Finding of extended-spectrum beta-lactamase (ESBL)-producing Enterobacterales in wild game meat originating from several European countries: predominance of Moellerella wisconsensis producing CTX-M-1, November 2021

IntroductionMeat can be a vehicle for food-borne transmission of antimicrobial resistant bacteria and antimicrobial resistance genes. The occurrence of extended‐spectrum beta‐lactamase (ESBL) producing Enterobacterales has been observed in meat from livestock production but has not been well studied in meat from wild game.AimWe aimed to investigate, particularly in central Europe, to what extent ESBL-producing Enterobacterales may be present in wild game meat.MethodsA total of 111 samples of different types of game meat supplied by butchers, hunters, retail stores and a large game-processing establishment in Europe were screened for ESBL-producing Enterobacterales using a selective culture medium. Isolates were genotypically and phenotypically characterised.ResultsThirty-nine samples (35% of the total) yielded ESBL-producing Enterobacterales, with most (35/39) supplied by the game-processing establishment. Isolates included 32 Moellerella wisconsensis, 18 Escherichia coli and one Escherichia marmotae. PCR screening identified bla _CTX-M-1 (n = 31), bla _CTX-M-32 (n = 8), bla _CTX-M-65 (n = 4), bla _CTX-M-15 (n = 3), bla _CTX-M-8 (n = 1), bla _CTX-M-14 (n = 1), bla _CTX-M-55 (n = 1), and bla _SHV-12 (n = 2). Most E. coli belonged to phylogenetic group A (n = 7) or B1 (n = 9), but several isolates belonged to extraintestinal pathogenic E. coli (ExPEC) sequence types (ST)58 (n = 4), ST68 (n = 1) and ST540 (n = 1). Whole genome sequencing of six selected isolates localised bla _CTX-M-1 on megaplasmids in four M. wisconsensis and bla _CTX-M-32 on IncN_1 plasmids in one M. wisconsensis and one E. marmotae. Forty-eight isolates (94%) exhibited a multidrug-resistance phenotype.ConclusionWe found a high occurrence of ESBL-producing Enterobacterales in wild game meat, suggesting wildlife habitat pollution and possible microbial contamination events occurring during skinning or cutting carcasses.     

Protein A adsorption of acute myelogenous leukemia serum induces in vitro blast lysis

We studied cytotoxic activity of acute myelogenous leukemia (AML) sera for AML blasts before and after immunoadsorption with Staphylococcus aureus, Cowan I (SAC), which contains protein A. We found in vitro that incubation with treated AML sera reduced viability to 42.7% of control (p less than 0.01) for autologous and 21.0% of control (p less than 0.01) for allogeneic blasts. Normal peripheral blood cells were not killed by treated AML sera. Wood 46 strain of Staphylococcus aureus, which does not contain protein A, did not significantly reduce AML blast viability (94.8%, p greater than 0.4), while Sepharose-bound protein A reduced viability to 63.8% (p less than 0.01). Cytotoxicity does not appear to be complement-mediated, byt cytotoxic activity is trypsin-sensitive and is contained in the immunoglobulin fraction. This model for study of the tumoricidal action of protein A adsorption should be useful for predicting utility of plasma adsorption as a therapeutic adjunct in the future.     

Chromosomal abnormalities in untreated patients with non-Hodgkin's lymphoma: associations with histology, clinical characteristics, and treatment outcome. The Nebraska Lymphoma Study Group

We describe the chromosomal abnormalities found in 104 previously untreated patients with non-Hodgkin's lymphoma (NHL) and the correlations of these abnormalities with disease characteristics. The cytogenetic method used was a 24- to 48-hour culture, followed by G- banding. Several significant associations were discovered. A trisomy 3 was correlated with high-grade NHL. In the patients with an immunoblastic NHL, an abnormal chromosome no. 3 or 6 was found significantly more frequently. As previously described, a t(14;18) was significantly correlated with a follicular growth pattern. Abnormalities on chromosome no. 17 were correlated with a diffuse histology and a shorter survival. A shorter survival was also correlated with a +5, +6, +18, all abnormalities on chromosome no. 5, or involvement of breakpoint 14q11-12. In a multivariate analysis, these chromosomal abnormalities appeared to be independent prognostic factors and correlated with survival more strongly than any traditional prognostic variable. Patients with a t(11;14)(q13;q32) had an elevated lactate dehydrogenase (LDH). Skin infiltration was correlated with abnormalities on 2p. Abnormalities involving breakpoints 6q11-16 were correlated with B symptoms. Patients with abnormalities involving breakpoints 3q21-25 and 13q21-24 had more frequent bulky disease. The correlations of certain clinical findings with specific chromosomal abnormalities might help unveil the pathogenetic mechanisms of NHL and tailor treatment regimens.     

