Second generation knockout sickle mice: the effect of HbF.

Sickle transgenic mice expressing exclusively human globins are desirable for studying pathophysiology and testing gene therapy strategies, but they must have significant pathology and show evidence of amelioration by antisickling hemoglobins. Mice were generated that expressed exclusively human sickle hemoglobin with 3 levels of HbF using their previously described sickle constructs (cointegrated human miniLCRalpha2 and miniLCRbeta(S) [PNAS 89:12150, 1992]), mouse alpha- and beta-globin-knockouts, and 3 different human gamma-transgenes. It was found that, at all 3 levels of HbF expression, these mice have balanced chain synthesis, nearly normal mean corpuscular hemoglobin, and, in some cases, F cells. Mice with the least adult HbF expression were the most severe. Progressive increase in HbF from less than 3% to 20% to 40% correlated with progressive increase in hematocrit (22% to 34% to 40%) and progressive decrease in reticulocyte count (from 60% to 30% to 13%). Urine concentrating ability was normalized at high HbF, and tissue damage detected by histopathology and organ weight were ameliorated by increased HbF. The gamma-transgene that produces intermediate levels of HbF was introduced into knockout sickle mice described by Pàszty and coworkers that express the miniLCRalpha1(G)gamma(A)gammadeltabeta(S) transgene and have fetal but not adult expression of HbF. It was found that the level of HbF required to ameliorate low hematocrit and normalize urine concentrating defect was different for the miniLCRalpha2beta(S) and miniLCRalpha1(G)gamma(A)gammadeltabeta(S) mice. We conclude that knockout mice with the miniLCRalpha2beta(S) transgene and postnatal expression of HbF have sufficiently faithful sickle pathology to serve as a platform for testing antisickling interventions.

In vivo silencing of the human gamma-globin gene in murine erythroid cells following retroviral transduction.

Increased expression of fetal hemoglobin can ameliorate the clinical severity of sickle cell disease. Whereas temporary induction of fetal hemoglobin can be achieved by pharmacologic therapy, gene transfer resulting in high-level expression of the fetal gamma-globin gene may provide a permanent cure for sickle cell disease. We had previously developed a high-titer, genetically stable retroviral vector in which the human gamma-globin gene was linked to HS-40, the major regulatory element of the human alpha-globin gene cluster. Based on experience in transgenic mice, the truncated promoter of the gamma-globin gene of this vector should be active in adult erythroid cells. Our earlier studies demonstrated that this retroviral vector can give rise to high-level expression of the human gamma-globin gene in murine erythroleukemia (MEL) cells. We have now utilized this vector to transduce murine bone marrow cells that were transplanted into W/W(v) recipient mice. Analysis of transduction of murine BFU-e's in vitro and peripheral blood cells from transplanted mice in vivo demonstrated efficient transfer of the human gamma-globin gene. However, in contrast to the high level of expression of the human gamma-globin gene of this vector in MEL cells, the gene was completely silent in vivo in all transplanted mice. These observations confirm that all the necessary regulatory elements responsible for the developmental stage-specific expression of the human gamma-globin gene reside in its proximal sequences. They also emphasize the differences between gene regulation in MEL cells, transgenic mice, and retroviral gene transfer vectors. For this form of globin gene therapy to succeed, the proximal regulatory elements of the human gamma-globin gene may have to be replaced with different regulatory elements that allow the expression of the gamma-globin coding sequences in adult red cells in vivo.

Malignant cells can be sensitized to undergo growth inhibition and apoptosis by arsenic trioxide through modulation of the glutathione redox system.

