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.

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.

Maize genotypes with favourable dgat1-2 and fatb alleles possess stable high kernel oil and better fatty acid health and nutritive indices.

Diverse uses of maize oil attracted various stakeholders, including food, feed, and bioenergy, highlighting the increased demand for sustainable production. Here, 48 diverse sub-tropical maize genotypes varying for dgat1-2 and fatb genes governing oil attributes, were evaluated in three diverse locations to assess trends of oil content, fatty acid (FA) profile, the effect of environment on oil attributes, the impact of different gene combinations and determine FA health and nutritional properties. The genotypes revealed wide variation in oil content (OC: 3.4-6.8 %) and FA compositional traits, namely palmitic (PA, 11.3-24.1 %), oleic (OA, 21.5-42.7 %), linoleic (LA, 36.6-61.7 %), and linolenic (ALA, 0.7-2.3 %) acids. Double-mutants with both favourable alleles (dd/ff) exhibited 51.6 % higher oil, 33.2 % higher OA, and 30.2 % reduced PA compared to wild-types (d+d+/f+f+) across locations. These double-mutants had lower saturated FA (12.2 %), and higher unsaturated FA (87.0 %), indicating reduced susceptibility to autooxidation, with lower atherogenicity (0.14), thrombogenicity (0.27) and peroxidisability (48.15), higher cholesterolemic index (7.16), optimum oxidability (5.27) and higher nutritive-value-index (3.35) compared to d+d+/f+f+, making them promising for significant health and nutritional benefits. Locally adapted stable novel double-mutants with high-oil and better FA properties identified here can expedite the maize breeding programs, meeting production demands and addressing long-standing challenges for breeders.

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.

T gamma (T gamma) cells suppress growth of erythroid colony-forming units in vitro in the pure red cell aplasia of B-cell chronic lymphocytic leukemia.

In vitro studies were performed in two patients with B-cell chronic lymphocytic leukemia who developed pure red cell aplasia (CLL-PRCA). During the active phase of their red cell aplasia, there was a marked reduction in the numbers of erythroid colony-forming units (CFU-E). Unfractionated sera or separated IgG fractions from these patients did not impair CFU-E proliferation from either autologous or allogeneic marrows. Increased numbers of T lymphocytes were present in marrow aspirates of these patients. Analysis of these T cells indicated that 90 and 35%, respectively, bore Fc receptors for IgG (T gamma cells). Removal of T cells by E-rosetting techniques augmented CFU-E growth in CLL-PRCA 10-fold. Similar treatment of normal marrows did not cause similar enhanced growth of CFU-E. Co-cultures of marrow T cells or T gamma cells obtained during the active phase of CLL-PRCA suppressed CFU-E growth from autologous or allogeneic marrows. After achieving drug-induced remission of the PRCA, marrow T cells were no longer inhibitory. In contrast, BFU-E (erythroid burst-forming units) or granulocyte proliferation in diffusion chambers were not suppressed by CLL-PRCA T cells. These findings suggest that the development of PRCA in B-cell CLL may result from suppression of CFU-E proliferation by T gamma cells.

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.

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.

Differences in the sensitivity of normal human peripheral blood and bone marrow granulocytic-macrophagic and eosinophilic colony forming cells (CFC) to a source of colony stimulating factor.

