgA) class switching, maintain intestinal mucus secretions, affect intestinal microbial composition, and promote the development of Peyer's patches. Eosinophil-deficient mice showed reduced expression of mediators of secretory IgA production, including intestinal interleukin 1β (IL-1β), inducible nitric oxide synthase, lymphotoxin (LT) α, and LT-β, and reduced levels of retinoic acid-related orphan receptor gamma t-positive (ROR-γt(+)) innate lymphoid cells (ILCs), while maintaining normal levels of APRIL (a proliferation-inducing ligand), BAFF (B cell-activating factor of the tumor necrosis factor family), and TGF-β (transforming growth factor β). GI eosinophils expressed a relatively high level of IL-1β, and IL-1β-deficient mice manifested the altered gene expression profiles observed in eosinophil-deficient mice and decreased levels of IgA(+) cells and ROR-γt(+) ILCs. On the basis of these collective data, we propose that eosinophils are required for homeostatic intestinal immune responses including IgA production and that their affect is mediated via IL-1β in the small intestine.

Efficient immune defenses are facilitated by the organized microarchitecture of lymphoid organs, and this organization is regulated by the compartmentalized expression of lymphoid tissue chemokines. Mouse cytomegalovirus (MCMV) infection induces significant remodeling of splenic microarchitecture, including loss of marginal zone macrophage populations and dissolution of T and B cell compartmentalization. MCMV preferentially infected the splenic stroma, targeting endothelial cells (EC) as revealed using MCMV-expressing green fluorescent protein. MCMV infection caused a specific, but transient transcriptional suppression of secondary lymphoid chemokine (CCL21). The loss of CCL21 was associated with the failure of T lymphocytes to locate within the T cell zone, although trafficking to the spleen was unaltered. Expression of CCL21 in lymphotoxin (LT)-alpha-deficient mice is dramatically reduced, however MCMV infection further reduced CCL21 levels, suggesting that viral modulation of CCL21 was independent of LTalpha signaling. Activation of LTbeta-receptor signaling with an agonistic antibody partially restored CCL21 mRNA expression and redirected transferred T cells to the splenic T cell zone in MCMV-infected mice. These results indicate that virus-induced alterations in lymphoid tissues can occur through an LT-independent modulation of chemokine transcription, and targeting of the LT cytokine system can counteract lymphoid tissue remodeling by MCMV.

Escherichia coli labile toxin (LT) was assessed as mucosal immunogen and as adjuvant for tetanus toxoid (TT) in mice. After oral administration of LT, C57BL/6 (H-2b) and BALB/c(H-2d) mice were high mucosal and serum antibody responders, while C3H/HeN (H-2k) mice were low responders. High responders exhibited mainly serum IgG (including IgG1, IgG2a, and IgG2b), as well as IgM and IgA, while mucosal responses were IgA. Analysis of LT-B-specific CD4+ T helper (Th) cells from Peyer's patches (PP) or from spleen revealed a mixed Th1 (interferon-gamma) and Th2 (interleukin-4 and -5) cell pattern. Oral LT given with TT induced TT-specific response patterns identical to LT-B. Analysis of mRNA from TT-specific PP CD4+ Th cells also revealed a mixed Th1- and Th2- type response. Thus, antibody response profiles induced by LT are regulated by both CD4+ Th1 and Th2 cell types.

A subgroup of common variable immunodeficiency (CVID) patients have distinct clinical features, particularly granulomata splenomegaly, characteristic blood lymphocyte phenotype, and elevated circulating TNF levels. To investigate the genetic basis for this phenotype, 150 CVID patients and 200 controls were genotyped for six biallelic TNF and lymphotoxin-alpha (LT alpha) polymorphisms and eight class I and II HLA loci using PCR and sequence specific primers (PCR-SSP) sequence-specific primers. Clinical and immunophenotypic data were collected for 90 patients to examine associations with CVID patient subgroups. The presence of granulomata (22% of patients) was strongly associated with splenomegaly, T and B lymphopenia, reduced CD4+ CD45RA+ T cells, and CD8+ CD57+ lymphocytosis, confirming the concept of a subgroup of patients with distinct clinical and laboratory features. The uncommon TNF +488A allele was strongly associated with this subgroup (p = 0.0005). The association between "granulomatous" CVID and TNF +488A was independent of HLA class I and II associations. We postulate that the presence of the TNF +488A allele, or alleles in linkage disequilibrium with it, contributes to the high levels of TNF and granulomatous complications characteristic of this subgroup of patients.

