Complement Factor H-Related Protein 3 Serum Levels Are Low Compared to Factor H and Mainly Determined by Gene Copy Number Variation in CFHR3

The major human complement regulator in blood, complement factor H (FH), has several closely related proteins, called FH-related (FHR) proteins. As all FHRs lack relevant complement regulatory activity, their physiological role is not well understood. FHR protein 3 (FHR-3) has been suggested to compete with FH for binding to Neisseria meningitidis, thereby affecting complement-mediated clearance. Clearly, the in vivo outcome of such competition greatly depends on the FH and FHR-3 concentrations. While FH levels have been established, accurate FHR-3 levels were never unequivocally reported to date. Moreover, CFHR3 gene copy numbers commonly vary, which may impact the FHR-3 concentration. Hence, we generated five anti-FHR-3 mAbs to specifically measure FHR-3 in human healthy donors of which we determined the gene copy number variation at the CFH/CFHR locus. Finally, we examined the acute-phase response characteristics of FHR-3 in a small sepsis cohort. We determined FHR-3 levels to have a mean of 19 nM and that under normal conditions the copy number of CFHR3 correlates to a very large extent with the FHR-3 serum levels. On average, FHR-3 was 132-fold lower compared to the FH concentration in the same serum samples and FHR-3 did not behave as a major acute phase response protein.     

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Recombinant human colony stimulating factor-granulocyte/macrophage and -granulocyte, but not macrophage induce the development of a respiratory burst in primary human myeloid leukemic cells in vitro

Respiratory burst develops in myeloid blast cells if they differentiate functionally along the monocytic or granulocytic lineage. Using the nitroblue tetrazolium (NBT) assay we studied the effects of recombinant human granulocyte/macrophage colony stimulating factor (rhuGM-CSF), rhuG-CSF and rhuM-CSF on development of respiratory burst activity in primary blast cells from patients with myeloid leukemia. Assessing suspension cultures containing cells from patients with acute myeloid leukemia (AML, n = 13) or myeloid-blast crisis (myBC) of chronic myeloid leukemia (CML, n = 5) it was found that the percentage of NBT positive cells was increased by at least 20% as compared to control cultures by rhuGM-CSF in 6/17 cases, by rhuG-CSF in 7/17 cases and by rhuM-CSF in 0/16 cases, representing in 'responders' a mean increase of 267% and 270% in the absolute number of NBT positive cells by rhuGM-CSF and rhuG-CSF, respectively. Morphological examination of cultured cells from 'responders', as compared to controls, showed decreased blast cell content but generally no evidence of terminal differentiation. The demonstration of Auer rods in NBT positive cells indicates that respiratory burst developed in a leukemic clone. These findings may be of physiological, pathophysiological and clinical relevance.     

CFU-gm assay, cytochemical and electron microscopic studies in agar in patients with preleukemic syndrome and aplastic anemia

Thirty-seven patients with chronic cytopenia were studied using a CFU-gm assay in agar. Cell proliferation was evaluated on days 2, 3, 5, 7, and 10 of incubation. Growth patterns were different in cultures of hematologically healthy persons versus patients with preleukemic syndrome (PL) and aplastic anemia (AA). Three types of PL syndrome and two types of AA (C1 and C2) were distinguished. Bone marrow dysfunction was evaluated further using cytochemistry and electron microscopy to morphologically study cell proliferation in vitro. Cytochemical staining performed in agar demonstrated well-defined maturation defects in myelopoietic precursor cells from the bone marrow of PL patients. Electron microscopic findings of Auer-body-like inclusions in "statu nascendi" in the vacuoles of preleukemic cells supported our results. PL patient groups at high risk for development of overt leukemia and patients with grave prognosis in AA were distinguished. Our results are relevant for the clinical diagnosis and prognosis of patients with cytopenia.     

Influence of purified recombinant human macrophage colony stimulating factor in mice

The endotoxin-resistant strain of mouse, C3H/Hej, was assessed for hematological responsiveness to multiple injections of high dosages of purified recombinant human macrophage colony stimulating factor (rhu-M-CSF). Mice were administered the rhu M-CSF i.p. at dosages of 40 micrograms per injection, 2 or 3 times per day for 4 days. This resulted in significant increases in circulating leukocytes compared to control mice given sterile pyrogen-free saline. Assessment of the marrow and spleen of these mice on the 5th day noted a significant reduction in the numbers of marrow hematopoietic progenitor cells, with no change in their cycling rates. In contrast, splenic granulocyte-macrophage and erythroid progenitor cell numbers were markedly increased and the cycling rates of these progenitors plus those of multipotential progenitors were significantly enhanced. Marrow and splenic early myeloid cells (blasts, promyelocytes, and myelocytes) and macrophages were increased, while marrow and splenic PMN were decreased. The results suggest that multiple injections of high dosages of rhu-M-CSF to previously untreated mice for a short period of time has a modest enhancing effect on blood leukocyte levels. This is associated with a shift of hematopoietic cell activity from the marrow to the spleen.     

