Blood feeding in juvenile Paragnathia formica (Isopoda: Gnathiidae): biochemical characterization of trypsin inhibitors, detection of anticoagulants, and molecular identification of fish hosts.

The 3 post-marsupial juvenile stages of the gnathiid isopod, Paragnathia formica, are haematophagous ectoparasites of fishes that may, in heavy infestations, cause host mortality. Protein digestion in fed stage 3 juveniles is accomplished by cysteine proteinases, but what bioactive compounds attenuate host haemostatic, inflammatory and immunological responses during feeding is unknown. Trypsin inhibitory activity and anticoagulant activity were detected in crude extracts of unfed P. formica stage 1 juveniles; fractionation of stage 1 crude extracts by ion exchange chromatography resulted in 3 preparations each displaying these bioactivities. Further characterization revealed anti-thrombin activity in 2 of these preparations, whilst the third displayed the strongest anticoagulant activity that targeted a factor of the intrinsic coagulation pathway. Three trypsin inhibitors (18 kDa, 21 kDa, and 22 kDa) were also detected using reverse zymography. In parallel, homogenates of fed stage 2 and 3 juveniles were used to identify their fish hosts by amplifying the 16S mitochondrial rDNA and 18S genomic rDNA vertebrate gene regions. Blood from at least 4 fish families had been ingested by separate individuals during feeding. This study demonstrates that trypsin inhibitors and anticoagulants are present in P. formica juveniles which could suppress host haemostatic, inflammatory and immunological responses during feeding, and that juveniles are not host specific.

Early but reversible haemostatic changes in a-symptomatic females expressing COVID-19 antibodies.

The coronavirus, COVID-19 pandemic spread across the globe in 2020, with an initial high case mortality in those requiring intensive care treatment due to serious complication. A vaccine programme was quickly developed and currently the UK is one of highest double vaccinated and boosted countries in the world. Despite tremendous efforts by the UK, new cases of COVID-19 are still occurring, due to viral mutation. A major problem associated with COVID-19 is the large a-symptomatic spread within the population. Little investigation into the a-symptomatic population has been carried out and therefore we pose that the residual effects of a-symptomatic infection is still largely unknown. Prior to mass vaccination, a multi-phased single cohort study of IgM and IgG COVID-19 antibody prevalence and the associated haemostatic changes were assessed in a Welsh cohort of 739 participants, at three time points. Positive antibody participants with age and gender matched negative antibody controls were assessed at 0, 3 and 6 months. Antibody positive females appeared to have lower antibody responses in comparison to their a-symptomatic male counterparts. Despite this initial testing showed a unique significant increase in TRAP-6-induced platelet aggregation, prothrombin time (PT) and clot initiation time. Despite coagulation parameters beginning to return to normal at 3 months, significant decreases are observed in both haemoglobin and haematocrit levels. The production of extracellular vesicles (EV) was also determined in this study. Although the overall number of EV does not change throughout the study, at the initial 0 months' time point a significant increase in the percentage of circulating pro-coagulant platelet derived EV is seen, which does not appear to be related to the extent of platelet activation in the subject. We conclude that early, but reversible changes in haemostatic pathways within the a-symptomatic, female, antibody positive COVID-19 individuals are present. These changes may be key in identifying a period of pro-coagulative risk for a-symptomatic female patients.

Changes to colour vision on exposure to high altitude.

OBJECTIVES: Several studies have shown deterioration in colour vision at altitudes above 3,000m. These studies have been conducted in photopic (bright daylight) conditions, whereas many military operations take place in mesopic (dim light) conditions. Data suggests that the tritan colour vision axis (blue cones, TA) are more susceptible to hypoxic insult than protan axis (red cones, PA). The objective of this study was to examine colour vision at high altitude, in mesopic conditions, and using a novel method of assessment to discriminate between the tritan and protan axis. METHODS: We examined 42 eyes (21 subjects, mean age 44, range 22-71), at sea level and within 12-36 hours of exposure to 3300m. This was done in a darkened room, with refractive error correction. Colour vision was studied using ChromaTest, a software programme that analyzes colour contrast threshold (CCT) of both TA and PA. We planned to repeat CCT measurement at 4,392m, but technology failure prevented this. Non-parametric paired data was examined using the Wilcoxon signed rank test. RESULTS: There was found to be no change to either the PA (p = 0.409) or the TA (p = 0.871) upon ascent. Within the PA 16 eyes had a lower CCT at high altitude, whilst 26 were higher. In the TA 20 eyes had a lower CCT and 22 were higher. At sea level, mean CCT for PA was 4.21 (SD 2.29) TA was 7.06 (SD 1.77). At 3,300m mean CCT for PA was 4.36 (SD 2.86) and TA was 6.93 (SD 2.39). CONCLUSIONS: This experiment revealed no changes to colour vision with exposure to 3,300m. This may be below the threshold altitude for cone dysfunction, alternatively colour vision deterioration may be less significant in mesopic conditions.