Proliferation of dendritic cells (DC) in the spleen is regulated by positive growth signals through the lymphotoxin (LT)-beta receptor; however, the countering inhibitory signals that achieve homeostatic control are unresolved. Mice deficient in LTalpha, LTbeta, LTbetaR, and the NFkappaB inducing kinase show a specific loss of CD8- DC subsets. In contrast, the CD8alpha- DC subsets were overpopulated in mice deficient in the herpesvirus entry mediator (HVEM) or B and T lymphocyte attenuator (BTLA). HVEM- and BTLA-deficient DC subsets displayed a specific growth advantage in repopulating the spleen in competitive replacement bone marrow chimeric mice. Expression of HVEM and BTLA were required in DC and in the surrounding microenvironment, although DC expression of LTbetaR was necessary to maintain homeostasis. Moreover, enforced activation of the LTbetaR with an agonist Ab drove expansion of CD8alpha- DC subsets, overriding regulation by the HVEM-BTLA pathway. These results indicate the HVEM-BTLA pathway provides an inhibitory checkpoint for DC homeostasis in lymphoid tissue. Together, the LTbetaR and HVEM-BTLA pathways form an integrated signaling network regulating DC homeostasis.

Anthrax lethal toxin (LT) activates the NLRP1b (NALP1b) inflammasome and caspase-1 in macrophages from certain inbred mouse strains, but the mechanism by which this occurs is poorly understood. We report here that similar to several NLRP3 (NALP3, cryopyrin)-activating stimuli, LT activation of the NLRP1b inflammasome involves lysosomal membrane permeabilization (LMP) and subsequent cytoplasmic cathepsin B activity. CA-074Me, a potent cathepsin B inhibitor, protects LT-sensitive macrophages from cell death and prevents the activation of caspase-1. RNA interference knockdown of cathepsin B expression, however, cannot prevent LT-mediated cell death, suggesting that CA-074Me may also act on other cellular proteases released during LMP. CA-074Me appears to function downstream of LT translocation to the cytosol (as assessed by mitogen-activated protein kinase kinase cleavage), K(+) effluxes, and proteasome activity. The initial increase in cytoplasmic activity of cathepsin B occurs at the same time or shortly before caspase-1 activation but precedes a larger-scale lysosomal destabilization correlated closely with cytolysis. We present results suggesting that LMP may be involved in the activation of the NLRP1b inflammasome.

