<p><div>(<em>b</em>)OBJECTIVE</<em>b</em>)</div>Faecal micro<em>b</em>iota transplant (FMT) effectively treats recurrent <i>Clostridioides difficile</i> infection (rCDI), <em>b</em>ut its mechanisms of action remain poorly defined. Certain <em>b</em>ile acids affect <i>C. difficile</i> germination or vegetative growth. We hypothesised that loss of gut micro<em>b</em>iota-derived <em>b</em>ile sa<em>lt</em> hydrolases (BSHs) predisposes to CDI <em>b</em>y pertur<em>b</em>ing gut <em>b</em>ile meta<em>b</em>olism, and that BSH restitution is a key mediator of FMT's efficacy in treating the condition.</p><p><div>(<em>b</em>)DESIGN</<em>b</em>)</div>Using stool collected from patients and donors pre-FMT/post-FMT for rCDI, we performed 16S rRNA gene sequencing, u<em>lt</em>ra performance liquid chromatography mass spectrometry (UPLC-MS) <em>b</em>ile acid profiling, BSH activity measurement, and qPCR of <i><em>b</em>sh</i>/<i><em>b</em>ai</i>CD genes involved in <em>b</em>ile meta<em>b</em>olism. Human data were validated in <i>C. difficile</i> <em>b</em>atch cu<em>lt</em>ures and a C57BL/6 mouse model of rCDI.</p><p><div>(<em>b</em>)RESULTS</<em>b</em>)</div>From metataxonomics, pre-FMT stool demonstrated a reduced proportion of BSH-producing <em>b</em>acterial species compared with donors/post-FMT. Pre-FMT stool was enriched in taurocholic acid (TCA, a potent <i>C. difficile</i> germinant); TCA levels negatively correlated with key <em>b</em>acterial genera containing BSH-producing organisms. Post-FMT samples demonstrated recovered BSH activity and <i><em>b</em>sh</i>/<i><em>b</em>ai</i>CD gene copy num<em>b</em>er compared with pretreatment (p&<em>lt</em>;0.05). In <em>b</em>atch cu<em>lt</em>ures, supernatant from engineered <i><em>b</em>sh</i>-expressing <i>E. coli</i> and naturally BSH-producing organisms (<i>Bacteroides ovatus, Collinsella aerofaciens, Bacteroides vulgatus</i> and <i>Blautia o<em>b</em>eum</i>) reduced TCA-mediated <i>C. difficile</i> germination relative to cu<em>lt</em>ure supernatant of wild-type (BSH-negative) <i>E. coli. C. difficile</i> total via<em>b</em>le counts were ~70% reduced in an rCDI mouse model after administration of <i>E. coli</i> expressing highly active BSH relative to mice administered BSH-negative <i>E. coli</i> (p&<em>lt</em>;0.05).</p><A<em>b</em>stractText>Restoration of gut BSH functionality contri<em>b</em>utes to the efficacy of FMT in treating rCDI.</A<em>b</em>stractText>

<A<em>b</em>stractText>S-1 is a standard postoperative adjuvant chemotherapy for patients with stage II or III gastric cancer in Asia. Neoadjuvant or perioperative strategies dominate in Western countries, and docetaxel has recently shown significant survival <em>b</em>enefits when com<em>b</em>ined with other standard regimens in advanced cancer and perioperative settings.</A<em>b</em>stractText><A<em>b</em>stractText>This randomized phase III study was designed to prove the superiority of postoperative S-1 plus docetaxel over S-1 alone for R0 resection of pathologic stage III gastric cancer. The sample size of 1,100 patients was necessary to detect a 7% increase in 3-year relapse-free survival as the primary end point (hazard ratio, 0.78; 2-sided α = .05; <em>β</em> = .2).</A<em>b</em>stractText><p><div>(<em>b</em>)RESULTS</<em>b</em>)</div>The second interim analysis was conducted when the num<em>b</em>er of events reached 216 among 915 enrolled patients (median follow-up, 12.5 months). Analysis demonstrated the superiority of S-1 plus docetaxel (66%) to S-1 (50%) for 3-year relapse-free survival (hazard ratio, 0.632; 99.99% CI, 0.400 to 0.998; stratified log-rank test, <i>P</i> &<em>lt</em>; .001), and enrollment was terminated as recommended <em>b</em>y the independent data and safety monitoring committee. Incidences of grade 3 or greater adverse events, particularly neutropenia and leukopenia, were higher in the S-1 plus docetaxel group, <em>b</em>ut all events were managea<em>b</em>le.</p><A<em>b</em>stractText>Addition of docetaxel to S-1 is effective with few safety concerns in patients with stage III gastric cancer. The present findings may also <em>b</em>e applica<em>b</em>le in countries in which perioperative adjuvant chemotherapy or chemoradiation is not standard.</A<em>b</em>stractText>

Objective: To explore whether hospitalized patients with SARS-CoV-2 and neurologic symptoms have evidence of CNS infection, inflammation and injury using CSF biomarker measurements.