The relative sensitivity of normal human peripheral blood (PB) and bone marrow (BM) granulocytic-macrophagic and eosinophilic committed stem cells (CFC) to a source of colony stimulating factor (CSF) was evaluated. In this study, PB and BM cells depleted of mature granulocytes, monocytes (Mo), thymus dependent lymphocytes (T cells), and, in some studies, bone marrow derived lymphocytes (B cells) were cultured in soft agar medium. Human placental conditioned medium (HPCM) was used as a source of CSF. The time sequence evaluation of cultures of PB and BM cells in the presence of a constant amount of HPCM confirmed the results of earlier culture studies of unseparated PB and BM nucleated cells which showed that the proliferation of PB CFC is maximum at or after days 14 to 15 and that of BM CFC at days 7 to 8. Culture of a constant number of PB and BM cells with variable amounts of HPCM indicated that PB CFC require an exogenous source of CSF for proliferation whereas BM CFC proliferate to a modest degree in its absence. The rate of increase in the number of CFC proliferated with the addition of increasing amounts of HPCM is higher for BM than for PB. These data indicate that the BM CFC are more sensitive to a source of CSF than are the PB CFC. Also, the results of this study, along with those in the literature, support the concept that the PB and BM CFC comprise distinct populations, and, further, suggest that the PB CFC is more primitive than the day 7 BM CFC.

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.

Kinetics of proliferation and differentiation of human hemopoietic cells in a diffusion chamber system.

Proliferation and differentiation of human granulocytes were studied in a diffusion chamber (DC) culture system. Evaluation of the nucleated marrow cells of normal volunteers revealed that the granulocytic precursors continue differentiation in the DC as they do in vivo. Generation time of the granulocytes in the multiplicative pool in DC was determined by the autoradiographic mean grain count halving method. With tritiated thymidine (3HTdR) the half-time was 67 h, where as with 125Iododeoxyuridine it was 47 h. The longer half-time with 3HTdR supports the notion that a fraction of nuclear 3HTdR from dead cells is reutilized by the newly proliferating cells. The 3HTdR labeling index of granulocytic precursors in the multiplicative pool during the first 10 days in DC is not constant, suggesting a variation in the rate of proliferation. The DNA synthesis time in DC, determined by double isotope labeling technique, showed that it was relatively constant. Simultaneous evaluation of rate of transition of granulocytes in DC from one stage to the next of one patient with chronic myelocytic leukemia and another with myeloid metaplasia with myelofibrosis indicated a more rapid transition in the DC than in vivo in man.

A triple stain technique to evaluate monocyte, neutrophil, and eosinophil proliferation in soft agar cultures.

A useful cytochemical technique has been developed which facilitates the identification of the types of cell aggregates which proliferate in soft agar cultures of hematopoietic stem cells. Staining of fixed agar discs for alpha-naphthyl acetate esterase (ANAE), naphthol AS-D chloroacetate esterase (CAE) and with Luxol fast blue (LFB) in sequence results in permanent preparations in which monocytic, neutrophilic and eosinophilic aggregates can readily be distinguished on the same slide. This represents an improvement over the currently used technique which relies on the removal of a representative sample of aggregates from the agar discs for cell typing. Whole plate staining results in more accurate estimates of eosinophil growth since the frequency of occurrence of these cell aggregates is low under many of the culturing conditions used. The technique provides a useful tool for studying normal and abnormal hematopoiesis.

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.

Evidence for reduced erythroid burst (BFUE) promoting function of T lymphocytes in the pure red cell aplasia of chronic lymphocytic leukemia.

T cells stimulate the proliferation of BFUE (burst forming units-erythroid) from normal blood null cells in an in vitro culture system in the presence of erythropoietin. To determine whether abnormal BFUE proliferating effect of T cells could explain the pure red cell aplasia in chronic lymphocytic leukemia (CLL-PRCA), we investigated the erythropoietic function of T and null cells in four patients with CLL-PRCA and compared results to three patients with idiopathic pure red cell aplasia (I-PRCA) and normals. Sera from I-PRCA patients (P greater than 0.05) but not CLL-PRCA patients (P less than 0.1) inhibited erythroid stem cell proliferation in the presence of complement. BFUE in null cells of all PRCA patients were barely detectable or absent (P less than 0.0025). Normal or I-PRCA T cells increased BFUE proliferation from PRCA null cells of six patients (P less than 0.001). In contrast, CLL-PRCA T cells were poor stimulators of BFUE from autologous (P less than 0.001) or allogeneic null cells (P less than 0.02). Treatment with cyclophosphamide and prednisone induced reticulocytosis in all four CLL-PRCA patients. After treatment, in two cases, the burst promoting function of T lymphocytes was normal. Analysis of T cell subpopulations in two CLL-PRCA patients, suggested that the reduced burst promoting function was due to decreased numbers and/or function of T cells bearing Fc receptors for IgM (TM cells). These findings suggest that reduced generation of a burst promoting activity by CLL-PRCA T cells may contribute to the pathogenesis of PRCA in chronic lymphocytic leukemia.

