Genetic Diversity and Population Structure of Maize (Zea mays L.) Inbred Lines in Association with Phenotypic and Grain Qualitative Traits Using SSR Genotyping.

Maize (Zea mays L.) is an important cereal and is affected by climate change. Therefore, the production of climate-smart maize is urgently needed by preserving diverse genetic backgrounds through the exploration of their genetic diversity. To achieve this, 96 maize inbred lines were used to screen for phenotypic yield-associated traits and grain quality parameters. These traits were studied across two different environments (Anand and Godhra) and polymorphic simple sequence repeat (SSR) markers were employed to investigate the genetic diversity, population structure, and trait-linked association. Genotype-environment interaction (GEI) reveals that most of the phenotypic traits were governed by the genotype itself across the environments, except for plant and ear height, which largely interact with the environment. The genotypic correlation was found to be positive and significant among protein, lysine and tryptophan content. Similarly, yield-attributing traits like ear girth, kernel rows ear-1, kernels row-1 and number of kernels ear-1 were strongly correlated to each other. Pair-wise genetic distance ranged from 0.0983 (1820194/T1 and 1820192/4-20) to 0.7377 (IGI-1101 and 1820168/T1). The SSRs can discriminate the maize population into three distinct groups and shortlisted two genotypes (IGI-1101 and 1820168/T1) as highly diverse lines. Out of the studied 136 SSRs, 61 were polymorphic to amplify a total of 131 alleles (2-3 per loci) with 0.46 average gene diversity. The Polymorphism Information Content (PIC) ranged from 0.24 (umc1578) to 0.58 (umc2252). Similarly, population structure analysis revealed three distinct groups with 19.79% admixture among the genotypes. Genome-wide scanning through a mixed linear model identifies the stable association of the markers umc2038, umc2050 and umc2296 with protein, umc2296 and umc2252 with tryptophan, and umc1535 and umc1303 with total soluble sugar. The obtained maize lines and SSRs can be utilized in future maize breeding programs in relation to other trait characterizations, developments, and subsequent molecular breeding performances for trait introgression into elite genotypes.

Multilocus functional characterization of indigenous and exotic inbreds for dgat1-2, fatb, ge2 and wri1a genes affecting kernel oil and fatty acid profile in maize.

Demand for maize oil is progressively increasing due to its diverse industrial applications, aside from its primary role in human nutrition and animal feed. Oil content and composition are two crucial determinants of maize oil in the international market. As kernel oil in maize is a complex quantitative trait, improving this trait presents a challenge for plant breeders and biotechnologists. Here, we characterized a set of 292 diverse maize inbreds of both indigenous and exotic origin by exploiting functional polymorphism of the dgat1-2, fatb, ge2, and wri1a genes governing kernel oil in maize. Genotyping using gene-based functional markers revealed a lower frequencies of dgat1-2 (0.15) and fatb (0.12) mutant alleles and a higher frequencies of wild-type alleles (Dgat1-2: 0.85; fatB: 0.88). The favorable wri1a allele was conserved across genotypes, while its wild-type allele (WRI1a) was not detected. In contrast, none of the genotypes possessed the ge2 favorable allele. The frequency of favorable alleles of both dgat1-2 and fatb decreased to 0.03 when considered together. Furthermore, pairwise protein-protein interactions among target gene products were conducted to understand the effect of one protein on another and their responses to kernel oil through functional enrichments. Thus, the identified maize genotypes with dgat1-2, fatb, and wri1a favourable alleles, along with insights gained through the protein-protein association network, serve as prominent and unique genetic resources for high-oil maize breeding programs. This is the first comprehensive report on the functional characterization of diverse genotypes at the molecular and protein levels.

Role of morphological traits and cell wall components in imparting resistance to pink stem borer, Sesamia inferens Walker in maize.