Methods: We assessed CSF SARS-CoV-2 RNA along with CSF biomarkers of intrathecal inflammation (CSF white blood cell count, neopterin, β₂-microglobulin (β₂M) and immunoglobulin G-index), blood-brain-barrier (BBB) integrity (albumin ratio), and axonal injury (CSF neurofilament light chain protein [NfL]) in 6 patients with moderate to severe COVID-19 and neurologic symptoms who had undergone a diagnostic lumbar puncture. Neurologic symptoms and signs included features of encephalopathies (4/6), suspected meningitis (1/6) and dysgeusia (1/6). SARS-CoV-2 infection was confirmed by rtPCR analysis of nasopharyngeal swabs.

Results: SARS-CoV-2 RNA was detected in the plasma of 2 patients (Cycle threshold [Ct] value 35.0-37.0) and in CSF at low levels (Ct 37.2, 38.0, 39.0) in 3 patients in one but not in a second rtPCR assay. CSF neopterin (median, 43.0 nmol/L) and β₂-microglobulin (median, 3.1 mg/L) were increased in all. Median IgG-index (0.39), albumin ratio (5.35) and CSF white blood cell count (<3 cells/µL) were normal in all, while CSF NfL was elevated in 2 patients.

Conclusion: Our results on patients with COVID-19 and neurologic symptoms suggest an unusual pattern of marked CSF inflammation in which soluble markers were increased but white cell response and other immunologic features typical of CNS viral infections were absent. While our initial hypothesis centered on CNS SARS-CoV-2 invasion, we could not convincingly detect SARS-CoV-2 as the underlying driver of CNS inflammation. These features distinguish COVID-19 CSF from other viral CNS infections, and raise fundamental questions about the CNS pathobiology of SARS-CoV-2 infection.

Autologous T cells engineered to express a CD19-specific chimeric antigen receptor (CAR) have produced impressive minimal residual disease-negative (MRD-negative) complete remission (CR) rates in patients with relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL). However, the factors associated with durable remissions after CAR T-cell therapy have not been fully elucidated. We studied patients with relapsed/refractory B-ALL enrolled in a phase 1/2 clinical trial evaluating lymphodepletion chemotherapy followed by CD19 CAR T-cell therapy at our institution. Forty-five (85%) of 53 patients who received CD19 CAR T-cell therapy and were evaluable for response achieved MRD-negative CR by high-resolution flow cytometry. With a median follow-up of 30.9 months, event-free survival (EFS) and overall survival (OS) were significantly better in the patients who achieved MRD-negative CR compared with those who did not (median EFS, 7.6 vs 0.8 months; P &lt; .0001; median OS, 20.0 vs 5.0 months; P = .014). In patients who achieved MRD-negative CR by flow cytometry, absence of the index malignant clone by IGH deep sequencing was associated with better EFS (P = .034). Stepwise multivariable modeling in patients achieving MRD-negative CR showed that lower prelymphodepletion lactate dehydrogenase concentration (hazard ratio [HR], 1.38 per 100 U/L increment increase), higher prelymphodepletion platelet count (HR, 0.74 per 50 000/μL increment increase), incorporation of fludarabine into the lymphodepletion regimen (HR, 0.25), and allogeneic hematopoietic cell transplantation (HCT) after CAR T-cell therapy (HR, 0.39) were associated with better EFS. These data allow identification of patients at higher risk of relapse after CAR T-cell immunotherapy who might benefit from consolidation strategies such as allogeneic HCT. This trial was registered at www.clinicaltrials.gov as #NCT01865617.

<A<em>b</em>stractText>Repurposing <em>b</em>road-spectrum antivirals is an immediate treatment opportunity for 2019 coronavirus disease (COVID-19). Favipiravir is an antiviral previously indicated for influenza and E<em>b</em>ola, which has shown some promise in early trials for treatment of COVID-19. We aim to review existing favipiravir safety evidence, which is vital to informing the potential future use of favipiravir in COVID-19.</A<em>b</em>stractText><A<em>b</em>stractText>A search was conducted across EMBASE and MEDLINE data<em>b</em>ases, supplemented <em>b</em>y relevant grey-literature and ClinicalTrials.gov. All studies assessing the use of favipiravir in humans <em>b</em>y 27 March 2020 were considered for inclusion. Further analysis of availa<em>b</em>le safety data from phase 2 and 3 studies was undertaken. Data extracted were adverse events (AEs) grade 1-4, serious AEs and discontinuation for AEs. Specific AEs of interest highlighted in early-phase studies, including gastrointestinal AEs and hyperuricaemia, were also examined.</A<em>b</em>stractText><p><div>(<em>b</em>)Resu<em>lt</em>s</<em>b</em>)</div>Twenty-nine studies were identified as potential sources of evidence of the clinical safety of favipiravir. Six were phase 2 and 3 studies reporting relevant safety data for statistical comparison, representing a total of 4299 participants, an estimated 175 person-years-of-follow-up (PYFU). Comparator drugs were ose<em>lt</em>amivir, umifenovir, lopinavir/ritonavir or place<em>b</em>o. Study follow-up was <em>b</em>etween 5 and 21 days. The proportions of grade 1-4 AEs on favipiravir was 28.2% <i>vs</i> 28.4% (<i>P</i> = n.s.) in the comparison arms. The proportion of discontinuations due to AEs on favipiravir was 1.1% <i>vs</i> 1.2% (<i>P</i> = n.s.) in the comparison arms. For serious AEs the proportion was 0.4% in <em>b</em>oth arms (<i>P</i> = n.s.). There were significantly fewer gastrointestinal AEs occurring on favipiravir <i>vs</i> comparators [8.7% <i>vs</i> 11.5%; <i>P</i> = 0.003]. Favipiravir showed significantly more uric acid elevations than comparators [5.8% <i>vs</i> 1.3%; <i>P</i>&<em>lt</em>;0.0001].</p><A<em>b</em>stractText>Favipiravir demonstrates a favoura<em>b</em>le safety profile regarding total and serious AEs. However, safety concerns remain: hyperuricaemia, teratogenicity and QTc prolongation have not yet <em>b</em>een adequately studied. Favipiravir may <em>b</em>e safe and tolera<em>b</em>le in short-term use, <em>b</em>ut more evidence is needed to assess the longer-term effects of treatment. Given the limitations of the evidence and unresolved safety concerns, caution is warranted in the widespread use of favipiravir against pandemic COVID-19.</A<em>b</em>stractText>