Biochemical, immunological, and in vivo functional characterization of B-domain-deleted factor VIII

Coagulation factor VIII (FVIII) is a cofactor in the intrinsic pathway of blood coagulation for which deficiency results in the bleeding disorder hemophilia A. FVIII contains a domain structure of A1-A2-B-A3- C1-C2 of which the B domain is dispensable for procoagulant activity in vitro. In this report, we compare the properties of B-domain-deleted FVIII (residues 760 through 1639, designated LA-VIII) to wildtype recombinant FVIII. In transfected Chinese hamster ovary (CHO) cells, LA- VIII was expressed at a 10- to 20-fold greater level compared with wildtype FVIII. The specific activity of purified LA-VIII was indistinguishable from wild-type recombinant FVIII and both exhibited similar thrombin activation coefficients. Wildtype recombinant-derived FVIII and LA-VIII also displayed similar timecourses of thrombin activation and heavy chain cleavage. However, compared with wildtype recombinant-derived FVIII, the light chain of LA-VIII was cleaved fivefold more rapidly by thrombin. Addition of purified von Willebrand factor (vWF) did not alter the kinetics of thrombin cleavage or activation of either wildtype recombinant-derived FVIII or LA-VIII. The immunogenicity of LA-VIII was compared with wildtype FVIII in a novel model of neonatal tolerance induction in mice. The results did not detect any immunologic differences between wildtype FVIII and LA-VIII, suggesting that LA-VIII does not contain significant new epitopes that are absent in wildtype FVIII. LA-VIII was tolerated well on infusion into FVIII-deficient dogs and was able to correct the cuticle bleeding time similar to wildtype recombinant factor VIII. In vivo, LA-VIII was bound to canine vWF and exhibited a half-life similar to wildtype recombinant FVIII. These studies support that B-domain-deleted FVIII may be efficacious in treatment of hemophilia A in humans.     

Production of granulocyte-macrophage colony-stimulating factor by primary cultures of unstimulated rat microvascular endothelial cells

Small vessel (microvascular) endothelial cells are in close contact with hematopoietic progenitor cells in the bone marrow and therefore may have an important role in hematopoiesis. Although other studies have shown that endothelial cells produce various colony-stimulating factors (CSFs), these studies examined large vessel endothelial cells, which are different in many respects from microvascular endothelial cells and which do not contact cells in the bone marrow. We show in this study that primary cultures of unstimulated rat fat capillary endothelial cells grown in serum-free medium produce a substantial amount of granulocyte-macrophage CSF (GM-CSF). The medium conditioned by these cells stimulated proliferation of two different lines of GM- CSF-responsive cells--PT-18 mast cells and FDC-P1 cells--and supported the growth of cells of the granulocyte and macrophage lines in cultures of rat bone marrow cells. The factor responsible for this activity had physical properties consistent with those of GM-CSF, namely, a similar apparent mol wt by gel filtration, resistance to repeated freeze-thaws, resistance to boiling for ten minutes but not for 30 minutes, and resistance to heating to 56 degrees C for one hour. The factor causing target cell stimulation was not B cell-stimulating factor-1 (BSF-1, or IL 4), since it failed to stimulate a BSF-1-responsive cell line HT2- JH, and target cells (PT-18) did not respond appreciably to recombinant BSF-1. Northern blot analysis of mRNA from rat fat capillary endothelial cells showed high levels of expression of GM-CSF, confirming that this factor is produced by microvascular endothelial cells. This is the first report of CSF production by unstimulated microvascular endothelial cells, demonstrating that these ubiquitous cells are capable of producing sizable amounts of at least one growth factor for hematopoietic progenitor cells.     

Irradiation induces release of von Willebrand protein from endothelial cells in culture

Human umbilical vein endothelial cells in tissue culture were irradiated with doses between 0 and 40 Gy, and the released von Willebrand (vW) protein and that which remained associated with the cells was quantitated. Doses of 20 Gy and higher produced a statistically significant increase in amount of vW protein secreted. This release was present whether the cells were labeled continuously throughout the experiment or just prelabeled before irradiation. An increase in fibronectin secretion was not observed. The release response to radiation was slow, reaching significance close to 24 hours after irradiation. The release of vW protein was not due to cell lysis, because the secreted vW protein contained very little of the large 260- kilodalton vW precursor subunit present in cell lysates and the cells appeared intact by immunofluorescence staining.     