Host Plant Resistance (HPR) is the most important component for sustainable management of insect pests. The purpose of the present work was to understand the role of various morphological and biochemical factors as defense mechanism and their interaction on different biological parameters attributed to survival and development of pink stem borer (PSB), Sesamia inferens Walker in maize. The resistant and moderately resistant genotypes (DMRE 63, CM 500 and WNZ Exotic pool) suffered least leaf injury rating (LIR), dead hearts (DH%), percentage stem tunneling (ST%), number of entry/exit holes (E/EH) and showed deleterious effects on biological parameters of pink stem borer as compared to susceptible ones (CM 202 and BML 6). Resistance index among the genotypes varied from 0.11 to 0.46. The variation in morphological traits such as number of nodes, internode distance and stem diameter could not distinguish all the resistant genotypes from that of susceptible genotypes in terms of its mean value. Higher levels of biochemical constituents, viz., p-Coumaric acid (p-CA), ferulic acid (FA), acid detergent fibre (ADF) and acid detergent lignin (ADL) were observed in resistant genotypes compared to susceptible ones. Antibiosis was expressed in terms of reduced pupal weight when fed on WNZ Exotic pool, whereas larval weight and larval survival affected when fed on DMRE 63. Higher concentration of p-CA content in pith of resistant maize genotypes prolonged the pupal period of pink stem borer. Higher concentration of p-CA and FA contents in rind reduced the adult emergence, as they showed significant negative correlation between them. The larval period was prolonged with higher levels of ADF and ADL contents in maize genotypes either in rind or both rind and pith as both ADF and ADL content showed a significant positive correlation with the larval period. The Pearson correlation analysis of most of the biochemical constituents revealed significant negative correlation with damage parameters. The correlation coefficients between p-CA with DH (%), ST (%) and E/EH were r= -0.9642**, r= -0.9363**, and r= -0.9646**, respectively. Similarly, the correlation coefficients between FA with DH (%), ST (%) and E/EH were r= -0.9217*, r= -0.9563**, and r= -0.9434**, respectively and ADF with DH (%), ST (%) and E/EH were r= -0.9506**, r= -0.9611**, and r= -0.9709**, respectively. The study confirms that stem damage parameters can also be used as selection criteria along with LIR to identify resistant genotypes against pink stem borer. Based on the correlation analysis it was concluded that resistance to pink stem borer in maize is the result of interaction of several morphological and biochemical traits rather than a single factor. The findings obtained from the present study can be utilised in pink stem borer resistance breeding programmes to enhance and diversify the basis of resistance.

Leaf Damage Based Phenotyping Technique and Its Validation Against Fall Armyworm, Spodoptera frugiperda (J. E. Smith), in Maize.

Globally, maize is an important cereal food crop with the highest production and productivity. Among the biotic constraints that limit the productivity of maize, the recent invasion of fall armyworm (FAW) in India is a concern. The first line of strategy available for FAW management is to evaluate and exploit resistant genotypes for inclusion in an IPM schedule. Screening for resistant maize genotypes against FAW is in its infancy in India, considering its recent occurrence in the country. The present work attempts to optimize screening techniques suited to Indian conditions, which involve the description of leaf damage rating (LDR) by comparing injury levels among maize genotypes and to validate the result obtained from the optimized screening technique by identification of lines potentially resistant to FAW under artificial infestation. Exposure to 20 neonate FAW larvae at the V5 phenological stage coupled with the adoption of LDR on a 1-9 scale aided in preliminary characterize maize genotypes as potentially resistant, moderately resistant, and susceptible. The LDR varies with genotype, neonate counts, and days after infestation. The genotypes, viz., DMRE 63, DML-163-1, CML 71, CML 141, CML 337, CML 346, and wild ancestor Zea mays ssp. parviglumis recorded lower LDR ratings against FAW and can be exploited for resistance breeding in maize.

Induction of cell wall phenolic monomers as part of direct defense response in maize to pink stem borer (Sesamia inferens Walker) and non-insect interactions.

