Clonal response of K562 leukemic cells to exogenous p21WAF1.

The p21WAF1 protein is involved in the control of cell differentiation and proliferation. We have previously shown that p21WAF1 is upregulated in normal, proliferating hematopoietic cells undergoing differentiation. Exogenous p21WAF1 has been reported to increase colony-formation by normal hematopoietic progenitors. We examined the effects of exogenous p21WAF1 on proliferation, differentiation, gene expression and colony-formation by K562 cells using an inducible p21WAF1 expression construct. Expression of the stathmin (oncoprotein 18) gene decreased within 24 h of p21WAF1 expression; Hox B4 expression increased. Four K562 subclones were derived which differed in their response to equivalent induction of p21WAF1. All four subclones exhibited growth arrest in response to p21WAF1 in liquid culture. Three of four clones developed cytoplasmic granulation and partial morphologic differentiation after p21WAF1 induction. One clone exhibited fewer morphologic features of differentiation following p21WAF1 induction and unlike other clones, colony formation in methlycellulose was not decreased by p21WAF1 expression in this clone. This indicates that additional cell-specific factors influence cellular fate in the presence of elevated p21WAF1.

Influence of cytokines on the growth kinetics and immunophenotype of daughter cells resulting from the first division of single CD34(+)Thy-1(+)lin- cells.

There is a need to determine whether culture conditions may exist for ex vivo expansion of hematopoeitic stem cells (HSC), which favor solely proliferative self-renewal of HSC as opposed to proliferation with differentiation. Using single cells, we studied the effects of individual and combinations of cytokines in serum-free medium on the kinetics of the first cell doubling and the resulting phenotype of each of individual daughter cell. CD34(+)Thy-1(+)lin- cells were plated 1 cell per well in Terasaki plates in serum-free medium containing cytokines. Each well containing a single cell was monitored daily over 7 days for maintenance, division, or death. When division occurred in an individual well, the phenotype of the daughter cells was determined by staining with anti-CD34 fluorescein isothiocyanate (FITC)- and phycoerythrin (PE)-conjugated lineage specific antibodies. The cumulative percent of wells with an undivided single cell, wells in which the cell had divided, and wells in which the cell had died were scored. The number of doublets with conserved phenotype (CD34(+)lin-) was compared to those wells with one or more differentiated daughter cells (CD34(+)lin+). Over 7 days, cells cultured in single factors showed that between 13% (interleukin-6 [IL-6]) and 29% (thrombopoietin [TPO]) of the cells were undivided, between 13% (IL-1) and 35% (TPO) of the cells doubled, and between 35% (TPO) and greater than 60% (IL-11, IL-1, or hepatocyte growth factor [HGF]) died. When combinations of cytokines were used over 7 days, between 5% (FLT-3 ligand [FLT-3L], stem cell factor [SCF], IL-3, IL-6, granulocyte colony-stimulating factor [G-CSF], beta nerve growth factor [betaNGF]) and 22% (FLT-3L + HGF) of the cells remained undivided, between 15% (HGF, IL-1, IL-11, G-CSF) and 68% (SCF + TPO) of the cells had doubled and between 27% (FLT-3L + TPO) and 70% (HGF, IL-1, IL-11, G-CSF) died. The combination of FLT-3L + TPO induced the highest total percent (64. 6%) of cells with conserved phenotype (percent conserved doublets + percent with 1 cell conserved), followed by SCF + TPO, (50%) and the combination of FLT-3L, SCF, IL-3, IL-6, G-CSF, betaNGF (53%). These combinations also produced the highest yield of cells with conserved phenotype after one division (FLT-3L + TPO - 81 cells/100 initial cells, SCF + TPO - 68 cells/100 initial cells) (P =.01). Observation of the time of the initial cell division and phenotype of the daughter cells allowed us to identify candidate combinations of cytokines that promote maintenance of lin- cells (TPO), or recruit the primitive cells to divide and undergo phenotypic self-renewal (FLT-3L + TPO, SCF + TPO).

Effects of recombinant cytokines on colony formation by irradiated human cord blood CD34+ hematopoietic progenitor cells.