Arsenic trioxide (As2O3) induces clinical remission in acute promyelocytic leukemia (APL) with minimal toxicity and apoptosis in APL-derived NB4 cells at low (1 to 2 micromol/L) concentration. We examined the basis for NB4 cell sensitivity to As2O3 to identify experimental conditions that would render other malignant cells responsive to low concentrations of As2O3. The intracellular glutathione (GSH) content had a decisive effect on As2O3-induced apoptosis. Highly sensitive NB4 cells had the lowest GSH and the sensitivity of other cell lines was inversely proportional to their GSH content. The t(14;18) B-cell lymphoma cell line had low GSH levels and sensitivity to As2O3 at levels slightly higher than in APL cells. Experimental upmodulation of GSH content decreased the sensitivity to As2O3. Ascorbic acid and buthionine sulfoxide (BSO) decreased GSH to a greater extent, and rendered malignant cells more sensitive to As2O3. As2O3-induced apoptosis was not enhanced by ascorbic acid in normal cells, suggesting that the combination of ascorbic acid and As2O3 may be selectively toxic to some malignant cells. Ascorbic acid enhanced the antilymphoma effect of As2O3 in vivo without additional toxicity. Thus, As2O3 alone or administered with ascorbic acid may provide a novel therapy for lymphoma.

Effect of antiviral drugs and hematopoietic growth factors on in vitro erythropoiesis.

BACKGROUND: The purpose of these studies was to improve our understanding of nucleoside analog, antiviral, drug-induced anemia in HIV infection. METHODS: Peripheral blood erythroid progenitor cells (BFU-E) from HIV-positive (HIV+) patients and normal donors were compared in methylcellulose cultures with erythropoietin, with and without antiviral drugs, and with and without the hematopoietic growth factors, stem cell factor (SCF), hemin, and interleukin-3 (IL-3). RESULTS: Normal numbers of BFU-E-derived colonies were observed in cultures from HIV+ patients (mean +/- 1 SD BFU-E/10(5) cells plated: normal = 14.1 +/- 7.9, HIV+ = 17.2 +/- 14.2, p = 0.39). The antiviral drugs zidovudine (AZT), dideoxyinosine (ddI), and didehydrodideoxythymidine (d4T) all inhibited erythroid colonies in HIV+ and normal cultures. AZT was the most erythropoietically inhibitory drug (AZT ID50, mean +/- 1 SD for normal cultures = 2.64 +/- 4.15 microM, for HIV+ cultures = 6.28 +/- 10.79 microM, p = 0.24). Hematologic toxicity was less with ddI and d4T. However, doses of ddI and d4T < or = 10 microM inhibited colony growth in 9/14 and 8/12 cultures, respectively, from HIV+ patients. CONCLUSIONS: Stem cell factor (SCF), hemin, and interleukin-3 (IL-3) increased colony growth in HIV+ and normal cultures. In control cultures, hematopoietic growth factors added singly increased growth 1.3- to 8-fold. Hematopoietic growth factors increased growth even in cultures containing antiviral drugs. In some instances growth factors restored growth to control levels. SCF, hemin, and IL-3 were most effective when combined.

Retroviral gene transfer into cord blood stem/progenitor cells using purified vector stocks.

Cord blood (CB) progenitor/stem cells (P/SC) are ideal targets for early gene therapy in individuals prenatally diagnosed with genetic disorders. Most retroviral transduction protocols were developed using adult peripheral blood stem cells (PBSC) and bone marrow (BM). Less is known about retroviral transduction of CB P/SC. We examined how timing, multiplicity of infection (MOI), and polycations in the transduction media affect transduction efficiency. Rates of transduction were determined in recently isolated CD34+ enriched CB cells and in colonies derived after various times in liquid cultures (LC). CB mononuclear cells (MNC) were separated by ficoll-hypaque centrifugation and enriched for CD34+ cells. Purity was assessed by flow cytometry. Transduction were performed with clinical-grade retroviral stocks at MOIs of 1-20. Transduction was performed with fetal bovine serum (FBS) or autologous plasma, IL-3, GM-CSF, IL-6, and SCF. The retroviral vector contained LacZ and neomycin resistance (neo) reporter genes. Transduction was determined by X-gal stain and by PCR amplification of the reporter genes. No drug selection was used. Twenty-five experiments were done. CB volumes ranged from 35-150 ml. MNC and CD34+ cell counts ranges were: 0.14-840 x 10(6) and 0.1-4.2 x 10(6), respectively. Transduction efficiency in liquid cultures ranged from 4-63%. Higher rates were seen using MOI > or = 10, 2 microg/ml polybrene, and 10% autologous CB plasma. In colonies, transduction rates were 63 to 72% by PCR and 32% by X-gal staining. In LTC-IC derived colonies, transduction was 7% by PCR. Short incubations of CD34+ CB cells with purified retroviral stocks, polybrene, and autologous sera result in high transduction rates of committed progenitors and moderately low efficiencies of transduction of LTC-IC in the absence of drug selection.