Pink stem borer (PSB) causes considerable yield losses to maize. Plant-insect interactions have significant implications for sustainable pest management. The present study demonstrated that PSB feeding, mechanical wounding, a combination of mechanical wounding and PSB regurgitation and exogenous application of methyl jasmonate have induced phenolic compound mediated defense responses both at short term (within 2 days of treatment) and long term (in 15 days of treatment) in leaf and stalk tissues of maize. The quantification of two major defense related phenolic compounds namely p-Coumaric acid (p-CA) and ferulic acid (FA) was carried out through ultra-fast liquid chromatography (UFLC) at 2 and 15 days after imposing the above treatments. The p-CA content induced in leaf tissues of maize genotypes were intrinsically higher when challenged by PSB attack at V3 and V6 stages in short- and long-term responses. Higher p-CA content was observed in stalk tissues upon wounding and regurgitation in short- and long-term responses at V3 and V6 stages. Significant accumulation of FA content was also observed in leaf tissues in response to PSB feeding at V3 stage in long-term response while at V6 stage it was observed both in short- and long-term responses. In stalk tissues, methyl jasmonate induced higher FA content in short-term response at V3 stage. However, at V6 stage PSB feeding induced FA accumulation in the short-term while, wounding and regurgitation treatment-induced defense responses in the long-term. In general, the resistant (DMRE 63, CM 500) and moderately resistant genotypes (WNZ ExoticPool) accumulated significantly higher contents of p-CA and FA content than susceptible ones (CM 202, BML 6) in most of the cases. The study indicates that phenolic mediated defense responses in maize are induced by PSB attack followed by wounding and regurgitation compared to the other induced treatments. Furthermore, the study confirmed that induced defense responses vary with plant genotype, stage of crop growth, plant tissue and short and long-term responses. The results of the study suggested that the Phenolic acids i.e. p-CA and FA may contribute to maize resistance mechanisms in the maize-PSB interaction system.

Anti-leukemia effect of resveratrol.

Resveratrol is a phytoalexin naturally present in fruits, medicinal plants and wines. It has a diversity of biological activities. While its role in the protection against coronary heart disease (CHD) in people with moderate wine consumption, remains unclear, resveratrol preferentially inhibits the growth of leukemia cells in culture. Potential mechanisms for its anti-leukemia effect include induction of leukemia cell differentiation, apoptosis, and cell cycle arrest at S-phase; and inhibition of DNA synthesis by inhibiting ribonucleotide reductase or DNA polymerase. Preliminary results suggest that resveratrol also inhibits the viability of freshly isolated leukemia cells, especially promyelocytic leukemia cells. Because of its low in vivo toxicity, resveratrol deserves further investigation as an anti-leukemia agent.

Hydrocortisone inhibits granulocyte-macrophage colony-stimulating factor production from normal human peripheral blood mononuclear cells and CD3+ T cells.

Normal human peripheral blood mononuclear cells (MNCs), particularly T lymphocytes (T cells), are a rich source of granulocyte-macrophage colony-stimulating factor (GM-CSF). Glucocorticoids are known to inhibit GM-CSF production in in vitro cultures of a human fibroblast cell line and in normal human blood monocytes and alveolar macrophages. To determine whether glucocorticoids also inhibit GM-CSF production from normal human MNCs and T cells, we set up cultures of normal human MNCs and T cells in a liquid system in the presence and absence of 5, 50, and 250 microg/dL of hydrocortisone, and an hour later, a constant dose of 50-ng/mL Escherichia coli lipopolysaccharide (LPS) or 10-microg/mL phytohemagglutinin (PHA) was added. After three days, cell counts and GM-CSF levels were determined. Administering 50- and 250-microg/dL hydrocortisone decreased lymphocyte recovery from MNC cultures with LPS (p < or = 0.01), and 250 microg/dL of hydrocortisone decreased lymphocyte recovery from MNC and T-cell cultures with PHA (p < or = 0.03). The amount of GM-CSF produced from PHA-stimulated MNCs was about 100-fold higher than that produced from LPS-stimulated MNCs. The magnitude of GM-CSFs produced in MNC and T-cell cultures stimulated by PHA was comparable (p=0.88). Administering hydrocortisone at 5, 50, and 250 pg/dL decreased GM-CSF production (p < 0.003) in LPS- or PHA-stimulated MNC cultures and in PHA-stimulated T-cell cultures. PHA (not tested with LPS)-stimulated GM-CSF messenger RNA (mRNA) expression was blocked by hydrocortisone. These results indicate that lower concentrations of hydrocortisone inhibit GM-CSF production from normal human blood MNCs and T cells entirely by inhibiting the expression of GM-CSF mRNA, and higher concentrations of hydrocortisone inhibit by a combined effect of inhibiting the expression of GM-CSF mRNA and decreasing the lymphocyte count.

Induction of Mn SOD in human monocytes without inflammatory cytokine production by a mutant endotoxin.