The role of recombinant hematopoietic growth factors in radiation repair has become a subject of increasing interest in both clinical and basic radiobiology. Combinations of cytokines such as hepatocyte growth factor, interleukin (IL)-3, IL-11, kit ligand, GM-CSF and erythropoietin were used to study the in vitro radiation dose response of human cord blood CD34+ hematopoietic progenitor cells using clonogenic survival assays. CD34+ cells were isolated by immunomagnetic selection and irradiated at 8 cGy/min. Irradiated cells were plated in methylcellulose with or without added cytokines, and hematopoietic colonies including CFU-GM, BFU-E and CFU-GEMM were scored on day 14. The radiation response characteristics of BFU-E and CFU-GEMM were similar for all culture conditions tested. The D0 values for BFU-E ranged between 1.29 and 2.40 Gy and n between 1.0 and 1.4. The D0 values for CFU-GEMM ranged from 86 cGy to 2.02 Gy and n between 1.0 and 1.5. The D0 for CFU-GM grown without added factors was 1.03 Gy. With single cytokine stimulation (IL-3, IL-11 or varying concentrations of HGF), D0 values ranged from 1.11 to 1.44 Gy. With the combination of IL-3, GM-CSF, kit ligand and HGF, D0 values were not significantly altered and ranged between 1.61 and 2.60 Gy. In contrast, the combination of IL-11 and HGF produced an increase in the shoulder of the radiation survival curve (n = 3.35). No increase in the shoulder was detected for any of the other conditions tested (n = 1.0-1.7). Thus the combination of HGF and IL-11 increased the radiation survival of hematopoietic progenitor cells forming CFU-GM. Understanding the mechanism by which combinations of early-acting growth factors support postirradiation recovery of primitive clonogenic hematopoietic cells may be relevant to the design of clinical protocols for improving hematopoietic recovery after total-body irradiation.

Synergistic effects of hepatocyte growth factor on human cord blood CD34+ progenitor cells are the result of c-met receptor expression.

Hepatocyte growth factor (HGF) is a pleiotropic growth factor which, in addition to its mitogenic potency for primary hepatocytes, also has a role in the regulation of cell motility, cell growth and morphogenesis. In the present study, we show that c-met, the high-affinity receptor for HGF, is expressed on human cord blood (CB) CD34+ progenitor cells and CD34+Thy-1+ Lin-(lin-) cells. We have investigated the capacity of HGF to synergize with other growth factors to induce colony formation by CB CD34+ progenitor cells. CD34+ cells were cultured in semisolid medium containing serum with increasing concentrations of GM-CSF, G-CSF, macrophage colony-stimulating factor (M-CSF), stem cell factor (SCF), interleukin 3 (IL-3) and IL-11 alone or in combination with HGF. HGF acted as a potent synergist and enhanced, up to fourfold, colony formation induced by GM-CSF, G-CSF or M-CSF. HGF in combination with SCF, IL-3 or IL-11 did not induce proliferation of colony forming units-granulocyte macrophage (CFU-GM) above control levels. In serum-deprived cultures, HGF was only detectably synergistic with IL-11, and all other culture combinations showed no proliferation. To determine whether the stimulatory effect of IL-11 and the synergistic effect of HGF in the absence of serum could be attributed to the effect of these two cytokines on stem cells, IL-11-stimulated and unstimulated lin- cells were analyzed for expression of c-met. CD34+Thy-1+Lin- cells were positive for c-met, both in the presence and absence of IL-11 stimulation, and Northern analysis indicated that c-met RNA expression was upregulated in response to IL-11 compared to unstimulated controls. These results provide strong evidence for upregulation of the HGF receptor on primitive hematopoietic cells by IL-11, and for the synergistic role of HGF in colony formation by hematopoietic stem cells.

Radiosensitization and hypoxic cell toxicity of NLA-1 and NLA-2, two new bioreductive compounds.

