The Interaction between Bradyrhizobium Japonicum E109 and Azospirillum Brasilense Az39 Improves Bradyrhizobium-Soybean Symbiosis: The Secrets Behind Co-Inoculation

Aims The aim of this work was evaluating the interaction between two of the most widely used strains for soybean inoculation in Argentina, B. japonicum E109 (BjE109) and A. brasilense Az39 (AbAz39). Methods Five treatments were performed: (i) uninoculated seeds; (ii) seeds inoculated with BjE109; (iii) seeds inoculated with AbAz39; (iv) seeds co-inoculated with BjE109 and AbAz39 in a 1:1 proportion (BjE109 + AbAz39) at the seeds sowing and (v) seeds inoculated with a 1:1 proportion of BjE109 and AbAz39 (BjE109-AbAz39) 24 h before seeds sowing. Each treatment was assessed through a seed recovery assay, glasshouse assays and field assays. Results The combination between the two strains improved the ability of BjE109 to survive on soybean seeds after inoculation partially explained by AbAz39’s capacity to produce diverse biologically active molecules. As a result of the greater rhizobial survival on seeds the nodulation values and symbiosis parameters like nodule number, size and biomass and nodulation percentage also increased. In agreement with these observations, combining BjE109 and AbAz39 at strains the grain yield under field conditions were 13,3 and 17,3% greater than single BjE109 inoculation. Conclusions These results here show that the pre-culture combining BjE109 and AbAz39 before the inoculation to the soybean seeds has benefits in plant nodulation and hence production, more than individual inoculation with BjE109 or AbAz39, or the immediate co-inoculation of both strains.

Microbial-Derived Metabolites Induce Epithelial Recovery Via the Sting Pathway in Mice and Men and Protect from Graft-Versus-Host Disease

Background: Graft-versus-host disease (GVHD) is a dreaded complication after allogeneic stem-cell transplantation (ASCT). Previously, we and others showed that activation of type I interferon (IFN-I) inducing pathways such as RIG-I/MAVS or cGAS-STING can promote the integrity of the intestinal barrier and limit GVHD (Fischer et al., Sci Transl Med, 2017). However, the signals that drive these protective IFN-I responses are poorly understood. Commensal microbiota can (i) have distant effects on immune responses through modulation of IFN-I signaling (Steed et al, Science, 2017; Swimm et al, Blood, 2018) and (ii) predict mortality in ASCT patients (Peled et al., N Engl J Med, 2020). We hypothesized that microbiota-derived products such as microbial metabolites engage IFN-I signaling in immune and non-immune cells poising them for induction of protective responses. We established a prospective, multi-centric clinical study in patients newly diagnosed with acute leukemia and performed longitudinal stool sampling to track changes in microbial community composition and metabolites expression levels. Submitted as a separate abstract to ASH 2021 (Orberg & Meedt et al.), we show that patients with high metabolite expression going into ASCT are less likely to develop GVHD. In this study, we translate our clinical observations to mouse models of acute GVHD and human and mouse intestinal organoids to uncover the molecular mechanisms via which metabolites protect the intestinal barrier during ASCT. Methods: Stool samples from ASCT patients were obtained in Munich and at Regensburg in accordance to IRB-approved study protocols. Patients were sampled at initial diagnosis (Dx), prior to conditioning and weekly after ASCT up to day 28. We analyzed samples by 16S rRNA sequencing and mass spectrometry to obtain a complete picture of microbiome composition and function. Next, we tested metabolites which we detected in patients (desaminotyrosine [DAT], indole-3-carboxaldehyde [ICA]) as treatment in preclinical models in ASCT and GVHD mouse models. Outcomes were assessed by a novel organoid recovery assay in addition to established read-outs. To obtain a mechanistic understanding of the signaling pathways involved, we stimulated WT or IFN-I-signaling-impaired mouse (incl. STING -/-, MAVS -/-, IFNαR -/-) as well as human intestinal crypt-derived organoids with metabolites. Results: Here, we present a 64-year-old female patient diagnosed with AML who received a 9/10 HLA-matched ASCT. At day 7, i.v. antibiotics were started due to fever and Enterococcus bacteraemia. At day 15, the patient developed skin and GI GVHD (Glucksberg III). At the timepoint initial diagnosis (Dx), i.e. the timepoint when we diagnosed AML but before therapy was initiated, we detected rich alpha diversity (Panel 1a) in the patient's stool. Flavonifractor plautii, a producer of the metabolite DAT, was detectable (Panel 1b). We observed high-level expression of metabolites including short-chain fatty acids, DAT, ICA and secondary bile acids (Panel 1c). Following ASCT, and especially at the early time-points day 0 and day 7, alpha diversity and metabolite expression declined drastically. We confirmed this trend in our multi-centric cohort of ASCT patients by comparing levels of DAT and ICA sampled at admission to the transplantation ward (Conditioning) versus at clinical diagnosis of GVHD: in patients with GVHD, metabolite levels were drastically reduced (Panel 2). Next, we prophylactically administered metabolites in a major mismatch mouse ASCT model. Metabolite-treated mice showed significantly showed better outcomes in our organoid recovery assay, which measures the ability of intestinal stem cells to recover after allogeneic injury (Panel 3). This effect that was abrogated in STING -/- recipients. Metabolite stimulation of mouse small intestinal organoids promoted organoid numbers and size, and required intact STING signaling (Panel 4a). Human colon organoids also responded to DAT and ICA, however the metabolite effect was lost when co-administered with the STING-inhibitor H151. Conclusions: We identify that microbial-derived metabolites detected in patients can engage the STING pathway in humans and mice to confer resistance from immune damage. Thus, prophylactic administration of metabolite cocktails or bacterial consortia that can produce these metabolites may reduce occurrence of GVHD in ASCT patients. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Neutralizing Anti-interferon-γ Autoantibodies; An Ameliorating Factor in COVID-19 Infection?