<A<em>b</em>stractText>The heterogeneity of metastatic <em>b</em>reast cancer (MBC) necessitates novel <em>b</em>iomarkers allowing stratification of patients for treatment selection and drug development. We propose to use the prognostic utility of circulating tumor cells (CTCs) for stratification of patients with stage IV disease.</A<em>b</em>stractText><p><div>(<em>b</em>)METHODS</<em>b</em>)</div>In a retrospective, pooled analysis of individual patient data from 18 cohorts, including 2436 MBC patients, a CTC threshold of 5 cells per 7.5 ml was used for stratification <em>b</em>ased on molecular su<em>b</em>types, disease location, and prior treatments. Patients with ≥ 5 CTCs were classified as Stage IV<su<em>b</em>)aggressive</su<em>b</em>), those with &<em>lt</em>; 5 CTCs as Stage IV<su<em>b</em>)indolent.</su<em>b</em>) Survival was analyzed using Kaplan-Meier curves and the log rank test.</p><p><div>(<em>b</em>)RESULTS</<em>b</em>)</div>For all patients, Stage IV<su<em>b</em>)indolent</su<em>b</em>) patients had longer median overall survival than those with Stage IV<su<em>b</em>)aggressive</su<em>b</em>) (36.3 months vs. 16.0 months, P &<em>lt</em>; 0.0001) and similarly for de novo MBC patients (41.4 months Stage IV<su<em>b</em>)indolent</su<em>b</em>) vs. 18.7 months Stage IV<su<em>b</em>)aggressive</su<em>b</em>), p &<em>lt</em>; 0.0001). Moreover, patients with Stage IV<su<em>b</em>)indolent</su<em>b</em>) disease had significantly longer overall survival across all disease su<em>b</em>types compared to the aggressive cohort: hormone receptor-positive (44 months vs. 17.3 months, P &<em>lt</em>; 0.0001), HER2-positive (36.7 months vs. 20.4 months, P &<em>lt</em>; 0.0001), and triple negative (23.8 months vs. 9.0 months, P &<em>lt</em>; 0.0001). Similar resu<em>lt</em>s were o<em>b</em>tained regardless of prior treatment or disease location.</p><p><div>(<em>b</em>)CONCLUSIONS</<em>b</em>)</div>We confirm the identification of two su<em>b</em>groups of MBC, Stage IV<su<em>b</em>)indolent</su<em>b</em>) and Stage IV<su<em>b</em>)aggressive</su<em>b</em>), independent of clinical and molecular varia<em>b</em>les. Thus, CTC count should <em>b</em>e considered an important tool for staging of advanced disease and for disease stratification in prospective clinical trials.</p>