Very low density lipoprotein hematopoiesis inhibitor from rat plasma.

Although the stimulatory effect of specific glycoproteins on bone marrow cell proliferation is acknowledged, little attention has been directed toward growth inhibitors. In this report we have explored the role of plasma lipoproteins in regulating the proliferation of hematopoietic cells. Lipoproteins were isolated from the plasma of normal rats and rats with cancer by density gradient ultracentrifugation. Lipoprotein fractions were then added to cell cultures to assess their effect on: 1) erythropoietin (Ep) stimulated rat marrow DNA and protein synthesis, 2) Ep and colony stimulating factor induced marrow colony formation (CFU(E), CFU(C)), and 3) phytohemagglutinin (PHA) stimulated lymphocyte DNA synthesis. The results indicated that very low density lipoproteins (VLDL) completely inhibited CFU(E) and CFU(C) formation. VLDL inhibited (> 80%) the synthesis of DNA by marrow cells cultured with Ep and lymphocytes cultured with PHA. VLDL from rats with Walker-256 cancer had a greater inhibitory effect than normal rat VLDL. Chylomicrons had moderate growth inhibitory effect, and plasma LDL and HDL were inactive. VLDL, however, did not inhibit the proliferation of rat fibroblasts. We conclude that physiologic concentrations of plasma VLDL have a significant inhibitory effect on the proliferation of erythroid, granulocytic and lymphocytic cells. A pathophysiologic role for VLDL in the impairment of erythropoiesis and immune function in cancer is suggested.

Pure red-cell aplasia in patients with chronic lymphocytic leukemia.

Four cases of pure red-cell aplasia (PRCA) in association with chronic lymphocytic leukemia (CLL) are summarized. In addition, all reported cases of CLL-PRCA (27, including our 4 cases) are reviewed to consolidate their clinical features and compare them with those of 26 patients with CLL who were anemic (Stage III) and 52 who were not anemic (Stages O-II). The incidence of PRCA in our patients with CLL is about 6%. Eighty-seven percent of patients with PRCA had B-lymphocyte (B-cell) CLL and 13% had T-lymphocyte (T-cell) CLL. In 4 of 17 patients PRCA was the presenting feature, and in 13 of 17 it developed in patients previously diagnosed as having CLL. In the latter group, the average time from the diagnosis of CLL to the development of PRCA was 50.2 months (range, 1 to 96). The average age at presentation with PRCA was 61.6 years (range, 37 to 79), and the male to female ratio was 1.6 to 3:1. Eighty-one percent had splenomegaly and 65% had lymphadenopathy. The average hematocrit was 17 +/- 3.6% with zero reticulocyte count, and the lymphocyte count was 134 X 10(3)/microliter. Normoblasts were absent in the marrow and lymphocytes constituted 50 to 100% of the marrow cells. In patients with B-cell CLL and those with T-cell CLL, the T cells were found to inhibit the growth of the marrow erythroid progenitors in in vitro cultures. These abnormal T cells (Tr cells) possessed IgG Fc receptors on their surface, which may be responsible for induction of PRCA in these patients. Continued administration of cyclophosphamide and prednisone in moderate doses appears to induce remission from PRCA, but not until the leukemic mass and abnormal Tr cells are markedly reduced. Daily or alternate-day cyclophosphamide administration in small to moderate doses seems to be essential to maintain remission.

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.

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.

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.