Manganese status,gut endogenous losses of manganese,and antioxidant enzyme activity in rats fed varying levels of manganese and fat

We hypothesized that manganese deficient animals fed high vs moderate levels of polyunsaturated fat would either manifest evidence of increased oxidative stress or would experience compensatory changes in antioxidant enzymes and/or shifts in manganese utilization that result in decreased endogenous gut manganese losses. Rats (females in Study 1, males in Study 2,n = 8/treatment) were fed diets that contained 5 or 20% corn oil by weight and either 0.01 or 1.5 μmol manganese/g diet. In study 2,⁵⁴Mn complexed to albumin was injected into the portal vein to assess gut endogenous losses of manganese. The manganese deficient rats:

Particle size influences decay rates of environmental DNA in aquatic systems

Environmental DNA (eDNA) analysis is a powerful tool for remote detection of target organisms. However, obtaining quantitative and longitudinal information from eDNA data is challenging, requiring a deep understanding of eDNA ecology. Notably, if the various size components of eDNA decay at different rates, and we can separate them within a sample, their changing proportions could be used to obtain longitudinal dynamics information on targets. To test this possibility, we conducted an aquatic mesocosm experiment in which we separated fish-derived eDNA components using sequential filtration to evaluate the decay rate and changing proportion of various eDNA particle sizes over time. We then fit four alternative mathematical decay models to the data, building towards a predictive framework to interpret eDNA data from various particle sizes. We found that medium-sized particles (1–10 μm) decayed more slowly than other size classes (i.e., <1 and > 10 μm), and thus made up an increasing proportion of eDNA particles over time. We also observed distinct eDNA particle size distribution (PSD) between our Common carp and Rainbow trout samples, suggesting that target-specific assays are required to determine starting eDNA PSDs. Additionally, we found evidence that different sizes of eDNA particles do not decay independently, with particle size conversion replenishing smaller particles over time. Nonetheless, a parsimonious mathematical model where particle sizes decay independently best explained the data. Given these results, we suggest a framework to discern target distance and abundance with eDNA data by applying sequential filtration, which theoretically has both metabarcoding and single-target applications.     

Expression of wild-type GBSS transgenes in the off-spring of partially and fully complementedamylose-free transformants of potato

Theamylose-free (amf) potato mutant can easily be complemented through introduction of the wild-type gene coding for granule-bound starch synthase (GBSS). After iodine staining the starch of theamf mutant is red whereas that of the wild type and the complementedamf mutant is blue. The level of complementation of selected transformants and their sexual off-spring after backcrossing withamf was investigated using sporophytic tuber cells and gametophytic microspore cells. Two diploid and two tetraploid transformants with full complementation demonstrated the expected segregation patterns of 1:1 (one active insert) or 3:1 (two independently segregating active inserts) in the microspores and in the F1 offspring based on staining of tubers. All expected genotypes in the F1 generation were found, based on microspore segregation patterns of the individual F1 plants. Two transformants with partial complementation (mixed phenotypes) were investigated. One of them, B1, was tetraploid and duplex for the GBSS insert, which had originated through mitotic doubling of the transformed diploid cells. In the F1 generation three phenotypic classes were found:amf, fully complemented and partially complemented. The latter two classes exist independently of a simplex or duplex gene status. The second transformant with partial complementation, B10, appeared to have a complex molecular composition. One cluster of five transgenes caused the partial complementation. Fully and partially complemented phenotypic classes were found after crossing B10 with theamf mutant. Indications were found that the ploidy level of the tissue in which the genes were introduced and expressed played an important role. Firstly, partial complementation was found after transformation of the diploid and not of the tetraploidamf genotypes. Secondly, the level of complementation was higher in tissue with lower ploidy levels, as illustrated by the colour of the starch inin vitro tubers (2x–4x cells) versus field-grown tubers (16x–64x).