<p><div>(<em>b</em>)BACKGROUND</<em>b</em>)</div>Myocardial utilization of 3-hydroxy<em>b</em>utyrate (3-OHB) is increased in patients with heart failure and reduced ejection fraction (HFrEF). However, the cardiovascular effects of increased circulating plasma-3-OHB levels in these patients are unknown. Consequently, the authors' aim was to modulate circulating 3-OHB levels in HFrEF patients and evaluate: (1) changes in cardiac output (CO); (2) a potential dose-response relationship <em>b</em>etween 3-OHB levels and CO; (3) the impact on myocardial external energy efficiency (MEE) and oxygen consumption (MVO<su<em>b</em>)2</su<em>b</em>)); and (4) whether the cardiovascular response differed <em>b</em>etween HFrEF patients and age-matched volunteers.</p><p><div>(<em>b</em>)METHODS</<em>b</em>)</div>Study 1: 16 chronic HFrEF patients (left ventricular ejection fraction: 37±3%) were randomized in a crossover design to 3-hour of 3-OHB or place<em>b</em>o infusion. Patients were monitored invasively with a Swan-Ganz catheter and with echocardiography. Study 2: In a dose-response study, 8 HFrEF patients were examined at increasing 3-OHB infusion rates. Study 3 to 4: 10 HFrEF patients and 10 age-matched volunteers were randomized in a crossover design to 3-hour 3-OHB or place<em>b</em>o infusion. MEE and MVO<su<em>b</em>)2</su<em>b</em>) were evaluated using 11C-acetate positron emission tomography.</p><p><div>(<em>b</em>)RESULTS</<em>b</em>)</div>3-OHB infusion increased circulating levels of plasma 3-OHB from 0.4±0.3 to 3.3±0.4 mM ( P&<em>lt</em>;0.001). CO rose <em>b</em>y 2.0±0.2 L/min ( P&<em>lt</em>;0.001) <em>b</em>ecause of an increase in stroke volume of 20±2 mL ( P&<em>lt</em>;0.001) and heart rate of 7±2 <em>b</em>eats per minute (<em>b</em>pm) ( P&<em>lt</em>;0.001). Left ventricular ejection fraction increased 8±1% ( P&<em>lt</em>;0.001) numerically. There was a dose-response relationship with a significant CO increase of 0.3 L/min already at plasma-3-OHB levels of 0.7 mM ( P&<em>lt</em>;0.001). 3-OHB increased MVO<su<em>b</em>)2</su<em>b</em>) without a<em>lt</em>ering MEE. The response to 3-OHB infusion in terms of MEE and CO did not differ <em>b</em>etween HFrEF patents and age-matched volunteers.</p><A<em>b</em>stractText>3-OHB has <em>b</em>eneficial hemodynamic effects in HFrEF patients without impairing MEE. These <em>b</em>eneficial effects are detecta<em>b</em>le in the physiological concentration range of circulating 3-OHB levels. The hemodynamic effects of 3-OHB were o<em>b</em>served in <em>b</em>oth HFrEF patients and age-matched volunteers. 3-OHB may potentially constitute a novel treatment principle in HFrEF patients.</A<em>b</em>stractText>

Despite the indication that the hypothalamo-pituitary-adrenal (HPA) axis is activated during treadmill running, there have not been any studies focusing on the relationship between exercise intensity and region-specific neural activities in hypothalamus. To address this, rats were subjected to 30 min of running, either at middle (supra-LT, 25 m min(-1)) or low speeds (sub-LT, 15 m min(-1)), and c-Fos-(+) cells were counted and compared with control rats. Significant increases in blood glucose and lactate levels, and plasma ACTH and osmolality levels were observed during supra-LT running. Only supra-LT running significantly increased c-Fos induction in various hypothalamic regions, namely, the medial preoptic area (MPO), periventricular nucleus (Pe), suprachiasmatic nucleus (SCN), supraoptic nucleus (SON), parvocellular division of the paraventricular nucleus (pPVN), anterior hypothalamic area (AH), arcuate nucleus (ARC) and posterior hypothalamic nucleus (PH). However, sub-LT caused no effect on c-Fos accumulation. This indicates that the hypothalamus responds uniquely to running in a threshold-like pattern distinct from the speed-dependent pattern previously reported for the medulla oblongata [Ohiwa et al., 2006a,b]. In addition, these results showed a physiologic basis for mild exercise useful for establishing our minimum running stress (MRS) rat model, or the running conditions that minimize the activation of the HPA axis.

Radiolabeled leukotriene (LT) E4 was infused into three healthy subjects in order to assess the production and elimination of sulfidopeptide leukotriene metabolites in urine. Three different radiolabeled tracers were employed, [14,15-3H]LTE4, [35S]LTE4, and [14C] LTE4 in five separate infusion studies. There was a rapid disappearance of radioactivity from the vascular compartment in an apparent two-phase process. The first elimination phase had an apparent half-life of approximately 7 min. Radioactivity quickly appeared in the urine with 10-16% eliminated during the first 2 h following intravenous infusion; 7%, 2-5 h; 4%, 5-8 h; 4%, 8-15 h; and 1.5%, 15-24 h from the [14C] LTE4 experiments. Unmetabolized LTE4 was the major radioactive component in the first urine collection, but at later times two more polar compounds predominated. After extensive purification by normal phase-solid phase extraction and reverse-phase high performance liquid chromatography, these compounds were characterized by UV spectroscopy, co-elution with synthetic standards, negative ion electron capture gas chromatography/mass spectrometry, and tandem mass spectrometry. The two major urinary metabolites were structurally determined to be 14-carboxy-hexanor-LTE3 and the conjugated tetraene, 16-carboxy-delta 13-tetranor-LTE4. Three other minor metabolites were detectable in the first urine collection only and were characterized by co-elution with synthetic standards as 16-carboxy-tetranor-LTE3, 18-carboxy-dinor-LTE4, and 20-carboxy-LTE4. omega-Oxidation and subsequent beta-oxidation from the methyl terminus appeared to be the major metabolic fate for sulfidopeptide leukotrienes in man. The accumulation of the 14-COOH-LTE3 and 16-COOH-delta 13-LTE4 may reflect a rate-limiting step in further oxidation of these compounds which places a conjugated triene or conjugated tetraene, respectively, two carbons removed from the CoA ester moiety. Also in the first urine collection there was another minor metabolite identified as N-acetyl-LTE4, however, no subsequent beta-oxidation of this metabolite was observed. The major metabolites of LTE4 might be useful in assessing in vivo production of sulfidopeptide leukotrienes in humans.

