Function of wild-type or mutant Rac2 and Rap1a GTPases in differentiated HL60 cell NADPH oxidase activation

Studies of neutrophil nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation in a cell-free system showed that the low molecular-weight guanosine triphosphatase (GTPase) Rac was required, and that Rap1a may participate in activation of the catalytic complex. Full-length posttranslationally modified Rac2 was active, whereas only the 1-166 truncated form of Rap1a was functional in the cell-free system, and thus, clarification of the function of Rap1a and Rac2 in intact human phagocytes is needed to provide further insight into their roles as signal transducers from plasma membrane receptors. In the present studies, oligonucleotide-directed mutagenesis was used to introduce a series of mutations into human rap1a or rac2 in the mammalian expression vector pSR alpha neo. HL60 cells transfected with wild-type or mutated rac2 or rap1a cDNA constructs and control HL60 cells transfected with the pSR alpha neo vector containing no inserted cDNA were selected in G418-containing media, then subclones were isolated. Compared with the parent HL60 cells, each of the stable transfected cell lines differentiated similarly into neutrophil-like cells and expressed comparable levels of NADPH oxidase components p47- phox, p67-phox and gp91-phox. The differentiated vector control cell line produced O2. in response to receptor stimulation at rates that were not significantly different from parent HL60 cells. O2-. production by differentiated cell lines expressing mutated N17 Rap1a or N17 Rac2 dominant-negative proteins was inhibited, whereas O2-. production by the subline overexpressing wild-type Rap1a was increased by fourfold. O2-. production by the differentiated cell line expressing GTPase-defective V12 Rap1a was also significantly inhibited, a finding that is consistent with a requirement for cycling between guanosine diphosphate- and GTP-bound forms of Rap1a for continuous NADPH oxidase activation in intact neutrophils. A model is proposed in which Rac2 mediates assembly of the p47 and p67 oxidase components on the cytosolic face of the plasma membrane via cytoskeletal reorganization, whereas Rap1a functions downstream as the final activation switch involving direct physical interaction with the transmembrane flavocytochrome component of the NADPH oxidase.     

Unrelated donor marrow transplantation in children

Eighty-eight children 0.5 to 17 years of age (median, 9 years of age) received an unrelated donor marrow transplant for treatment of chronic myeloid leukemia (CML; n = 16), acute lymphoblastic leukemia (ALL) in first or second remission (n = 15) or more advanced stage (n = 28), acute myeloid leukemia (AML; n = 13), or other hematologic diseases (n = 16) between June 1985 and April 1993. All patients were conditioned with cyclophosphamide and total body irradiation and received a combination of methotrexate and cyclosporine as graft-versus-host disease (GVHD) prophylaxis. Fourty-six patients received transplants from HLA-identical donors and 42 patients received transplants from donors who were minor-mismatched at one HLA-A or B or D/DRB1 locus. The Kaplan-Meier estimates of disease-free survival and relapse were 75% and 0% for patients with CML, 47% and 20% for ALL in first or second remission, 10% and 60% for ALL in relapse or third remission, 46% and 46% for AML in first remission (n = 1) or more advanced disease (n = 12), and 29% and 69% for other diseases. HLA disparity was not significantly associated with lower disease-free survival, but the results suggest more relapses in HLA-matched recipients and there was significantly more transplant-related mortality in mismatched recipients (51% v 24%, P = .04). Most deaths were due to infections associated with acuteor chronic GVHD and occurred within the first 2 years after transplantation. Granulocyte engraftment occurred in all evaluable patients. Sixty-three percent of HLA-matched and 57% of HLA- mismatched recipients were discharged home disease-free at a median of 98 and 103 days, respectively, after transplantation (P = not significant [NS]). The incidence of grades II-IV acute GVHD was 83% in HLA-matched and 98% in HLA-mismatched recipients (P = .009). The incidence of chronic GVHD was 60% in HLA-matched and 69% in HLA- mismatched recipients (P = NS). One or multiple late adverse events such as cataracts, osteonecrosis of the hip or knee, restrictive or obstructive pulmonary disease, and hypothyroidism have occurred in 11 of 33 (33%) surviving patients. Immunosuppression was discontinued in 58% of surviving patients, including all 12 patients surviving more than 3.2 years, all of whom have a Lansky or Karnofsky score of 100%.(ABSTRACT TRUNCATED AT 400 WORDS)     