Endotoxin selectively induces monocyte Mn superoxide dismutase (SOD) without affecting levels of Cu,Zn SOD, catalase, or glutathione peroxidase. However, little is known about the structure-activity relationship and the mechanism by which endotoxin induces Mn SOD. In this study we demonstrated that a mutant Escherichia coli endotoxin lacking myristoyl fatty acid at the 3' R-3-hydroxymyristate position of the lipid A moiety retained its full capacity to coagulate Limulus amoebocyte lysate compared with the wild-type E. coli endotoxin and markedly stimulated the activation of human monocyte nuclear factor-kappaB and the induction of Mn SOD mRNA and enzyme activity. However, in contrast to the wild-type endotoxin, it failed to induce significant production of tumor necrosis factor-alpha and macrophage inflammatory protein-1alpha by monocytes and did not induce the phosphorylation and nuclear translocation of mitogen-activated protein kinase. These results suggest that 1) lipid A myristoyl fatty acid, although it is important for the induction of inflammatory cytokine production by human monocytes, is not necessary for the induction of Mn SOD, 2) endotoxin-mediated induction of Mn SOD and inflammatory cytokines are regulated, at least in part, through different signal transduction pathways, and 3) failure of the mutant endotoxin to induce tumor necrosis factor-alpha production is, at least in part, due to its inability to activate mitogen-activated protein kinase.

Chronic relapsing thrombotic thrombocytopenic purpura: role of therapy with cyclosporine.

Relapsing thrombotic thrombocytopenic purpura (TTP) is a rare disorder with most individuals experiencing 1 to 5 relapses. We report a patient with 18 episodes of thrombotic thrombocytopenic purpura (TTP), the highest number of relapses thus far described. The last 11 episodes were treated with regimens containing cyclosporine. The patient's medical record was reviewed for pertinent clinical, laboratory, and treatment data. We summarized various parameters for each episode and compared characteristics of relapses treated with vs. without cyclosporine. The initial episode of TTP was unusual in that it failed to respond to plasmapheresis, glucocorticoids, and fresh frozen plasma (FFP). It remitted only following splenectomy. Episodes 2-7 responded to FFP plus prednisone. Episode 8 failed to respond to prednisone plus FFP but remitted promptly with cyclosporine plus prednisone. Subsequently, 2 relapses responded to cyclosporine alone, 2 to cyclosporine plus FFP, 4 to cyclosporine plus prednisone +/- FFP, and 2 to cyclosporine, FFP, prednisone, and plasma exchange. There was no difference in remission duration, or in severity or duration of relapses treated with vs. without cyclosporine. Use of cyclosporine, however, significantly decreased the requirement for prednisone and the length of maintenance therapy; thus it is effective mainly as an adjunctive therapy for TTP.

Bone marrow biopsy imprints (touch preparations) for assessment of iron stores.

Noninvasive evaluation for iron deficiency is compromised in many individuals due to the presence of chronic inflammatory processes and/or malignancy, thus necessitating bone marrow examination for definitive diagnosis. However, bone marrow aspiration is not obtainable or is inadequate for interpretation (dry tap) in some individuals, and decalcified bone marrow biopsies require 24-48 hr to prepare, and may falsely indicate absence of iron. We evaluated the accuracy of bone marrow biopsy imprints (touch preparations) compared with aspirate particle smears for semiquantitation of bone marrow iron stores. Results indicate that Prussian blue-stained bone marrow biopsy imprints accurately reflect the quantity of iron, compared with bone marrow aspirate particle smears, allowing for rapid determination of iron stores in individuals in whom a bone marrow aspirate cannot be obtained.

Lipoxygenase products regulate proliferation of granulocyte-macrophage progenitors.