c-kit ligand (KL) activated mouse bone marrow-derived mast cells (BMMC) for the dose- and time-dependent release of arachidonic acid from cell membrane phospholipids, with generation of leukotriene (LT) C4 in preference to prostaglandin (PG)D2. KL at concentrations of 10 ng/ml elicited half-maximal eicosanoid generation and at concentrations of>> 50 ng/ml elicited a maximal generation of approximately 15 ng LTC4 and 1 ng PGD2 per 10(6) cells, with 20% net beta-hexosaminidase release 10 min after stimulation. Of the other cytokines tested, none, either alone or in combination with KL, elicited or modulated the immediate phase of mediator release by BMMC, indicating strict specificity for KL. Activation of BMMC in response to KL was accompanied by transient phosphorylation of cytosolic phospholipase A2 and reversible translocation of 5-lipoxygenase to a cell membrane fraction 2-5 min after stimulation, when the rate of arachidonic acid release and LTC4 production were maximal. BMMC continuously exposed to KL in the presence of IL-10 and IL-1 beta generated LTC4 in marked preference to PGD2 over the first 10 min followed by delayed generation of PGD2 with no LTC4 over several hours. Pharmacologic studies revealed that PGD2 generation in the immediate phase depended on prostaglandin endoperoxide synthase (PGHS)-1 and in the delayed phase on PGHS-2. Thus, KL provided a nonallergic stimulus for biphasic eicosanoid generation by mast cells. The immediate phase is dominated by LTC4 generation with kinetics and postreceptor biosynthetic events similar to those observed after cell activation through the high affinity IgE receptor, whereas the delayed phase of slow and selective PGD2 production is mediated by induction of PGHS-2.

A comparative study of gnd genes from Escherichia coli strains isolated from natural populations and laboratory strains and from Salmonella typhimurium was undertaken. In the accompanying paper (G. J. Barcak and R. E. Wolf, Jr., J. Bacteriol. 170:365-371, 1988), we showed that the growth-rate-dependent regulation of gnd expression was conserved among four natural E. coli isolates and E. coli B/r in a manner qualitatively similar to that of the gene from E. coli K-12. Here, we report the DNA sequence of the 5' regulatory region and the first 125 codons of the structural gene for the five E. coli gnd genes and the gnd gene from S. typhimurium LT-2. The sequences differed from one another by 5% on the average. All sequences defined putative secondary structures of the mRNA leader, which were previously proposed to be important in the regulation of the K-12 gene. In addition, a sequence between codons 69 and 74, which is highly complementary to the ribosome-binding site of the mRNA, was conserved in all the genes. The sequence data are discussed with respect to potential regulatory consequences.

Anthrax is the disease caused by the Gram-positive bacterium Bacillus anthracis. Two toxins secreted by B. anthracis - lethal toxin (LT) and oedema toxin (OT) - contribute significantly to virulence. Although these toxins have been studied for half a century, recent evidence indicates that LT and OT have several roles during infection not previously ascribed to them. Research on toxin-induced effects other than cytolysis of target cells has revealed that LT and OT influence cell types previously thought to be insensitive to toxin. Multiple host factors that confer sensitivity to anthrax toxin have been identified recently, and evidence indicates that the toxins probably contribute to colonisation and invasion of the host. Additionally, the toxins are now known to cause a wide spectrum of tissue and organ pathophysiologies associated with anthrax. Taken together, these new findings indicate that anthrax-toxin-associated pathogenesis is much more complex than has been traditionally recognised.

The 8th step in the 10-step heme biosynthetic pathway of Salmonella typhimurium is the oxidation of coproporphyrinogen III to protoporphyrinogen IX. On the basis of genetic studies, we have suggested that this reaction may be catalyzed by either of two different enzymes, an oxygen-dependent one encoded by hemF or an oxygen-independent enzyme encoded by hemN. Here, we report the cloning of the S. typhimurium hemF gene and its DNA sequence. The predicted amino acid sequence of the HemF protein is 44% identical to that of the coproporphyrinogen oxidase encoded by the yeast HEM13 gene. The wild-type S. typhimurium strain LT-2 produces an oxygen-dependent coproporphyrinogen oxidase activity detectable in crude extracts, which is not found in hemF mutants and is overproduced in strains carrying the hemF gene on a multicopy plasmid. the hemF gene is the second gene in an operon with an upstream gene with an unknown function, whose amino acid sequence suggests a relation to amidases involved in cell wall synthesis or remodeling. The upstream gene and hemF are cotranscribed from a promoter which was mapped by primer extension. A weaker, hemF-specific promoter is inferred from the behavior of an omega-Cm insertion mutation in the upstream gene. Although this insertion decreases expression of beta-galactosidase about 7.5-fold when placed upstream of a hemF-lacZ operon fusion, it still allows sufficient HemF expression from an otherwise wild-type construct to confer a Hem+ phenotype. The hemF operon is transcribed clockwise with respect to the genetic map.