The capacity of mitogen-responsive T cells to "home" to their tissue of origin, analyzed by means of chromosome markers.

Lance and Taub (1) showed that when radioactively labeled lymphocytes were injected into a syngeneic mouse and the lymph node cells of this animal transferred to a second syngeneic recipient, the proportion of radioactivity found in the lymph node relative to the amount present in the spleen of the secondary recipient had increased markedly. The interpretation of this result was that some lymphocytes have the capacity to "home" to their organ of origin. The purpose of the experiments described here was to test the "homing" copacity of T cells by a method that did not involve radioactive labeling. It has been shown elsewhere that some or all mouse T cells are stimulated to divide in culture by the mitogens phytohemagglutinin (PHA) and concanavalin A (Con A) (2). We therefore elected to inject karyotypically distinct lymphocytes into syngeneic recipients and to follow their subsequent distribution by culture of lymph node and spleen cells of the recipient with PHA or Con A. In this manner the homing capacities of spleen and lymph node T cells could be determined, and furthermore, the effects of labeling with chromium-51 ((51)Cr) could be assayed with respect to the persistence of mitogen responsiveness in the injected cells.

3-D ocean particle tracking modeling reveals extensive vertical movement and downstream interdependence of closed areas in the northwest Atlantic.

Novel 3-D passive particle tracking experiments were performed in the northwest Atlantic to elucidate connectivity among areas closed to protect vulnerable marine ecosystems. We examined (1) the degree of vertical movement of particles released at different depths and locations; (2) the location of potential source populations for the deep-sea taxa protected by the closures; and (3) the degree of functional connectivity. A long-term oceanographic dataset (EN4) was queried to characterize the temperature and salinity regimes in each of the closed areas as a basis for interpreting recently published climate change projections. Using the Parcels Lagrangian particle tracking framework and the BNAM hydrodynamic model, we found enhanced connectivity over previously developed 2-D models and unexpected, current-driven, strong (to a maximum of about 1340 m) downward displacement at depth (450, 1000 and 2250 m), with weaker upward displacement except for the release depth of 2250 m which showed upward movement of 955 m with a drift duration of 3 months. The current velocities create down-stream interdependence among closed areas and allow redundancy to develop in some of the areas of the network, with some of the larger areas also showing retention. Source populations for sponges in the upstream closure are likely in adjacent waters of the Canadian continental shelf. Collectively this information can be used to inform management decisions related to the size and placement of these closed areas, and vertical velocity surfaces have potential for use in species distribution modeling of benthic species and habitats.

First record of a Kabatana sp. microsporidium infecting fish in the Atlantic Ocean.

Two-spotted goby Gobiusculus flavescens from the Swedish Gullmarsfjord regularly present subcutaneous creamy-white patches in the body musculature, associated with Kabatana sp. infection. Analysis of the 16S rRNA gene of the microsporidium showed 98.54% homology with Kabatana newberryi infecting a marine goby from California, indicating that the Swedish microsporidium is either a different strain of K. newberryi or a closely related species. This represents the first record of a Kabatana species in the Atlantic Ocean. The genetic similarity of the 2 microsporidia was paralleled by close infection phenotypes. Infected muscle fibres were swollen compared to adjacent non-infected fibres, and mature spore masses were found throughout the skeletal musculature. No xenoma formation was detected. Since G. flavescens is an established model species in behavioural ecology, the host-parasite system is ideally suited for testing how microsporidian infections affect host behaviour and fitness.

Radioimmunotherapy for B-cell lymphoma: Y90 ibritumomab tiuxetan and I(131) tositumomab.

Radioimmunotherapy, targeting the CD20 antigen, in B-cell lymphoma has clearly demonstrated efficacy and tolerability over the preceding 15 years. As a result, two products are available with Food and Drug Administration approval for marketing - Y(90) ibritumomab tiuxetan and I(131) tositumomab, given as the Zevalin and Bexxar therapeutic regimens, respectively. Both demonstrate high-response rates and durability of remission in the relapsed/refractory disease setting. Data are emerging regarding their utility as initial therapy, and furthermore, they are been investigated for use sequentially with chemotherapy, and in the myeloablative setting. As yet however, how to best use these agents in the clinical disease course remains uncertain.