Disparate lympho-erythroid donor to recipient chimaerism in a beta(0)-thalassaemia bone marrow transplant recipient with red cell indices indicative of apparent full engraftment.

A 4-year-old girl with transfusion-dependent beta(0)-thalassaemia received an HLA-identical bone marrow transplant (BMT) from her beta(0)-thalassaemia trait sister. Prior to BMT, chromosomal analysis revealed the recipient to have 46,XX,9qh+, a polymorphic variant of the heterochromatin region of chromosome 9, which her donor did not have. Within 1 month post-BMT, 89% of nucleated bone marrow cells were of donor origin. One year later, donor engraftment had decreased to 44% and 34% in nucleated bone marrow cells and blood lymphocytes, respectively. By 2 years, donor lymphocyte engraftment fell to 5%, raising concern of possible graft rejection. To examine erythroid chimaerism, globin synthesis by individual erythroid progenitor cell derived colonies (BFU-E) was analysed. On days 1000 and 1130 post-BMT, 79% and 77% of colonies, respectively, synthesized beta-globin and therefore were of donor origin.

A murine model for human cord blood transplantation: near-term fetal and neonatal peripheral blood cells can achieve long-term bone marrow engraftment in sublethally irradiated adult recipients.

The purposes of the research reported here were first to explore a murine model for human placental and umbilical cord blood transplantation and second to evaluate the engraftment ability of ex vivo cultured hematopoietic cells. Murine near-term fetal and neonatal peripheral blood (FNPB) cells, genetically marked with the human multiple drug resistance transgene (MDR1) were used for syngeneic transplants into sublethally irradiated adult mice. Donor cells were transplanted either fresh and untreated, or after ex vivo culture in the presence of the hematopoietic growth factors recombinant murine stem cell factor, recombinant human interleukin-3 (rHu IL-3), and rHu IL-6, in a liquid culture system. To evaluate, count, and characterize FNPB progenitor cell-derived colonies, neonatal mouse mononuclear cells were cultured directly in methylcellulose with growth factors. To assess their ex vivo expansion ability, FNPB mononuclear cells were first cultured in liquid medium for 3 to 8 days and then transferred to semisolid assay plates. Evaluation of the cell counts after liquid culture showed a 1.4- to 11.6-fold increase, and the numbers of colonies observed in methylcellulose were similar to those produced by fresh FNPB cells. Donor-type engraftment was demonstrated by polymerase chain reaction (PCR) amplification of the human MDR1 transgene in the peripheral blood of all surviving animals (5 of 7 recipients of the fresh, and 3 of 8 recipients of the ex vivo-cultured cells) 2 to 4 months after transplantation. The proportion of donor leukocytes in the peripheral blood of the recipients (chimerism) was evaluated using fluorescence in situ hybridization (FISH) analysis 4 to 6 months after transplantation and ranged from 2% to 26%. In addition, bone marrow cultures were obtained from two recipient animals: one had received fresh-untreated cells and was evaluated 8 months after transplant, the other had received ex vivo cultured cells and was tested 14 months after grafting. The derived hematopoietic colonies were tested by PCR and the transgene was detected, conclusively proving long-term engraftment of donor cells. These results indicate that FNPB transplants can be successfully performed in sublethally irradiated mice with and without ex vivo culture. Long-term donor-type engraftment with sustained chimerism has been demonstrated. Thus, murine neonatal blood grafts can be used as an animal model for cord blood transplantation for gene therapy studies where complete myeloablation is not desirable and partial replacement of defective marrow may be sufficient. Furthermore, the possibility of numerically expanding hematopoietic progenitor cells contained in neonatal blood without affecting their engraftment ability could facilitate use of cord blood grafts in adult recipients.