Leukotriene B(4) (LTB(4)) is a potent lipid mediator of inflammation formed by the 5-lipoxygenase (5-LO)-catalyzed oxidation of arachidonic acid. We have previously shown that (i) LTB(4) is generated during infection, (ii) its biosynthesis is essential for optimal antimicrobial host defense, (iii) LT deficiency is associated with clinical states of immunocompromise, and (iv) exogenous LTB(4) augments antimicrobial functions in phagocytes. Here, we sought to determine whether the administration of LTB(4) has therapeutic potential in a mouse model of pneumonia. Wild-type and 5-LO knockout mice were challenged with Streptococcus pneumoniae via the intranasal route, and bacterial burdens, leukocyte counts, and cytokine levels were determined. LTB(4) was administered via the intraperitoneal, intravenous, and intranasal routes prior to pneumococcal infection and by aerosol 24 h following infection. Leukocytes recovered from mice given S. pneumoniae and treated with aerosolized LTB(4) were evaluated for expression levels of the p47phox subunit of NADPH oxidase. Intrapulmonary but not systemic pretreatment with LTB(4) significantly reduced the lung S. pneumoniae burden in wild-type mice. Aerosolized LTB(4) was effective at improving lung bacterial clearance when administered postinoculation in animals with established infection and exhibited greater potency in 5-LO knockout animals, which also exhibited greater baseline susceptibility. Augmented bacterial clearance in response to LTB(4) was associated with enhanced monocyte recruitment and leukocyte expression of p47phox. The results of the current study in an animal model serve as a proof of concept for the potential utility of treatment with aerosolized LTB(4) as an immunostimulatory strategy in patients with bacterial pneumonia.

The distribution of JC virus (JCV) variants in the brain, lung, liver, kidney, spleen and lymph nodes collected at autopsy from AIDS patients with (Group A: 10 Ss) and without (Group B: 5 Ss) progressive multifocal leukoencephalopathy (PML) and from HIV-negative patients (Group C: 5 Ss), was examined by amplifying the JCV large T antigen (LT), the regulatory (R) and the VP1 regions. Among the samples from the PML patients, JCV DNA was detected in all of the demyelinating areas, in 60% of the lesion-free brain tissues, in 60% of the lung tissues and in 40% of the spleen and kidney tissues, whereas all liver and lymph node sections were negative. JCV DNA was also found in two of the five brain specimens, in two of the five kidney specimens, in one of the five lung specimens from the HIV-positive patients without PML and in the brain specimens from two of the five HIV-negative subjects. Nucleotide sequence analysis indicated that all of the R region amplified from extraneural tissues had rearrangements similar to those of the Mad-4 strain and that VP1-region amplified products were similar to the Mad-1 strain. In the brain specimens from two PML patients, we found a unique rearranged R region, along with a VP1 region of JCV type 2. In addition, an almost unique variant with multiple rearrangements in the R region and unusual base mutations in the VP1 region was detected in the brain sample from another PML patient. The data indicate that diffuse visceral involvement of JCV is particularly frequent in AIDS patients with PML. Moreover, the presence of rearrangements and mutations, involving different regions of the viral genome, observed in PML-affected brain tissues, could represent a risk factor for the development of PML in immunosuppressed individuals.

This study was to determine whether individual rotavirus capsid proteins could stimulate protection against rotavirus shedding in an adult mouse model. BALB/c mice were intranasally or intramuscularly administered purified Escherichia coli-expressed murine rotavirus strain EDIM VP4, VP6, or truncated VP7 (TrVP7) protein fused to the 42.7-kDa maltose-binding protein (MBP). One month after the last immunization, mice were challenged with EDIM and shedding of rotavirus antigen was measured. When three 9-microg doses of one of the three rotavirus proteins fused to MBP were administered intramuscularly with the saponin adjuvant QS-21, serum rotavirus immunoglobulin G (IgG) was induced by each protein. Following EDIM challenge, shedding was significantly (P = 0.02) reduced (i.e., 38%) in MBP::VP6-immunized mice only. Three 9-micrograms doses of chimeric MBP::VP6 or MBP::TrVP7 administered intranasally with attenuated E. coli heat-labile toxin LT(R192G) also induced serum rotavirus IgG, but MBP::VP4 immunization stimulated no detectable rotavirus antibody. No protection against EDIM shedding was observed in the MBP::TrVP7-immunized mice. However, shedding was reduced 93 to 100% following MBP::VP6 inoculation and 56% following MBP::VP4 immunization relative to that of controls (P = <0.001). Substitution of cholera toxin for LT(R192G) as the adjuvant, reduction of the number of doses to 1, and challenge of the mice 3 months after the last immunization did not reduce the level of protection stimulated by intranasal administration of MBP::VP6. When MBP::VP6 was administered intranasally to B-cell-deficient microMt mice that made no rotavirus antibody, shedding was still reduced to <1% of that of controls. These results show that mice can be protected against rotavirus shedding by intranasal administration of individual rotavirus proteins and that this protection can occur independently of rotavirus antibody.