Earlier studies demonstrated that dietary omega-3 polyunsaturated fatty acid (PUFA) supplementation attenuates the chemotactic response of neutrophils and the generation of leukotriene (LT) B4 by neutrophils stimulated with calcium ionophore; however, the mechanisms and relationship of these effects were not examined. Neutrophils and monocytes from eight healthy individuals were examined before and after 3 and 10 wk of dietary supplementation with 20 g SuperEPA daily, which provides 9.4 g eicosapentaenoic acid (EPA) and 5 g docosahexaenoic acid. The maximal neutrophil chemotactic response to LTB4, assessed in Boyden microchambers, decreased by 69% after 3 wk and by 93% after 10 wk from prediet values. The formation of [3H]inositol tris-phosphate (IP3) by [3H]inositol-labeled neutrophils stimulated by LTB4 decreased by 71% after 3 wk (0.033 +/- 0.013% [3H] release, mean +/- SEM) and by 90% after 10 wk (0.011 +/- 0.011%) from predict values (0.114 +/- 0.030%) as quantitated by beta-scintillation counting after resolution on HPLC. LTB4-stimulated neutrophil chemotaxis and IP3 formation correlated significantly (P < 0.0001); each response correlated closely and negatively with the EPA content of the neutrophil phosphatidylinositol (PI) pool (P = 0.0003 and P = 0.0005, respectively). Neither the affinities and densities of the high and low affinity LTB4 receptors on neutrophils nor LTB4-mediated diglyceride formation changed appreciably during the study. Similar results were observed in neutrophils activated with platelet-activating factor (PAF). The summed formation of LTB4 plus LTB5 was selectively inhibited in calcium ionophore-stimulated neutrophils and was also inhibited in zymosan-stimulated neutrophils. The inhibition of the summed formation of LTB4 plus LTB5 in calcium ionophore-stimulated neutrophils and in zymosan-stimulated neutrophils did not correlate significantly with the EPA content of the PI pool. The data indicate that dietary omega-3 PUFA supplementation inhibits the autoamplification of the neutrophil inflammatory response by decreasing LTB4 formation through the inactivation of the LTA epoxide hydrolase and independently by inhibiting LTB4- (and PAF) stimulated chemotaxis by attenuating the formation of IP3 by the PI-selective phospholipase C. This is the initial demonstration that dietary omega-3 PUFA supplementation can suppress signal transduction at the level of the PI-specific phospholipase C in humans.

CONCLUSIONS

Cytokines mediate key communication pathways essential for regulation of immune responses. Full activation of antigen-responding lymphocytes requires cooperating signals from the tumor necrosis factor (TNF)-related cytokines and their specific receptors. LIGHT, a lymphotoxin-beta (LTbeta)-related TNF family member, modulates T-cell activation through two receptors, the herpesvirus entry mediator (HVEM) and indirectly through the LT-beta receptor. An unexpected finding revealed a non-canonical binding site on HVEM for the immunoglobulin superfamily member, B and T lymphocyte attenuator (BTLA), and an inhibitory signaling protein suppressing T-cell activation. Thus, HVEM can act as a molecular switch between proinflammatory and inhibitory signaling. The non-canonical HVEM-BTLA pathway also acts to counter LTbetaR signaling that promotes the proliferation of antigen-presenting dendritic cells (DCs) within lymphoid tissue microenvironments. These results indicate LTbeta receptor and HVEM-BTLA pathways form an integrated signaling circuit. Targeting these cytokine pathways with specific antagonists (antibody or decoy receptor) can alter lymphocyte differentiation and activation. Alternately, agonists directed at their cell surface receptors can restore homeostasis and potentially reset immune and inflammatory processes, which may be useful in treating autoimmune and infectious diseases and cancer.

OBJECTIVE

Membrane lymphotoxin (LT) alpha/beta, a member of the tumor necrosis factor (TNF) family of immune regulatory molecules, is involved both in the development of secondary lymphoid tissues and the maintenance of organized lymphoid tissues in the adult. Defects observed in the mucosal immune system in animals with a genetically disrupted LTalpha/beta pathway coupled with the expression of LTalpha/beta in activated T cells motivated an examination of the importance of this pathway in experimental colitis.

METHODS

Soluble LTbeta receptor (LTbetaR) immunoglobulin fusion protein was used to inhibit the LTalpha/beta/light axis in two independent rodent models of colitis: CD45RBhi CD4(+)-reconstituted SCID mice and bone marrow-transplanted tg26 mice (BM ->> tg26).

RESULTS

Treatment with LTbetaR immunoglobulin attenuated the development of both the clinical and histological manifestations of the disease in these two murine models of colitis. Given the success of TNF inhibitors in the treatment of human Crohn's disease, the effects of LTbetaR immunoglobulin have been compared with antibody to TNF in the BM ->> tg26 model, and both treatments were equally efficacious.

CONCLUSIONS

The LT pathway plays a role in the development of colitis as important as that of the TNF system and, therefore, represents a potential novel intervention point for the treatment of inflammatory bowel disease.