Postnatal accumulation of intermediate filaments in the cat and human primary visual cortex

A principal characteristic of the mammalian visual system is its high capacity for plasticity in early postnatal development during a time commonly referred to as the critical period. The progressive diminution of plasticity with age is linked to the emergence of a collection of molecules called molecular brakes that reduce plasticity and stabilize neural circuits modified by earlier visual experiences. Manipulation of braking molecules either pharmacologically or though experiential alteration enhances plasticity and promotes recovery from visual impairment. The stability of neural circuitry is increased by intermediate filamentous proteins of the cytoskeleton such as neurofilaments and α‐internexin. We examined levels of these intermediate filaments within cat and human primary visual cortex (V1) across development to determine whether they accumulate following a time course consistent with a molecular brake. In both species, levels of intermediate filaments increased considerably throughout early postnatal life beginning shortly after the peak of the critical period, with the highest levels measured in adults. Neurofilament phosphorylation was also observed to increase throughout development, raising the possibility that posttranslational modification by phosphorylation reduces plasticity due to increased protein stability. Finally, an approach to scale developmental time points between species is presented that compares the developmental profiles of intermediate filaments between cats and humans. Although causality between intermediate filaments and plasticity was not directly tested in this study, their accumulation relative to the critical period indicates that they may contribute to the decline in plasticity with age, and may also constrain the success of treatments for visual disorders applied in adulthood. J. Comp. Neurol. 523:2111–2126, 2015. © 2015 Wiley Periodicals, Inc.     