High-dose therapy and autologous peripheral blood stem cell transplantation for patients with lymphoma

Forty patients with refractory Hodgkin's disease (24 patients) or non- Hodgkin's lymphoma (16 patients) who were considered for high-dose therapy but not for autologous bone marrow transplantation (ABMT) due to BM metastases, previous pelvic irradiation, a history of marrow involvement by tumor or hypocellular marrow in conventional harvest sites received high-dose therapy and autologous peripheral blood (PB) hematopoietic stem cell transplantation. Disappearance of circulating neutrophils and development of RBC and platelet transfusion-dependence was followed, in the evaluable patients, by reappearance of 0.5 x 10(9)/L circulating granulocytes and sufficient platelets to obviate the need for platelet transfusions at a median of 25 days after transplantation. Twenty-three patients experienced a clinical complete remission (CR). The projected 2-year event-free survival was 24% for all 40 patients and 49% for the non-Hodgkin's lymphoma patients. The projected 18-month event-free survival for the Hodgkin's disease patients was 15%. PB stem cell transplantation provided an opportunity to administer high-dose salvage therapy to patients with refractory lymphoma who otherwise were not candidates for such therapy. For some of those patients, the high-dose therapy produced prolonged survival, free of tumor progression.     

Autologous peripheral hematopoietic stem cell transplantation restores hematopoietic function following marrow ablative therapy

From ten patients with advanced malignant disease involving the bone marrow, autologous hematopoietic stem cells were collected from the peripheral blood during eight four-hour pheresis procedures and cryopreserved. No manipulations to increase the number of stem cells circulating in the blood were used during the collections. Following marrow ablative chemotherapy or chemoradiotherapy, the autologous cells were thawed and infused intravenously (IV). WBCs reappeared in the circulation at a median of eight days (range seven to 11 days) after stem cell infusion. Two patients died early, whereas the other eight reached normal numbers of circulating granulocytes that have persisted for up to greater than 20 months. These eight patients became independent of RBC transfusions (hemoglobin concentration greater than 10 g/dL) at a median of 27 days (range 11 to 58 days) after transplantation. One patient received platelet transfusions for counts less than 50 x 109)/L, one patient developed a clinical picture of idiopathic thrombocytopenic purpura, and six patients maintained a platelet count greater than 20 x 10(9)/L at a median of 23 days (range 14 to 25 days) following stem cell infusion. This technique allows patients ineligible for autologous bone marrow transplantation due to unacceptable anesthetic risks, prior pelvic irradiation, or bone marrow metastases to receive marrow ablative therapy.     

Blood cell dynamics in P-selectin-deficient mice

P-selectin is expressed on the surfaces of activated platelets and endothelium where it mediates binding to leukocytes. P-selectin- deficient mice were shown to exhibit peripheral neutrophilia (Mayadas et al: Cell 74:541, 1993). We now show that this is not caused by changes in bone marrow precursors nor by a lack of neutrophil margination. Both P-selectin-positive and -negative animals displayed similar increases in peripheral blood neutrophil numbers after injection of epinephrine. However, clearance of 51Chromium-labeled neutrophils is delayed in mice deficient for P-selectin, indicating that the neutrophilia is at least in part the result of delayed removal. We detected no obvious alterations in lymphocyte differentiation, distribution, or adhesion to high endothelial venules in peripheral lymph nodes. Through intravital microscopy, we examined the impact of P-selectin deficiency on leukocyte/endothelial interaction beyond the initial stages of inflammation. Four hours after the administration of an inflammatory irritant, leukocyte rolling was observed even in the absence of P-selectin. There were significantly fewer rolling cells relative to wild-type mice, and their velocity was reduced. Moreover, in the peritonitis model, the number of peritoneal macrophages in wild-type mice increased threefold at 48 hours, whereas the macrophages in the mutant mice remained near baseline levels. Thus, whereas P-selectin is known to be involved in early stages of an inflammatory response, our results indicate that it is additionally responsible for leukocyte rolling and macrophage recruitment in more prolonged tissue injury.