Lymphoid organ development and inflammation have previously been considered as distinct mechanistically and functionally. In recent years, it has been realized that these phenomena have much in common. This insight has been gained from the recognition that cytokines of the lymphotoxin (LT)/tumor necrosis factor (TNF) family are involved in both processes. The members of the family, LT-alpha, LT-beta, and TNF-alpha, and their multiple receptors participate combinatorially in lymphoid organ development and chronic inflammation. When inflammation that arises in microbial infection or autoimmune disease becomes chronic, it can take on the appearance of organized lymphoid tissue and has been called a tertiary lymphoid organ. Data with transgenic and knockout mice suggest that the process is cytokine-mediated and could be called "lymphoid neo-organogenesis." LT as LT-alpha3 and LT-alpha1betaLT influences these events through induction of adhesion molecules such as E-selectin adhesion molecule (ELAM), vascular cell adhesion molecule (VCAM), intercellular adhesion molecule (ICAM), mucosal addressin cellular adhesion molecule (MAdCAM), and peripheral node addressin (PNAd), and chemokines.

With the goal of examining the functional diversity of human immunodeficiency virus type 1 (HIV-1) env genes within the peripheral blood mononuclear cells of an asymptomatic individual, we substituted four complete env genes into the replication-competent NL4-3 provirus. Despite encoding full-length open reading frames for gp120 and gp41 and the second coding exon of tat and rev, each chimera was replication defective. Site-directed mutagenesis of codon 78 in the Rev activation domain (from a hitherto unique Ile to the subtype B consensus Leu) partially restored infectivity for two of three chimeras tested. Similarly, mutagenesis of rev codon 78 of NL4-3 from Leu to Ile partially attenuated this virus. Ile-78 was found in all 13 clones examined from samples taken from this asymptomatic subject 4.5 years after infection, including 9 from peripheral blood mononuclear cells and 4 from a virus isolate, as well as 4 additional clones each from peripheral blood mononuclear cells sampled 37 and 51 months later. We next examined conservation of the Rev activation domain within and among long-term survivors (LTS) and patients with AIDS, as well as T-cell-line-adapted strains of HIV-1. Putative attenuating mutations were found in a minority of sequences from all five LTS and two of four patients with AIDS. Of the 11 T-cell-line-adapted viruses examined, none had these changes. Among and within LTS virus population had marginally higher levels of diversity in Rev than in Env; patients with AIDS had similar levels of diversity in the two reading frames; and T-cell-line-adapted viruses had higher levels of diversity in Env. These results are consistent with the hypothesis that asymptomatic individuals harbor attenuated variants of HIV-1 which correlate with and contribute to their lack of disease progression.

Immunization with amyloid-beta (Abeta) peptide in mouse models of Alzheimer's disease has been reported to decrease cerebral Abeta levels and improve behavioral deficits. Several mechanisms have been proposed, including antibody-induced phagocytosis of Abeta by cerebral microglia and increased efflux of Abeta from the brain to the periphery. The latter mechanism was suggested in mice undergoing acute, passive transfer of an Abeta monoclonal antibody. Here, PSAPP transgenic mice were actively immunized by a single intraperitoneal injection of synthetic Abeta followed by chronic intranasal administration of Abeta with the mucosal adjuvant, Escherichia coli heat-labile enterotoxin, LT, twice weekly for 8 weeks. Serum from Abeta-immunized mice had an average of 240 microg/ml of anti-Abeta-specific antibodies; these antibodies had epitope(s) within Abetabetax-42 levels (P < 0.026) in brain, and gliosis and neuritic dystrophy were diminished. No pathological effects of the immunization were observed in kidney, spleen, or snout. Serum Abeta levels increased 28-fold in immunized mice (53.06 ng/ml) compared to controls (1.87 ng/ml). Most of the Abeta in the serum of the immunized mice was bound to antibodies. We conclude that following active immunization, anti-Abeta antibodies sequester serum Abeta and may increase central nervous system to serum Abeta clearance.