Immunological tolerance and the autoimmune response.

The discovery by McIntire and Faulk of masked autoantibodies calls into question the mechanisms of self recognition that exist in the human body. Their finding is reviewed in relation to three other facets of the interaction of humans with the foreign agencies with which they come in contact, innate immunity, microchimaerism and communicable disease-causing infections. It is concluded that the capacity to respond to foreign agencies may not primarily be defensive and that self recognition is probably an active process rather than dependent on elimination of self reactive capacity.

A limited role for TP53 mutation in the transformation of follicular lymphoma to diffuse large B-cell lymphoma.

The role of TP53 mutation in transformation of follicular lymphoma (FL) to diffuse large B-cell lymphoma (t-FL) was examined in a panel of 91 lymph node biopsies derived from 29 patients pre- and post-transformation. The entire TP53 coding sequence was screened and immunocytochemistry performed to determine expression of p53 and its key regulator MDM2. A total of 10 mutations were detected in eight patients (28%), although none were present at FL diagnosis. Mutations were not detected solely at the time of transformation; in three patients, mutated TP53 arose in at least one antecedent FL sample (6 months, 2.5 years and 4 years prior to transformation). Loss of heterozygosity at the TP53 locus occurred in 2/20 informative patients (only in t-FL samples). p53 staining was positive in 82% (9/11) of available biopsies with a missense mutation, and negative in 71% (45/63) with wtTP53. MDM2 expression was significantly higher in t-FL samples (mean 72% positive; 95% confidence interval (95% CI) 68-76%) than FL (mean 58% positive; 95% CI 54-62%) (P<0.001) but did not correlate with TP53 status. TP53 mutation has only a limited role in the transformation of FL, exerting a heterogeneous influence upon phenotypic change. In contrast, dysregulation of MDM2 is frequent and may provide a more rational therapeutic target..

Immune reactivity in the Moloney strain of murine sarcoma virus oncogenesis: requirement of thymus-derived lymphocytes for in vivo protection.

To study the function of different lymphocyte populations in the Moloney strain of murine sarcoma virus (M-MuSV) tumorigenesis, we gave M-MuSV injections to CBA mice selectively deprived of thymus (T) lymphocytes by thymectomy, X-rradiation, and syngeneic bone marrow injection. Although no tumors appeared in the control group, 80% of the derived mice had tumors that grew progressively and ultimately killed them. In deprived mice, grafted with a syngeneic thymus (reconstituted mice) before or after an M-MuSV injection, tumors regressed or did not develop. Histologically, the lymph nodes and spleens of reconstituted mice, compared to those of deprived animals, showed repopulation of the thymus-dependent areas and prominent follicles in the cortex. Moreover, tumor tissue of reconstituted mice was extensively infiltrated by lymphocytes. To evaluate the number of lymphoid cells needed to prevent or regress M-MuSV tumors, we injected varying amounts of lymphoid cells into deprived mice. Even low lymphocyte numbers (10(6) cells) were sufficient to exert, in some cases, protection against M-MuSV tumorigenesis. This effect was not abolished by subsequent splenectomy or antilymphocyte serum treatment. Finally, deprived mice, given repeated injections of antiserum (hyperimmune) against M-MuSV, had tumors which appeared only after a prolonged latency. From these results, it is concluded that T-cell population integrity is important in affording total host protection against the M-MuSV tumors.

Growth of single-layer boron nitride dome-shaped nanostructures catalysed by iron clusters.

We report on the growth and formation of single-layer boron nitride dome-shaped nanostructures mediated by small iron clusters located on flakes of hexagonal boron nitride. The nanostructures were synthesized in situ at high temperature inside a transmission electron microscope while the e-beam was blanked. The formation process, typically originating at defective step-edges on the boron nitride support, was investigated using a combination of transmission electron microscopy, electron energy loss spectroscopy and computational modelling. Computational modelling showed that the domes exhibit a nanotube-like structure with flat circular caps and that their stability was comparable to that of a single boron nitride layer.

Covalent attachment of horseradish peroxidase to the outer surface of liposomes.