In vitro erythropoiesis in polycythemia vera and other myeloproliferative disorders.

PV is a myeloproliferative disorder characterized by an elevated hematocrit and red blood cell mass. In vitro hematopoietic culture systems have been used extensively to characterize the cellular defect in PV. Erythroid progenitor cells from PV patients exhibit characteristic endogenous erythroid colony growth in serum-containing semisolid culture medium. These endogenous erythroid colonies can be used as a diagnostic tool to distinguish PV from other myeloproliferative disorders and secondary erythrocytosis. Both EPO independence and exquisite EPO sensitivity are mechanism which have been proposed to explain the growth of endogenous colonies. In contrast with normal erythroid progenitor cells which have both high and low affinity EPO-R, PV erythroid cells have only low affinity EPO-R, Molecular analyses did not reveal mutations in the PV EPO-R. These findings have failed to clarify the role of EPO in the etiology of PV. PV hematopoietic progenitor cells also exhibit increased sensitivity to the hematopoietic growth factors GM-CSF, IL-3, and SCF. As with EPO-R studies, examination of IL-3 and SCF receptors on PV erythroid cells has not identified mechanisms underlying the observed increased sensitivities to these hematopoietic growth factors. The recent development of a truly serum-free culture system has led to the observation that PV progenitor cells are more than 100-fold more sensitive to IGF-1 than are normal progenitor cells. In addition, the IGF-1 receptor on PV progenitor cells exhibits increased basal phosphorylation and a hypersensitivity and hyperresponsiveness to IGF-1 with respect to tyrosine phosphorylation. Thus in PV, hypersensitivity to several hematopoietic growth factors may result in hyperproliferation of hematopoietic cells. This hypersensitivity ma be due to a defective intracellular mechanism common to these hematopoietic growth factors.

Combined granular-lattice ('Avellino') corneal dystrophy.

BACKGROUND/PURPOSE: In 1988, a report was published describing the histopathologic examination of corneal buttons of 4 patients who had undergone unilateral keratoplasty because of decreased vision caused by what had been diagnosed clinically as granular dystrophy. But on pathologic examination, lesions characteristic of both granular dystrophy and lattice dystrophy were found in each of the 4 corneal buttons. The patients came from 3 different families, each of which traced its origin to the Italian province of Avellino. We studied the clinical and histopathological features of 4 corneas affected by combined granular-lattice dystrophy, adding thereby to the total of 12 other corneas that have been so-described in the literature. METHODS: Two women underwent bilateral penetrating keratoplasty for what was diagnosed clinically as Reis-Bücklers dystrophy in the first patient, and as granular dystrophy in the second patient. We studied all 4 corneas pathologically, using both conventional hematoxylin and eosin stains as well as special histochemical techniques. RESULTS: All 4 corneas contained lesions characteristic of both granular dystrophy and lattice dystrophy, a circumstance that has given rise to the name "combined granular-lattice dystrophy." The patients are not known to be of Italian ancestry. CONCLUSIONS: Three clinical signs characterize combined granular-lattice dystrophy: (1) anterior stromal discrete, grayish-white deposits; (2) lattice lesions located in mid-stroma to posterior stroma; and (3) anterior stromal haze. Both clinically and histopathologically, the lattice lesions are of greater diameter than are those that occur in lattice dystrophy type I. In the past few years, striking advances have been made in understanding the genetics of combined granular-lattice dystrophy. The most recent of these was published just 2 months before the 1997 meeting of the American Ophthalmological Society, and establishes a common molecular origin for granular dystrophy, lattice dystrophy type I, Avellino dystrophy, and Reis-Bücklers dystrophy.

Low oxygen enhances sickle and normal erythropoiesis and fetal hemoglobin synthesis in vitro.