Background: Elevated levels of interferon-gamma (IFNγ) have been found in COVID-19 infection, however its role in this setting remains poorly understood. Cases of non-tuberculous mycobacterium (NTM) infections due to anti-IFNγ autoantibodies (Ab) were first reported in 2004. NTM and COVID-19 co-infection in a patient with acquired IFNγ deficiency has not previously been described. The impact of anti-IFNγ Ab on the severity of COVID-19 has not been previously explored. Objective: We report a case of COVID-19 infection in a patient hospitalised with NTM infection due to acquired IFNγ deficiency caused by anti-IFNγ Ab. We also explore effects of IFNγ Ab on the severity of COVID-19 infection. Methods: Detailed immunological investigations were performed. Bio-rad, Bio-Plex methodology was used to detect anti-IFNγ Ab, titration, IFNγ recovery assay and SARS-CoV-2 serology. Anti-IFNγ Ab were tested in patients with severe (COV-Pat) and health care workers with mild/asymptomatic COVID-19 infection (COV-Asx). Results: Mycobacterium avium intracellulare was diagnosed following bone marrow examination and culture. High titre anti-IFNγ Ab were detected in patient’s serum. The autoantibodies neutralized both endogenously produced and exogenously administered IFNγ. SARS-COV-2 infection was identified during routine inpatient testing. Despite prolonged SARS-COV-2 infection the patient showed only minimal additional symptoms, never developed any significant inflammatory complications and eventually mounted an adequate IgG antibody response to the SARS-COV-2 trimeric S-protein. Elevated titres of anti-IFNγ Ab were detected in COV-Pat and COV-Asx, compared to non-infected healthy controls. The titres were broadly similar between COV-Pat and COV-Asx groups, but much lower compared to patients with acquired IFNγ Ab deficiency. Conclusion: IFN-γ is known to play a central role in hyperinflammatory disease states such as macrophage activation syndrome This study illustrates the potential value of inhibiting IFNγ to prevent pathological inflammatory response to COVID-19.

Ehl Factors at Lower Than Standard Dose Achieve Satisfactory Surgical Haemostatsis in Haemophilia

Patients and methods All patients with haemophilia A or B who underwent major surgery, either as elective or as emergency, with rFVIIIFc or rFIXFc as the clotting factor concentrate (CFC) for hemostasis were included in this study. None of them were positive for inhibitors before. The factor was administered once daily or once every 36-48 hours depending on the product. The factor assays were done with a one stage APTT method. The CFC support was continued till suture removal, usually 10 days. Other supportive measures included tranexamic acid,at physician discretion. Results A total of 57 patients underwent surgery. There were 45 patients with haemophilia A and 12 with haemophilia B. Of those with haemophilia A, - 37 (82.2%) had severe, 3 (11.1%) moderate and 5 (6.7%) mild disease. Of those with haemophilia B, 7 (58.3%) had severe,3 (25 %) moderate and 2 (16.7%) mild disease. The median age was 30 years (range: 1-70). Among those with haemophilia A, there were 4 with inhibitor titre <5 BU, 3 more developed inhibitor 3-7 days following surgery, 1 of >5 BU, requiring bypassing agents. None with haemophilia B had inhibitor. The surgical procedures included 40 (70.1%) orthopaedic, 6 (10.5%) abdomino-perineal, 4(7%) urological and 3(5.2%) neurosurgical and 1(1.7%) each - venricular septal defect repair, skin graft, tympanoplasty and coronary angioplasty. The orthopaedic surgeries included pseudotumour excision (n=9), bilateral total knee replacement (bilateral n=4, unilateral n=1), total hip replacement (n=3), open reduction and internal fixation of fracture (n=11), corrective osteotomies/soft tissue release (n=6), amputation (n=1), arthrodesis (n=1), laminectomy (n=1), implant removal (n=1),intra articular repair(n=1)and curettage (n=1). For those with haemophilia A, the median recovery assay pre-op was 108% (range: 50-200).The median trough levels for post- operative days +1,+3,+5 ,+7 were 40% (11-138), 36.5% (13-94), 36.4% (1.4-118), and 27%(1.6-77) respectively. Among 35 patients with severe haemophilia undergoing major surgery, the median total factor used was 305 (125-563)IU/kg. The median recovery assay for those with haemophilia B prior to surgery was 70.1%(50-165).The median trough levels for post operative days+1,+3,+5 ,+7 were 49.8% (28-70.7), 40.7%(31.6-56.2), 42.3%(11.6-58.2), and 40.5%(24.5-59.4) respectively. The median total factor used was 400 (134-1000)IU/kg. Seventeen of those with haemophilia A (37.8%) required transfusion of at least 1 packed red cell each as per expected blood loss in those surgeries. Two patients required FFP and 1 patient required platelet transfusion as well for DIC. One patient had sustained injury to axillary artery during surgery and the other one had undergone resection of extensive pseudotumour. There was no unexpected post-operative bleeding. In those with haemophilia B, 2(16.7%) patients required transfusion of packed red cell as per expected blood loss in those surgeries. No other blood product was required. There was no excessive bleed during the perioperative period. Conclusions Modest doses of rFVIIIFc and rFIXFc can be used safely for major surgery in haemophilia with effective haemostasis and monitored with one stage APTT based assays. The less frequent administration makes these agents a more convenient option. Disclosures No relevant conflicts of interest to declare.