We studied the role of lipoxygenase products on the proliferation and recovery of granulocyte-macrophage progenitors (CFU-GM) in liquid cultures of normal human blood mononuclear cells containing physiologic or slightly higher than physiologic concentrations of hydrocortisone (HC). Lipoxygenase blockade by addition of nordihydroguaiaretic acid (NDGA) resulted in enhanced recovery of CFU-GM (mean increase of 230%). The number of CFU-GM recovered from 14-day liquid cultures containing 1.0 microM HC plus 10 microM NDGA was a mean of six times higher than the number present in the inoculum. Effects of addition of selected 5-lipoxygenase products into the culture containing a lipoxygenase blocker on the CFU-GM recovery and proliferative activity were dose- and metabolite-specific. Leukotriene (LT) B4 and 5-hydroxy-eicosatetraenoic acid (5-HETE) decreased recovery of CFU-GM while LTC4 and LTD4 had biphasic effects--lower doses decreased while higher doses had no effect on CFU-GM recovery. Lipoxygenase blockade decreased the percent of CFU-GM in DNA synthesis phase. Readdition of LTB4 did not reverse this effect while LTD4 had a biphasic effect--low concentrations increased the percent of CFU-GM in DNA synthesis phase to levels equivalent to CFU-GM in cultures without NDGA while higher concentrations had no effect. In semisolid CFU-GM assays, lipoxygenase blockade with NDGA completely prevented CFU-GM colony formation, suggesting that NDGA inhibits proliferation and/or differentiation of CFU-GM in semisolid culture assays. The results of our studies suggest that 5-lipoxygenase metabolites are physiologically important in regulating the proliferation of CFU-GM and, thus, granulopoiesis.

Cyclosporine and prednisone therapy for pure red cell aplasia in patients with chronic lymphocytic leukemia.

We describe the characteristics of response to treatment with cyclosporine (CYA) plus prednisone in seven episodes of pure red cell aplasia (PRCA) in four patients with B cell chronic lymphocytic leukemia (CLL). Fourteen episodes of PRCA occurred in four patients with CLL. Eleven episodes were treated with conventional therapies which included an alkylating agent and prednisone. Four episodes that failed to respond to conventional therapies and an additional three episodes were treated with CYA and prednisone. Six of the seven episodes, including three of four which had failed conventional therapies, responded to CYA plus prednisone compared with six of eleven episodes treated with conventional therapies. Response to CYA and prednisone occurred without a reduction in leukemic mass. In contrast, PRCA remission did not occur until after leukemic mass reduction in three of four patients treated successfully with conventional therapies. Time to response was shorter (14 +/- 3 days) with CYA plus prednisone than with conventional therapies (154 +/- 97 days) in three of four patients. These results indicate that CYA plus prednisone is an effective therapy for the induction of remission from PRCA in patients with CLL.

Influence of hydrocortisone on the survival and cluster and colony forming characteristics of normal human granulocyte-macrophage progenitors.

This study was performed to determine the colony and cluster forming ability of granulocyte-macrophage (CFU-GM) progenitors of normal human blood low density cells cultured in a liquid culture system in the presence and absence of physiological doses of hydrocortisone (Hc). The CFU-GM recovered from the liquid cultures were assayed in soft agar medium. The results of the assays indicated that time-related development of clusters and colonies over 1-16 days, proliferative responsiveness to a source of colony stimulating activity, number of cells developed per colony, and the cellular composition of clusters and colonies produced from CFU-GM recovered from 14-day-old liquid cultures with 1.0 microM Hc, were all similar to those that developed from the normal human blood low density cells. However, a higher fraction of the CFU-GM in day 14 liquid cultures with 1.0 microM Hc were in DNA synthesis phase compared with the CFU-GM from the peripheral blood. This study confirmed the results of previous studies showing lower numbers of recognizable neutrophilic granulocytes and improved survival/proliferation of CFU-GM at day 14 in liquid cultures with 1.0 microM Hc compared with cultures without Hc. The present results suggest that the normal human blood CFU-GM which persists and proliferates under the influence of Hc in a liquid culture system is similar in ontogeny to the blood CFU-GM, and that the recovery of CFU-GM from liquid cultures under the influence of Hc appears to be exerted through stimulation of proliferation and controlled differentiation.

Hydrocortisone promotes survival and proliferation of granulocyte-macrophage progenitors via monocytes/macrophages.