We describe a method by which horseradish peroxidase may be attached covalently to the surface of liposomes under conditions which permit minimal non-covalent association of the enzyme with the lipids. The coupling method adopted does not allow the formation of homopolymers of liposomes or peroxidase. For phosphatidylelthanolamine/phosphatidylcholine and stearylamine/phosphatidylcholine and vesicles, minimal disruption of vesicular structure is observed, whilst for phosphatidylserine vesicles, the lipid-protein complex appears to form structures much smaller than 25 nm in diameter. Stearylamine/phosphatidylcholine vesicles have been shown to retain entrapped inulin, and activity measurements for the peroxidase suggest that it is located exclusively on the external surface of the liposome membrane. Peroxidase can be localized histochemically which has permitted the morpholo gical study of the coated liposomes and their interactions with cells.

A comparison of the in vitro and in vivo activities of conjugates of anti-mouse lymphocyte globulin and abrin.

Anti-mouse lymphocyte globulin and normal immunoglobulin have been conjugated to abrin using two procedures, one involving linkage through an amide bond and a piperazine ring and the other the introduction of two amide bonds flanking a disulphide bridge. The four conjugates produced were equipotent as inhibitors of protein synthesis in rabbit reticulocyte lysates. Each antibody-containing conjugate was a more effective inhibitor of protein synthesis in cultured cells than the equivalent normal immunoglobulin-containing conjugate. In addition the conjugates with disulphide linkage groups were ten times more potent than their counterparts. The disulphide conjugates were also twice as toxic to mice in an acute toxicity test but when used to suppress their immune responses to sheep red blood cells it was the non-disulphide-linked conjugates that were superior. In all instances antibody-containing conjugates were more powerful immunosuppressants than those containing normal IgG. The results are taken to indicate a relative lack of stability of the disulphide conjugates in the tissues.

Durable remissions of myelodysplastic syndrome and acute myeloid leukemia after reduced-intensity allografting.

PURPOSE: To evaluate the use of reduced-intensity (RI) conditioning with allogeneic hematopoietic stem cell transplantation (HSCT) from HLA-identical family donors in patients with myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). PATIENTS AND METHODS: Sixteen patients (median age, 54 years; range, 37 to 66 years) underwent RI-HSCT using a conditioning regimen of fludarabine 25 mg/m2 daily for 5 days and either cyclophosphamide 1 g/m2 daily for 2 days (14 patients) or melphalan 140 mg/m2 for 1 day (two patients). The median number of CD34+ cells and CD3+ cells infused per kilogram of recipient weight was 4.5 x 106 (range, 1.8 to 7.3 x 106 cells) and 2.9 x 108 (range, 0.1 to 9.6 x 108 cells), respectively. RESULTS: There was no transplant-related mortality (TRM) within 100 days of HSCT. Grade 1 to 2 acute graft-versus-host disease (GVHD) occurred in three patients, but neither grade 3 nor grade 4 disease was observed. Chronic GVHD occurred in 10 patients. One patient had cytomegalovirus (CMV) reactivation but did not develop CMV disease. With a median follow-up of 26 months (range, 15 to 45 months), 11 patients are alive (nine in continuous complete remission and one in complete remission after a second transplantation), and five have died (four from disease progression and one from bone-marrow aplasia induced by cyclosporine withdrawal). The 2-year actuarial overall and event-free survival rates were 69% (95% confidence interval [CI], 40% to 86%) and 56% (95% CI, 30% to 68%), respectively. CONCLUSION: This strategy of RI-HSCT resulted in reliable engraftment with low incidence of acute GVHD and TRM. Durable remissions were observed in patients with MDS and AML consistent with a graft-versus-leukemia effect.

Tositumomab and iodine I 131 tositumomab for recurrent indolent and transformed B-cell non-Hodgkin's lymphoma.