Two new bioreductive compounds, 9-[3-(2-nitro-1-imidazolyl)propylamino]acridine hydrochloride (NLA-1) and 9-[2-(2-nitro-1-imidazolyl)ethylamino]acridine hydrochloride (NLA-2), have been prepared. They feature an acridine ring to intercalate with DNA, a 2-nitroimidazole ring as the radiosensitizing moiety and an amino functionality for increased DNA-binding and hydrophilicity. Time and concentration dependent cytotoxicity as well as radiosensitization efficacy of the two compounds under hypoxic or aerobic conditions were determined in vitro using V-79 cells and an MTT colorimetric or clonogenic assay. The isosensitization point (ISP), defined as that drug concentration which results in the same survival decrement upon exposure of hypoxic or oxygenated cells to a given radiation dose, has been determined for both compounds at 7.5 Gy and the values are significantly lower than the ISPs of 5-[3-(2-nitro-1-imidazolyl)propyl]phenanthridinium bromide, 2-(2-nitro-1-imidazolyl)ethylamine or misonidazole (MISO). NLA-1 and NLA-2 are potent hypoxic cytotoxins and on a concentration basis, more potent than MISO as radiosensitizers in vitro. The sensitization enhancement ratios were significantly increased when 1 h drug preincubation under hypoxia at 37 degrees C was applied, before irradiation at room temperature.

Early treatment of Campylobacter jejuni enteritis.

The bacteriologic and clinical effects of early antibiotic treatment of Campylobacter jejuni enteritis were studied. Erythromycin rapidly eliminated C. jejuni from stools, whereas trimethoprim-sulfamethoxazole did not. Despite its bacteriologic effectiveness, erythromycin did not reduce the duration or severity of diarrhea, abdominal pain, or other symptoms.

Prolonged and recurring diarrhea in the northeast of Brazil: examination of cases from a community-based study.

From prospective daily surveillance of diarrhea in a poor rural area of northeastern Brazil, this study of prolonged diarrheal episodes identified the 3% of diarrheal episodes that lasted 15 days or longer. These episodes also defined a subpopulation of children who spent over 16% of their days with diarrhea. Such children warrant further attention in an attempt to define potentially treatable causes as well as to assure appropriate nutritional support. There was no single season for these prolonged illnesses, but they appeared to involve both the wet, slightly warmer season of peak enterotoxigenic Escherichia coli diarrhea as well as the dry, slightly cooler season of peak rotaviral infections. Limited etiologic data support this idea that multiple pathogens are found, often in combination with each other, that may work together to contribute to the important problem of chronic diarrhea. Future studies should focus attention on further defining risk factors, mechanisms, and appropriate therapy for the subset of children who experience prolonged diarrhea in this type of setting.

Emergence of sulfamethoxazole-trimethoprim resistant Shigella flexneri in Northeastern Brazil.

In contrast to prior experience in this setting, three of four Shigella flexneri strains recently isolated from patients in Northeastern Brazil with acute inflammatory diarrhea were found to be resistant to sulfamethoxazole, trimethoprim and the combination in vitro. We performed mating studies to determine if the resistance was transferable, and then isolated and characterized plasmid DNA from the resistant Shigella isolates, other resistant Enterobacteriaceae isolated simultaneously from the stools of these individuals, and transconjugant strains. Each of the resistant Shigella strains contained a large plasmid. These plasmids were of different molecular weights ranging from 30 to 50 Mdal in size. Two of these plasmids were transferred with sulfamethoxazole-trimethoprim resistance to E. coli K-12 recipient strains. These findings of transferable resistance to sulfamethoxazole-trimethoprim associated with plasmids in Shigella and in other Enterobacteriaceae raises concerns about the potential limitations of this widely used antimicrobial combination.

Evaluation and diagnosis of acute infectious diarrhea.