A single injection of syngeneic islet cells into the thymus of 4-week-old NOD/Lt female mice strongly retards diabetogenesis. The present study used the intrathymic route of antigen administration to compare the relative efficacy of peptides/proteins derived from two major candidate pancreatic beta-cell autoantigens, insulin and GAD65, to modulate diabetogenesis. Intrathymic administration of insulin B chain or recombinant human GAD65 significantly suppressed diabetogenesis during a 20-week follow-up period, whereas no protection was mediated by either insulin A chain or a synthetic peptide (A2) derived from it. Quite unexpectedly, two GAD65-derived peptides near the COOH-terminus (p34 and p35) accelerated diabetes onset. Semiquantitative reverse transcription-polymerase chain reaction analysis was performed on cDNAs from isolated islets or whole pancreases of NOD/Lt females 4 weeks after intrathymic injections. Protection mediated by intrathymic administration with either intact islet cells or GAD65 were correlated with an upregulation of mRNA for T-helper 2 (Th2)-associated cytokines (interleukin [IL]-4, IL-10), concomitant with downregulation of Th1-associated interferon (IFN) transcripts (all normalized to T-cell receptor Cbeta transcripts) in islet-infiltrating lymphocytes. Protection mediated by the intrathymic administration of insulin B chain, however, correlated only with a modest upregulation of IL-4 and IL-10 transcript levels, and no diminution in IFN-gamma transcripts. In contrast, the diabetes-accelerating GAD65 p34 and p35 peptides were not associated with an immune deviation, expressing levels of IFN-gamma characteristic of islet-infiltrating lymphocytes in vehicle-injected NOD controls. Hence, Th1-to-Th2 immune deviation provides only a partial explanation for peptide immunotherapy of diabetes in NOD mice. The finding that certain peptides can accelerate rather than retard diabetogenesis as a function of route and age of administration adds a cautionary note to this type of therapy.

Inhalation anthrax results in high-grade bacteremia and is accompanied by a delay in the rise of the peripheral polymorphonuclear neutrophil (PMN) count and a paucity of PMNs in the infected pleural fluid and mediastinum. Edema toxin (ET) is one of the major Bacillus anthracis virulence factors and consists of the adenylate cyclase edema factor (EF) and protective antigen (PA). Relatively low concentrations of ET (100 to 500 ng/ml of PA and EF) significantly impair human PMN chemokinesis, chemotaxis, and ability to polarize. These changes are accompanied by a reduction in chemoattractant-stimulated PMN actin assembly. ET also causes a significant decrease in Listeria monocytogenes intracellular actin-based motility within HeLa cells. These defects in actin assembly are accompanied by a >50-fold increase in intracellular cyclic AMP and a >4-fold increase in the phosphorylation of protein kinase A. We have previously shown that anthrax lethal toxin (LT) also impairs neutrophil actin-based motility (R. L. During, W. Li, B. Hao, J. M. Koenig, D. S. Stephens, C. P. Quinn, and F. S. Southwick, J. Infect. Dis. 192:837-845, 2005), and we now find that LT combined with ET causes an additive inhibition of PMN chemokinesis, polarization, chemotaxis, and FMLP (N-formyl-met-leu-phe)-induced actin assembly. We conclude that ET alone or combined with LT impairs PMN actin assembly, resulting in paralysis of PMN chemotaxis.

Genetic analysis of autoimmune insulin-dependent diabetes mellitus (IDDM) has focused on genes controlling immune functions, with little investigation of innate susceptibility determinants expressed at the level of target beta cells. The Alloxan (AL) Resistant (R) Leiter (Lt) mouse strain, closely related to the IDDM-prone nonobese diabetic (NOD)/Lt strain, demonstrates the importance of such determinants. ALR mice are unusual in their high constitutive expression of molecules associated with dissipation of free-radical stress systemically and at the beta-cell level. ALR islets were found to be remarkably resistant to two different combinations of beta-cytotoxic cytokines (IL-1beta, tumor necrosis factor alpha, and IFN-gamma) that destroyed islets from the related NOD and alloxan-susceptible strains. The close MHC relatedness between the NOD and ALR strains (H2-Kd and H2-Ag7 identical) allowed us to examine whether ALR islet cells could survive autoimmune destruction by NOD-derived Kd-restricted diabetogenic cytotoxic T lymphocyte clones (AI4 and the insulin-reactive G9C8 clones). Both clones killed islet cells from all Kd-expressing strains except ALR. ALR resistance to diabetogenic immune systems was determined in vivo by means of adoptive transfer of the G9C8 clone or by chimerizing lethally irradiated ALR or reciprocal (ALR x NOD)F1 recipients with NOD bone marrow. In all in vivo systems, ALR and F1 female recipients of NOD marrow remained IDDM free; in contrast, all of the NOD recipients became diabetic. In conclusion, the ALR mouse presents a unique opportunity to identify dominant IDDM resistance determinants expressed at the beta cell level.