Financial competitiveness of organic agriculture on a global scale

To promote global food and ecosystem security, several innovative farming systems have been identified that better balance multiple sustainability goals. The most rapidly growing and contentious of these systems is organic agriculture. Whether organic agriculture can continue to expand will likely be determined by whether it is economically competitive with conventional agriculture. Here, we examined the financial performance of organic and conventional agriculture by conducting a meta-analysis of a global dataset spanning 55 crops grown on five continents. When organic premiums were not applied, benefit/cost ratios (−8 to −7%) and net present values (−27 to −23%) of organic agriculture were significantly lower than conventional agriculture. However, when actual premiums were applied, organic agriculture was significantly more profitable (22–35%) and had higher benefit/cost ratios (20–24%) than conventional agriculture. Although premiums were 29–32%, breakeven premiums necessary for organic profits to match conventional profits were only 5–7%, even with organic yields being 10–18% lower. Total costs were not significantly different, but labor costs were significantly higher (7–13%) with organic farming practices. Studies in our meta-analysis accounted for neither environmental costs (negative externalities) nor ecosystem services from good farming practices, which likely favor organic agriculture. With only 1% of the global agricultural land in organic production, our findings suggest that organic agriculture can continue to expand even if premiums decline. Furthermore, with their multiple sustainability benefits, organic farming systems can contribute a larger share in feeding the world.Although agriculture provides growing supplies of food and other products, it is a major contributor to greenhouse gases, biodiversity loss, agrochemical pollution, and soil degradation (1–3). Concerns about the sustainability of conventional agriculture in particular have promoted interest in alternative farming systems that are more environmentally benign. These alternative systems are not widespread and include organic, integrated, conservation agriculture, mixed crop/livestock, and perennial grains (2, 4, 5). Organic agriculture is the most popular alternative farming system in the world, with global sales of organic foods and beverages growing 170% to $63 billion from 2002 to 2011 (6). The majority of these sales (90%) are concentrated in North America and Europe, with Asia, Latin America, and Africa being primarily export producers (6). Although certified organic farming is practiced in 162 countries, organic agriculture currently occupies only 1% of global cropland (6). Growth of organic agriculture is often limited by inexperience with production methods, inadequate marketing and technical infrastructure, low consumer spending power, and government policies (7, 8).Organic agriculture is contentious, with critics arguing that it is inefficient (9, 10), relying on more land to produce the same amount of food as conventional agriculture. In turn, adopting organic agriculture on too large a scale could potentially threaten the world’s forests, wetlands, and grasslands (9, 11). Additionally, skeptics contend that organic agriculture has too many shortcomings and poor solutions to agricultural problems (9, 12) and will become less relevant in the future (10). However, recent international agricultural reports recognize organic agriculture as an innovative farming system that balances multiple sustainability goals and will be of increasing importance in global food and ecosystem security (1, 4, 5). According to a National Academy of Sciences report (5), such multiple sustainable goals for any farming system should include producing adequate amounts of high-quality food, enhancing the natural resource base and environment, contributing to the well-being of farmers and their communities, and making farming financially viable.Reviews and meta-analyses have shown that although organic agriculture produces lower yields compared with conventional agriculture (13–15), it delivers equal or more nutritious foods (16–18) with less to no pesticide residues (17–19). Such aggregate studies generally support the perception that organic farming systems are more environmentally friendly than conventional farming systems. For example, environmental benefits include greater energy efficiency (20–22); enhanced soil carbon and quality (20–24); greater floral, faunal, and landscape diversity (21–23, 25–27); and less pesticide and nutrient pollution of ground and surface waters (20, 23, 25, 26). Although few studies have been conducted comparing the sociocultural aspects of organic and conventional agriculture, organic farming has been shown to have some sociocultural strengths, such as more humane animal production conditions (28), positive shifts in community economic development and social interactions (29, 30), and greater employment of farm workers and cooperation among farmers (31, 32).To be sustainable, organic agriculture must also be profitable (8). Moreover, whether organic agriculture can continue to expand globally will primarily be determined by its financial performance compared to conventional agriculture (7, 13). The main factors that determine the profitability of organic agriculture include crop yields, labor costs, price premiums for organic products, potential for reduced income during the organic transition period, and potential cost savings from the reduced use of nonrenewable resources and purchased inputs (33). Although individual studies have compared the financial performance of organic and conventional farms and addressed these factors, no studies have synthesized this information on a global scale. Here, we address this knowledge gap by comparing the financial performance of organic and conventional agriculture with a dataset spanning 55 crops grown on five continents. Using meta-analysis, we identified broad economic sustainability patterns not apparent in primary field research by examining total costs, variable costs, labor costs, fixed costs, gross returns, benefit/cost ratios, net present values, organic price premiums, and yields for organic and conventional agriculture.     

Studies on levamisole--induced agranulocytosis

Widespread clinical trials of leavo-tetramisole (levamisole) as an immunopotentiating agent in rheumatoid arthritis, metastatic carcinoma, and immunodeficiency states have been complicated by agranulocytosis (AGC) in 2.5%-13% of patients. Other than a relationship with prolonged high dosage, very little is known regarding the pathogenesis of levamisole-induced AGC. Whereas leukoagglutination was negative, fluorochromatic microgranulocytotoxicity (GCY) tests were positive with serum from 10 of 10 acutely neutropenic patients. The antibody was IgM, reacted with 100% of unrelated granulocytes, but not with T or B lymphocytes. Some sera also reacted with monocytes and the myeloid cell line, K-562. Tests for antigen-antibody complexes or cold autoantibodies were negative. Although clinical evidence strongly suggests a haptene (drug) mechanism, in vitro mixing experiments were also negative. An alternative choice parallels the model of aldomet- induced Coombs'-positive hemolytic anemia. Finally, GCY first became positive 2-3 mo prior to the onset of AGC on two patients, suggesting the possibility of identifying those at risk well before the onset of neutropenia.     

Fanconi's anemia treated by allogeneic marrow transplantation

Eight patients with Fanconi's anemia were given cyclophosphamide alone (seven patients) or combined with procarbazine and antithymocyte globulin (one patient) followed by marrow grafts from HLA-identical siblings. All patients had engraftment. Seven developed acute and three chronic graft-versus-host disease (GVHD). Three patients died with GVHD and infectious complications (days 19, 56, and 82) and one with an intracerebral hemorrhage (day 540). Four patients are surviving 647- 3435 days after grafting, two are well, and two have chronic GVHD that is improving. These results show that Fanconi's anemia can be treated successfully by allogeneic marrow transplantation.     