We have determined the mechanism by which hydrocortisone (Hc) promotes the survival and proliferation of normal human granulocyte-macrophage progenitors (granulocyte-macrophage colony-forming units, CFU-GM). Peripheral blood mononuclear cells were cultured in a liquid system with 0-10.0 microM Hc over 3 weeks. At 7-day intervals 50% of the culture media along with the cells (suspension cells) present in the media were removed and replaced with fresh media. No CFU-GM or very small numbers of CFU-GM were contained in the suspension cells of the 14- and 21-day-old liquid cultures without Hc; CFU-GM were present and increased with increasing concentrations of Hc. The CFU-GM content in suspension cells of 14- and 21-day-old liquid cultures with 1.0 microM Hc was at least threefold higher compared to liquid cultures without Hc. In a double-layer CFU-GM agar culture system, the suspension cells from liquid cultures with 1.0 microM Hc, but not from liquid cultures without Hc, supported CFU-GM proliferation from normal human bone marrow cells. The CFU-GM proliferation-inducing ability was confined to the monocytes/macrophages (Mo). CFU-GM colony inhibitory and stimulatory activities were detected in cell-free media recovered from liquid cultures without Hc, but only colony stimulatory activity was detected in the media from cultures with 1.0 microM Hc. These results indicate that greater than or equal to physiological concentrations of Hc (0.1-1.0 microM) are required for the persistence and proliferation of CFU-GM, and the effect of Hc is mediated through the Mo, probably by inhibiting the production of one or more of the CFU-GM colony inhibitory molecules.

Effect in vivo of multiple injections of purified murine and recombinant human macrophage colony-stimulating factor to mice.

Hematopoietic efficacy in vivo of multiple injections of purified murine L-cell and recombinant human macrophage colony-stimulating factors (M-CSF; specific activity, greater than 2 x 10(7) units/mg) was assessed in mice. Injections i.v. of sterile saline or 20,000 units of M-CSF were administered once (at 0 h), twice (at 0 and 12 h), or three times (at 0, 12, and 24 h) to C57BL/6 x DBA/2 F1 mice. Numbers and cycling rates of marrow and spleen granulocyte-macrophage, erythroid, and multipotential progenitor cells were assessed 32-36 h after the first injection. Marrow, spleen, and peripheral blood cellularity was assessed at intervals of up to 105 h. Progenitor cell cycling rates were significantly increased after one and two injections of M-CSF but were reduced to a slow or noncycling state after three injections. For marrow cells, the third injection resulted in a significant suppression of hematopoietic progenitor cell cycling compared to the control group. No significant changes were noted for number of progenitors per femur or spleen, for marrow, spleen, or peripheral blood cellularity, or for differential cell counts in these organs after any of the M-CSF treatment schedules. Suppression of progenitor cell proliferation noted after three injections of M-CSF may at least partially explain why repeated injections of 20,000 units of M-CSF fails to increase bone marrow, spleen, or blood cellularity even though one injection of M-CSF increases cycling rates of the hematopoietic progenitors.

Monocyte induces alkaline phosphatase synthesis in neutrophils of chronic myeloid leukemia and myelofibrosis with myeloid metaplasia patients.

Studies were done to test the neutrophil alkaline phosphatase (NAP) synthesizing capacity of the neutrophils of patients with chronic phase chronic myeloid leukemia (CML) and of patients with stable-phase myeloid metaplasia (MM) and to test the NAP synthesis-inducing capacity of monocytes (Mos) of patients with CML. Suspension cultures of the blood light density (LD) cells, LD cells depleted of T-cells and Mos (LD-T-Mo), and cocultures of LD-T-Mo with Mos were performed. NAP synthesis occurred in a normal fashion in LD cell cultures of five of seven patients with CML and of two patients with MM. The NAP synthesis was very slight or did not occur in cultures of LD-T-Mo cells of all patients with chronic-phase CML and MM. However, addition of allogeneic or autologous Mos to the LD-T-Mo cultures restored the NAP synthesis. These results confirm the previous finding that the low or absent NAP in CML is caused by a relative reduction in the monocyte mass and they further indicate the mechanism to be the same for the low or absent NAP in patients with MM. The results also indicate that the NAP-synthesizing capacity of neutrophils of CML and MM patients and the NAP synthesis-inducing capacity of the Mos of patients with CML are normal.

Granulocyte colony-stimulating factor (G-CSF) induces synthesis of alkaline phosphatase in neutrophilic granulocytes of chronic myelogenous leukemia patients.