Erythropoiesis is increased in cultures of human blood progenitors when oxygen tension is reduced from 20% (room air) to 5% (low oxygen, closer to physiological bone marrow levels). The effects of low oxygen on gamma-globin synthesis and colony growth in methyl cellulose cultures of blood mononuclear cells from normal individuals and patients with sickle cell diseases were examined. Low oxygen increased colony numbers by 1.5- to 2-fold and erythropoietin sensitivity by almost 2-fold. The interval required for maximal colony growth in cultures from patients with sickle cell disease (sickle colonies) was reduced from 17 days in 20% oxygen to 13 days in 5% oxygen. Relative synthesis of gamma-globin was examined by labeling with 3H-leucine and electrophoresis on Triton acid urea polyacrylamide gels. The % gamma was 1.7-fold higher in normal and 1.4-fold higher in sickle cultures on day 13 in low oxygen. On day 16 the expected temporal decline was not seen in low oxygen, and the % gamma was 2-fold higher in normal and 1.8-fold higher in the sickle studies. Hemin increased colony growth and gamma-globin synthesis in normal cultures in air, and the effects of hemin and low oxygen were additive. In sickle cultures, hemin and low oxygen had additive effects on colony growth, but only low oxygen increased gamma-globin synthesis. Interleukin-3 increased colony numbers on day 13, primarily by acceleration of peak growth. Interleukin-3 also increased gamma-globin synthesis in low oxygen in normal but not sickle cultures. Thus, low oxygen increases in vitro sensitivity to erythropoietin, colony numbers, and relative gamma-globin synthesis in normal and sickle cultures.

The effectiveness of psychological interventions in competitive sport.

The use of psychological interventions in competitive sport to enhance performance has become increasingly popular. However, the effectiveness of these interventions has been questioned by some sports psychologists. In general, educationally-based psychological interventions have produced significant increases in performance. Specifically, it was found that 38 of the 45 studies examined (85%) had found positive performance effects, although causality could only be inferred in 20 of these studies. These interventions could be classified as relaxation-based, cognitive, cognitive-behavioural or behavioural in nature. Although general support was provided for the effectiveness of psychological interventions in competitive sports, a number of methodological shortcomings limit the application of the findings. For example, future intervention research in competitive sport should employ more detailed manipulation checks, include follow-up assessments beyond a mere post-test, include placebo-control groups to control for expectancy effects and include more diverse samples. In addition to the empirical intervention research, recent research employing qualitative methodologies has greatly added to our understanding of the types of interventions and what type of sport psychology consultants most positively affect performance enhancement, as well as the personal growth of athletes. Continued quantitative and qualitative research needs to be conducted so that a better understanding is gained of how to conduct psychological interventions with athletes that will enhance performance as well as personal growth.

Goal setting and performance in sport and exercise settings: a synthesis and critique.

Although goal setting research in industrial and organizational settings has been proliferating rapidly over the past 20 yr, it is only recently that sport psychologists begun to systematically test its effects in sport and exercise settings. However, the recent empirical literature in sport and exercise examining the relationship between goals and performance has been equivocal. Thus, the purposes of the present review are fourfold: (a) briefly review the industrial/organizational literatures concerning the relationship between goals and task performance; (b) review the literature testing the relationship between goals and performance in sport and exercise settings; (c) discuss methodological and interpretive limitations including the impact of mediating variables; (d) offer future directions for research.

Effects of treatment with 5-azacytidine on the in vivo and in vitro hematopoiesis in patients with myelodysplastic syndromes.