PURPOSE: An open-label phase II study was conducted at two centers to establish the efficacy and safety of tositumomab and iodine I 131 tositumomab at first or second recurrence of indolent or transformed indolent B-cell lymphoma. PATIENTS AND METHODS: A single dosimetric dose was followed at 7 to 14 days by the patient-specific administered radioactivity required to deliver a total body dose of 0.75 Gy (reduced to 0.65 Gy for patients with platelets counts of 100 to 149 x 10(9)/L). Forty of 41 patients received both infusions. RESULTS: Thirty-one of 41 patients (76%) responded, with 20 patients (49%) achieving either a complete (CR) or unconfirmed complete remission [CR(u)] and 11 patients (27%) achieving a partial remission. Response rates were similar in both indolent (76%) and transformed disease (71%). The overall median duration of remission was 1.3 years. The median duration of remission has not yet been reached for those patients who achieved a CR or CR(u). Eleven patients continue in CR or CR(u) between 2.6+ and 5.2+ years after therapy. Therapy was well tolerated; hematologic toxicity was the principal adverse event. Grade 3 or 4 anemia, neutropenia, and thrombocytopenia were observed in 5%, 45%, and 32% of patients, respectively. Secondary myelodysplasia has occurred in one patient. Four patients developed human antimouse antibodies after therapy. Five of 38 assessable patients have developed an elevated thyroid-stimulating hormone; treatment with thyroxine has been initiated in one patient. CONCLUSION: High overall and CR rates were observed after a single dose of tositumomab and iodine I 131 tositumomab in this patient group. Toxicity was modest and easily managed.

Misplaced Macroplastique injection presenting as a vaginal nodule and a bladder mass.

We describe a very unusual presentation of misplaced Macroplastique injection. This incidentally showed up as bladder lesion and vaginal nodule during a tension-free vaginal tape surgery.

Thymus dependency of bone marrow stem cell proliferation in response to certain antigens.

The activity of mouse bone marrow CFU's following systemic injection of antigen has been investigated. In normal mice quiescent CFU's may be triggered into cycle after antigenic challenge. Prior adult thymectomy prevents stimulation of CFU if thymus-dependent antigens are injected but not when thymus-independent antigens or non-specific stimuli are used. Similar results were obtained in congenitally athymic mice. This seems to indicate an independent pathway for CFU triggering for each cell lineage and for T and B cells.

The intracellular movement and cycling of ricin.

The binding, internalization and recycling of the plant toxin ricin, was studied using electron microscopy and biochemical techniques. For the electron microscope study, ricin was visualized using a gold-labeled second antibody, in the cells of the EJ human bladder carcinoma line growing in monolayer culture. The labeled antibody/toxin complex was found to enter the cell in coated pits and to accumulate in endosomes and to a lesser extent in vesicles associated with the Golgi system. The complex recycled to the cell surface partly in uncoated vesicles, but largely in multivesicular bodies which appeared to exocytose their contents to the extracellular space. Twenty hours after the initial contact with ricin as much as 50% of the cellular label was found on the cell surface mainly associated with shed vesicles. When cells were treated with unlabeled ricin holotoxin and then after 20 h stained post-fixation, ricin molecules, partly associated with vesicles, were present on the cell surface. Biochemical studies showed that ricin was internalized by cells and then released in an intact form to the extracellular space. It was found that less than 10% of the released material had been degraded during its passage through the cells, which is in accord with the low level of label found in the lysosomal system during the morphological study.

The phosphorylation pattern of insulin-like growth factor-binding protein-1 in normal plasma is different from that in amniotic fluid and changes during pregnancy.

We have determined the phosphorylation pattern of circulating insulin-like growth factor-binding protein-1 (IGFBP-1) in normal subjects and assessed how this changes in pregnancy. Two RIAs employing different monoclonal antibodies (MAbs 6303 or 6305) were used to measure IGFBP-1. In normal subjects, RIA 6303 measured 11-fold higher levels than RIA 6305 (72.8 vs. 6.6 micrograms/L; P < 0.008). However, in amniotic fluid (AF), the two assays gave similar results. Immunoprecipitation of plasma and AF with MAb 6303 and 6305 before nonsodium dodecyl sulfate-electrophoresis and Western ligand blotting revealed different IGFBP-1 isoforms and differential antibody recognition as the cause of this discrepancy. In AF, both MAbs precipitated nonphosphorylated and phosphorylated isoforms, whereas in plasma, only a single highly phosphorylated species, not seen in AF, was observed. This form of IGFBP-1 was precipitated by MAb 6303 only. During pregnancy, the phosphorylation state of IGFBP-1 in the maternal circulation was altered, as nonphosphorylated IGFBP-1 and three lesser phosphoforms were also observed. The appearance of these other variants resulted in a significant increase in IGFBP-1 measured by RIA 6305 (37, 51, and 83 micrograms/L in first, second, and third trimesters, respectively; P < 0.0005 vs. controls). The changes in IGFBP-1 phosphorylation induced by pregnancy may influence the modulatory effects of IGFBP-1 on IGF bioavailability and, hence, fetal growth.