The appropriate approach to the diagnosis and management of acute infectious diarrhea is determined by the frequency and setting of the illness, the recognizable causes or syndromes, the cost and yield of available diagnostic tests, and the treatability of the disease. Acute diarrhea affects everyone throughout the world from one to more than six times each year, depending on age, location, and living conditions. The range of identifiable viral, bacterial, and parasitic etiologies is great, and the cost of indiscriminate use of etiologic studies for diagnosis is prohibitive. Because of its insensitivity for many organisms and poor selection of cases for testing, routine stool culture has been one of the most costly and ineffective microbiologic tests; the cost per positive result has traditionally exceeded $900 to $1,000. The appropriate treatment for the vast majority of cases (independent of their cause) is simple and effective: oral glucose- and electrolyte-containing rehydration solution. On the basis of an appropriate history and understanding of pathogenesis, fecal specimens can be selectively obtained and promptly examined for leukocytes and parasites, and the common noninflammatory diarrheas can be separated from the inflammatory infections in order to focus further studies on the latter group. The bacteria for which specific antimicrobial therapy should be considered usually cause inflammatory diarrhea in the United States. Therefore, only when the history or fecal leukocyte findings indicates an inflammatory process is it appropriate to culture for the routine invasive bacterial pathogens. In sporadic inflammatory diarrhea, culture methods should include those for Campylobacter jejuni as well as Salmonella and Shigella. Several special circumstances may prompt a consideration of parasites (including Giardia, Entamoeba, Strongyloides, Cryptosporidium), Vibrio, Yersinia, Clostridium difficile, enterotoxigenic Escherichia coli, food-borne agents, or sexually transmitted pathogens. The practical value of specific identification of rotaviruses (by enzyme-linked immunosorbent assay, Rotazyme, or electron microscopy) is primarily epidemiologic, particularly in hospitalized infants or young children. Using such a selective approach to fecal culture will greatly increase its yield and can reduce the cost per positive result from $1,000 to less than $150.

Sulfamethoxazole-trimethoprim-resistant Shigella flexneri in northeastern Brazil.

In contrast to prior experience in northeastern Brazil, three of four Shigella flexneri strains recently isolated from patients with acute inflammatory diarrhea in this setting were found to be resistant to sulfamethoxazole-trimethoprim. The resistant strains contained large, different plasmids, two of which were transferred with sulfamethoxazole-trimethoprim resistance to Escherichia coli K-12 recipient strains.

Prospective study of diarrheal illnesses in northeastern Brazil: patterns of disease, nutritional impact, etiologies, and risk factors.

Diarrhea is a leading cause of death in tropical countries. One of the highest childhood mortalities is in northeastern Brazil, where little is known about the morbidity, etiology, and risk factors of diarrhea. Prospective village surveillance over 30 months revealed diarrhea attack rates of more than seven episodes per child-year at six to 11 months of age among the children of the poorest families. Other risk factors included early weaning and the lack of toilets. Diarrhea led to weight loss and stunted growth. Enterotoxigenic Escherichia coli and rotaviruses were the most common pathogens, accounting for 21% and 19% of cases, respectively, followed by Shigella species (8.0%), Campylobacter jejuni (7.5%), Giardia species (6.7%), Strongyloides species (5.3%), and enteropathogenic E coli serotypes (4.6%). Most (84%) enterotoxigenic E coli were isolated during the rainy season of October to March (P less than 0.03), whereas 71% of rotaviral illnesses occurred during the drier months of June to October (P less than 0.03). In the present study, the early occurrence and nutritional impact of diarrhea and weaning, as well as the major etiologic agents of diarrhea and their different seasonal patterns have been defined for this region in which life-threatening diarrhea is endemic.

Comparison of paired whole milk and dried filter paper samples for anti-enterotoxin and anti-rotavirus activities.

Milk specimens, 75 from cows immunized against cholera toxin and 35 from a human population in which enterotoxigenic Escherichia coli and rotaviral infections are endemic, were collected as paired filter paper and frozen whole milk samples. Each pair was tested for antibody activity against heat-labile E. coli and Vibrio cholerae enterotoxins. Additionally, 12 of the 35 paired human milk samples stored as frozen whole milk and dried on filter paper were tested for anti-rotavirus immunoglobulin A. Anti-enterotoxin and anti-rotavirus immunoglobulin A titers in milk dried on filter paper compared favorably with those of their frozen whole milk pairs. Filter paper samples offered considerable advantages for field collection, transportation, and storage over frozen liquid samples.