A new genre of fluorescence recovery assay to evaluate polo-like kinase 1 ATP-competitive inhibitors

Using a probe consisting of a fluorescein-labeled variant of the potent polo-like kinase 1 (Plk1) inhibitor BI2536 , we determined the IC50 of ATP-competitive Type 1 inhibitors of Plk1 by means of a fluorescence recovery assay.

Effects of sublethal and realistic concentrations of the commercial herbicide atrazine in Pacu (Piaractus mesopotamicus): Long-term exposure and recovery assays

Background and Aim: The commercial formulations of the herbicide atrazine (cATZ) are widely employed in Brazilian agriculture, and, as a consequence, ATZ has been found at levels above that established by law in the river basins in Brazil. Although the toxicity of ATZ in fish is well documented, there are few studies on the recovery capacity after cATZ exposure. This work aimed to evaluate, using several biomarkers, the toxic effects of long-term exposure to the sublethal (3.57 mg/L) and nonlethal realistic (3.00 μg/L) cATZ concentrations followed by a recovery assay, in fingerlings of a Brazilian teleost, the Piaractus mesopotamicus (pacu). Materials and Methods: Pacu fingerlings were housed in glass tanks and divided into the following experimental groups (two tanks/group): Exposure control = EC, recovery control = RC, the sublethal groups exposed to 3.57 mg/L of cATZ, (sublethal exposure group = SLE and sublethal recovery group = SLR) and the nonlethal groups treated with 3.00 μg/L of cATZ (nonlethal exposure group = NLE and nonlethal recovery group = NLR). The exposure assay was semi-static with a duration of 30 days and the recovery assay (after cATZ withdrawal) lasted 14 days. Several biomarkers were evaluated in fingerlings from all groups: The swimming behavior, the body weight gain, the micronucleus formation and nuclear alterations in erythrocytes, and the hepatic and renal histopathology analyzed by qualitative and semi-quantitative morphological methods (using light and electron microscopy). Results: No significant difference in weight gain was observed among the groups after the exposure and recovery assays. The sublethal exposure induced impaired swimming movements, significant histopathological alterations, including necrosis in the liver and kidney, and a significant increase in the frequency of micronuclei in erythrocytes. The nonlethal exposure induced only subtle histopathological changes in the liver and kidney. After recovery assay, no genotoxic alteration was noted in pacu exposed to sublethal concentration, while the cATZ-induced kidney damage was partially reversed but not the hepatic injury. Conclusion: cATZ exhibits long-term toxic effects on pacu, even at relatively low concentrations, affecting mainly the liver and the kidney, and the effects of sublethal concentration are only partially reversed after cATZ withdrawal.

In vitro efficacy of Duddingtonia flagrans against nematodes of sheep based on in vivo calculations

Duddingtonia flagrans has been tested as an alternative parasite control, but data from in vitro experiments based on in vivo calculations describing nematophagous fungi predation in nematodes are restricted. The objective of this work was to determine the efficacy of D. flagrans against sheep nematode larvae in vitro using in vivo calculations. Fecal samples were introduced to fungi in different concentrations: 0.0/control; 0.05; 0.1; 0.2; 0.4; 0.8; 1.6; 3.2; and 6.4 g corresponding, respectively, to 583.000; 1.166.000; 2.332.000; 4.664.000; 9.328.000; 18.656.000; 37.312.000 and 74.624.000 chlamydospores/kg of body weight. The material was incubated for 14 days, before the larvae recovery (Assay 1). Assay 2 was carried out with the doses of 0.00625; 0.0125; and 0.025 g. The results showed a negative correlation between fungal concentrations and larval numbers for both assays. The fungus demonstrated an efficacy above 89% in both assays. Thus, we consider that the data from in vitro studies based on in vivo calculations may optimize the fungi quantities for field experiments.