Coincidence of B-cell chronic lymphocytic leukemia and cutaneous T-cell lymphoma (mycosis fungoides): immunologic characterization by monoclonal antibodies

Although rare cases of chronic lymphocytic leukemia (CLL) of the T-cell type have been reported, CLL is more commonly found to be a neoplastic lymphoproliferative disease of B-cell origin. In this article, we describe a patient with long-standing CLL that was immunologically shown to be of the B-cell type, who, during the course of his disease, developed cutaneous T-cell lymphoma (CTCL), which was shown to be of the helper/inducer subtype. The neoplastic lymphoid cells in the skin infiltrate differed morphologically and immunologically from those in the peripheral blood. The occurrence of CTCL during this patient's clinical course represents a second neoplasm arising from a different cell line, rather than a tissue manifestation of the patient's CLL. To our knowledge, this is the first report in which the occurrence of CTCL is documented in a patient with immunologically known B-cell CLL. In addition to establishing the presence of B-cell CLL and CTCL of the helper/inducer T-cell type in the same patient, this case report demonstrates the usefulness and necessity of evaluating lymphoproliferative disorders by means of a multidisciplinary approach.     

Second marrow transplants in patients with aplastic anemia rejecting the first graft: use of a conditioning regimen including cyclophosphamide and antithymocyte globulin

Sixteen (11%) of 146 consecutive patients with severe aplastic anemia prepared for engraftment with cyclophosphamide (200 mg/kg) rejected marrow grafts from their HLA-identical siblings. They were given a second marrow transplant from either the same (n = 13) or a second (n = 3) HLA-identical sibling between 23 and 743 (median 86) days after the first transplant. The preparation for the second transplant included cyclophosphamide, 50 mg/kg, on each of four successive days. Twelve hours after each of the first three doses of cyclophosphamide, antithymocyte globulin, 30 mg/kg/dose, was infused. One of the 16 patients died from infection too early after the second transplant to be evaluated, two had failure of engraftment and died with infection, one rejected the second graft and is surviving almost 5 years later with full autologous marrow recovery, and 12 had successful and sustained second grafts. Of these 12, six are surviving between 11 months and 7 3/4 years. Four of the six have no graft-v-host disease (GVHD), while two have chronic GVHD requiring treatment. Five have Karnofsky scores of 100% and one of 90%. Six of the 12 patients with sustained grafts died between 63 days and 38 months after transplantation, four with infections (related in two patients to chronic GVHD), one with acute GVHD, and one with hemorrhage. The average interval from first to second transplant was 308 days during the past five years, compared to 61 days in earlier patients. Five of seven recent patients are surviving, compared to two of nine earlier patients. In conclusion, successful second transplants after cyclophosphamide and antithymocyte globulin are possible in most patients with aplastic anemia who have rejected their first marrow grafts; however, mortality remains high, with only 40% of the patients becoming long-term survivors.     

Marrow transplantation from HLA-identical siblings for treatment of aplastic anemia: is exposure to marrow donor blood products 24 hours before high-dose cyclophosphamide needed for successful engraftment?

The present study in patients with aplastic anemia was undertaken to determine whether exposure of recipients to donor blood products 24 hr before preparation with cyclophosphamide (1) enhanced the rate of sustained engraftment of marrow from HLA-identical siblings as suggested by animal experiments, (2) increased the rejection rate, in particular in transfused patients who may already have been exposed to donor antigens by blood products, or (3) was of no relevance to the outcome of transplantation of marrow from HLA-identical siblings. One- hundred fifty-five patients were studied, of whom 78 received blood products from the marrow donor 24 hr before cyclophosphamide and 77 did not. A binary logistic regression analysis was applied to the data, simultaneously considering five previously known risk factors for rejection. Results showed that preceding transfusion of donor blood products had neither a significant beneficial nor detrimental effect on the incidence of sustained engraftment.