The monocyte, monocyte conditioned media (MoCM), giant cell tumor conditioned media (GCT) and a purified colony-stimulating factor (G-CSF) promote granulocyte-macrophage progenitors (CFU-GM) growth and differentiation along the neutrophil lineage and also induce alkaline phosphatase (NAP) synthesis in the neutrophilic cells of normal subjects and of patients with chronic phase chronic myelogenous leukemia (CML). However, it is not known if granulocyte-macrophage-CSF (GM-CSF), macrophage-CSF (CSF-1) or other cytokines can induce NAP synthesis from the neutrophilic cells of CML patients. The objective of this study were (a) to ascertain which of the three CFU-GM CSFs would induce NAP synthesis, and (b) to test if any of the other cytokines--interleukin-1 (IL-1), interleukin-2 (IL-2), alpha- and gamma-interferons (alpha-INF and r-INF), and phytohemagglutinin-stimulated T-cell conditioned media (TCM) would induce NAP synthesis. Light density cells obtained from the blood of patients with chronic phase CML were depleted of T cells and monocytes. These cells were cultured with various amounts of G-CSF, GM-CSF, CSF-1, IL-1, IL-2, alpha-INF, r-INF, MoCM, GCT and TCM in a suspension culture system over 6-7 days. Evaluation of the cultures indicated that G-CSF, MoCM and GCT, but not the other factors or cytokines, consistently induced NAP synthesis in a dose-dependent manner. Actinomycin-D and puromycin in separate cultures inhibited NAP synthesis without any significant reduction in cell counts. This indicated that NAP is not prepackaged in neutrophilic cells, and its synthesis occurs by a sequential transcription at the DNA level and translation at the ribosomal level. Our results suggest that the molecule which is responsible for promotion of CFU-GM growth and differentiation along the neutrophilic cell lineage is also responsible for derepression of NAP gene and initiation of NAP synthesis.

Cyclosporin-A for the treatment of pure red cell aplasia in a patient with chronic lymphocytic leukemia.

A 62-year-old man with B-cell chronic lymphocytic leukemia had three separate episodes of pure red cell aplasia (PRCA). The last episode was treated with cyclosporin-A (CyA) and prednisone. After the patient was on the therapy for 2 weeks, erythropoietic recovery was observed and with continued therapy the hematocrit (Hct) became normal. The PRCA remission was associated with a fall in the blood lymphocyte count, and a reduction in the spleen and lymph node size and bone marrow lymphocyte density. At diagnosis of PRCA the blood T-cells bearing IgG Fc receptors (T gamma cells) were increased, and the marrow contained very few or no late-stage erythroid progenitors. After remission of PRCA the T gamma cell fraction decreased, and the marrow erythroid progenitor's number became normal. We speculate that therapy with CyA and prednisone inhibited the production of interleukins-1 and -2 from monocytes and T-cells, respectively, and was responsible for the reduction of the T gamma cell fraction and B-cell leukemic mass in this patient. Further, we believe that normalization of T gamma cells in association with the therapy was responsible for the PRCA remission.

Organ distribution and fate of human platelets: studies of asplenic and splenomegalic patients.

Little is known about the organ distribution and fate of human platelets. We investigated the kinetics, organ distribution, and fate of autologous 111In-oxine-labeled platelets in 12 normal volunteers, four asplenic subjects, and four patients with splenomegaly. The initial recovery of infused 111In-platelets from the circulation was 97.8 +/- 9.8% (means +/- SD) for asplenic subjects and 26.3 +/- 5.9% for splenomegalic patients as compared to 59.2 +/- 9.3% for normal controls. The mean platelet survival times as derived from the multiple-hit model were 9.2 +/- 1.0 days for asplenics and 6.2 +/- 0.6 days for splenomegalic subjects (8.4 +/- 0.8 days for normals). At 30 min postinfusion, 79.4 +/- 19.2% of the infused 111In-platelets pooled in the spleen of splenomegalic subjects and 42.7 +/- 12.2% in normal controls. There was 7.1 +/- 2.0, 12.6 +/- 3.7, and 29.3 +/- 8.4% pooling in the liver of splenomegalic, normal, and asplenic subjects, respectively. At 10 days postinfusion, 37 and 24% of the 111In-platelets were sequestered in the spleen and liver of normal control subjects, respectively. Similar figures for splenomegalic subjects were 71 and 14%, respectively. In asplenic subjects, 89% was sequestered in the liver. We conclude that spleen and liver are the primary sites of platelet destruction, accounting for 61% of infused 111In-platelets in normal volunteers and 85% in splenomegalics, while the liver is the primary site of platelet destruction, accounting for 89% in asplenic subjects.