Tumor necrosis factor (TNF) is a 17-kDa protein produced by monocytes and a wide variety of other cell types in response to endotoxin and other cytokines. In contrast, lymphotoxin (LT) is a 25-kDa glycoprotein produced only by lymphocytes activated by mitogens. These two cytokines are 28% identical in their amino acid sequences. As they have common cell surface receptors, it is generally assumed that all cellular responses mediated through TNF are also mediated by LT and vice versa. In this report we tested this assumption, comparing the effect of TNF and LT on mediation of early (activation of the transcription factor NF-kappa B) and late (reduction of nitro blue tetrazolium, NBT) cellular responses in the human myelomonoblastic leukemic cell line ML-1a. Both qualitative and quantitative differences were found. LT was found to display 5-10 times more potent antiproliferative effects against murine fibroblasts than TNF. However, in ML-1a cells at concentrations wherein TNF activated NF-kappa B, LT did not. Higher concentrations (1,000-10,000 fold) of LT could activate NF-kappa B, but the activated complex was short lived (less than 1 h versus greater than 6 h when activated by TNF) and required longer treatment (15 min versus less than 5 min). TNF induced NBT-reducing activity in a dose-dependent manner, whereas LT was essentially inactive. Since both TNF and LT have been shown to bind to a common receptor, we tested whether the TNF-induced effects could be blocked by LT. LT inhibited both the early and late TNF-mediated cellular responses. By using receptor-blocking antibodies we found that both p60 and p80 forms of TNF receptors were functional for NBT-reducing activity, but TNF-dependent NF-kappa B activation required only the p60 receptor. Furthermore, we found that both TNF and LT bound with higher affinity to the p80 than to the p60 receptor. Thus, our overall results indicate that there are qualitative and quantitative differences in the action of TNF and LT, and these could be noted quite early in their signaling.

Liver transplantation (LT) has evolved rapidly since the first successful liver transplant performed in1967. Despite a humble beginning, this procedure gained widespread acceptance in the western world as a suitable option for patients with end stage liver disease (ESLD) by the beginning of the 1980s. At present, approximately 25,000 liver transplants are being performed worldwide every year with approximately 90% one year survival. The techniques of living donor liver transplantation (LDLT) developed in East Asia in the 1990s to overcome the shortage of suitable grafts for children and scarcity of deceased donors. While deceased donor liver transplantation (DDLT) constitutes more than 90% of LT in the western world, in India and other Asian countries, most transplants are LDLT. Despite the initial disparity, outcomes following LDLT in eastern countries have been quite satisfactory when compared to the western programs. The etiologies of liver failure requiring LT vary in different parts of the world. The commonest etiology for acute liver failure (ALF) leading to LT is drugs in the west and acute viral hepatitis in Asia. The most common indication for LT due to ESLD in west is alcoholic cirrhosis and hepatitis C virus (HCV), while hepatitis B virus (HBV) predominates in the east. There is a variation in prognostic models for assessing candidature and prioritizing organ allocation across the world. Model for end-stage liver disease (MELD) is followed in United States and some European centers. Other European countries rely on the Child-Turcotte-Pugh (CTP) score. Some parts of Asia still follow chronological order of listing. The debate regarding the best model for organ allocation is far from over.

Synthetically produced Escherichia coli heat-stable toxin (ST) was conjugated to the nontoxic B subunit of the heat-labile toxin (LT) by the carbodiimide reaction. Modifying the molar ratio of toxins mixed and the ratio of carbodiimide added to the toxins permitted synthesis of conjugates with any desired degree of proportional antigenicity for each toxin component. Immunization of rats by the parenteral/peroral routes with cross-linked vaccine containing 39% ST and 61% B subunit antigenicity, with 0.06% residual ST toxicity, evoked fourfold to sevenfold increases over control values of serum IgG and mucosal secretory IgA antitoxin titers to each of the component toxins, thus providing significant (P less than 0.001) protection against challenge with either LT or ST or with viable heterologous strains which produce these toxins. These observations show that cross-linking synthetic ST to the B subunit results in a nontoxic vaccine that provides protection against all types of enterotoxigenic E. coli.

The lipoxygenase-derived leukotrienes (LTs) are important proinflammatory lipid mediators. Lipoxins (LXs), more recently described lipoxygenase products, modulate many proinflammatory actions of LTs and have impressive proresolution properties. Mesangial cell (MC) proliferation is a central event in the pathogenesis of glomerulonephritis. LTD4-induced proliferation of mesangial cells is modulated by LXA4. Here, we demonstrate that LXA4 inhibits PDGF- and LTD4-stimulated proliferation through modulation of platelet-derived growth factor receptor beta (PDGFRbeta) activation. Specifically, we demonstrate that LTD4 transactivates the PDGFRbeta, a process associated with c-src recruitment and ras activation. We demonstrate expression of cysLT1 and cysLT2 receptors in MCs. LTD4-induced c-src activation was insensitive to pertussis toxin and the cysLT1 receptor antagonist Zafirlukast but was blocked by the nonselective antagonist Pobilukast. We show that LXA4 inhibits LTD4-stimulated activation of the PDGFRbeta and that LXA4 modulates PDGF-BB-stimulated tyrosine phosphorylation of the PDGFRb and subsequent mitogenic events. Furthermore, expression of recombinant LXA4 receptor (ALXR) in CHOK1 cells was associated with an attenuation of serum-stimulated proliferation. These data demonstrate that LXA4 receptor (ALXR) activation is accompanied by antimitogenic effects coupled with inactivation of growth factor receptors, highlighting the complex cross-talk between G protein-coupled receptors and receptor tyrosine kinases in an inflammatory milieu. These data elaborate on the profile of cell signaling events that underpin the anti-inflammatory and proresolution bioactions of LX.