The myelodysplastic syndrome (MDS) comprises a group of clonal hematopoietic disorders derived from an abnormality affecting a multipotent hematopoietic stem cell. Despite trials testing numerous agents in patients with MDS, no single drug has yet emerged as the accepted standard of treatment. Observation and supportive care with blood products and antibiotics, when necessary, continue to be the mainstays of therapy. We administered 5-azacytidine, a cell-cycle specific ring analog of the pyrimidine nucleoside cytosine, as a continuous intravenous infusion, 75 mg/m2 per day for 7 days every 4 weeks. Patients had refractory anemia with excess blasts (RAEB) or refractory anemia with excess blasts in transformation (RAEB-T). Responses were seen in 21 (49%) of 43 evaluable patients: five (12%) in complete remission (CR, complete normalization of bone marrow and peripheral blood counts); 11 (25%) in partial remission (PR, > or = 50% restoration of the deficit from normal of all three peripheral blood cell lines, elimination of transfusion requirements, and a decrease in percentage bone marrow blasts by > or = 50% from prestudy values); five (12%) improved (> or = 50% restoration in the deficit from normal of one or more peripheral blood cell lines and/or a > or = 50% decrease in transfusion requirements). A trilineage improvement (CR and PR) occurred in 37% of the patients. The median survival for all patients was 13.3 months and the median duration of remission for those with CR and PR was 14.7 months. Mild to moderate nausea and/or vomiting was the most common side effect (63%). Myelosuppression, either bone marrow hypoplasia or drug related cytopenias requiring a reduction in the dose of azacitidine, occurred in only 33% of the patients. Prior to treatment, bone marrow erythroid progenitor cells were assayed in vitro. Colonies derived from erythroid burst-forming units (BFU-e) were undetectable in one patient and reduced in two. The number of colonies derived from erythroid colony-forming units (CFU-e)) were also reduced in two of the three patients. In the two patients with detectable colony growth prior to treatment, colony number decreased by day 8 of the first cycle, followed by a subsequent increase. Continued treatment with azacitidine led to normalization of the number of CFU-e derived colonies as well as an increase in the number of BFU-e derived colonies. This improvement in erythroid colony number correlated with the spontaneous rise in hemoglobin levels and red cell transfusion independence.(ABSTRACT TRUNCATED AT 400 WORDS)

Stem cell factor amplifies newborn and sickle erythropoiesis in liquid cultures.

A two-phase liquid-culture system was used to substantially amplify and differentiate erythroblasts, starting with mononuclear cells from the blood of normal adults, newborn infants, and patients with sickle cell anemia. After the first 7 days (phase 1), in medium plus fetal bovine serum (FBS) alone, or in combination with stem cell factor (SCF) or conditioned medium (CM), the cell number was unchanged, and the cells all looked like lymphocytes. These cells were then diluted into medium with erythropoietin (Ep) alone, with Ep and either SCF or CM, or in methylcellulose with the same factors (phase 2). After 14 days in liquid phase 2 with SCF and Ep, the cell numbers increased an average of 30-fold in the sickle, 24-fold in the newborn, and 4-fold in the normal adult cultures; almost all the cells were erythroblasts and erythrocytes. SCF in phase 1 increased the number of late progenitors (CFU-E) assayed in methylcellulose, with the largest number in sickle, followed by newborn cultures and then adult cultures. We conclude that erythroid progenitor cells survive for at least 7 days without Ep (but with FBS). Progenitor cells are amplified, particularly with SCF. Later in culture, SCF with Ep increases the final number of differentiated erythroid cells. Both the early and the late effects of SCF are most effective in sickle, followed by newborn cultures and then adult cultures.

Effect of stem cell factor on in vitro erythropoiesis in patients with bone marrow failure syndromes.

Stem cell factor (SCF) enhances normal hematopoiesis. We examined its effect in vitro on bone marrow and blood progenitors from patients with inherited bone marrow failure syndromes, including 17 patients each with Diamond-Blackfan anemia (DBA) and Fanconi's anemia (FA), 3 with dyskeratosis congenita (DC), and 1 each with amegakaryocytic thrombocytopenia (amega) and transient erythroblastopenia of childhood (TEC). Mononuclear cells were cultured with erythropoietin (Ep) alone or combined with SCF or other factors. SCF increased the growth of erythroid progenitors in cultures from 50% of normal controls, 90% of DBA, 70% of FA, 30% of DC, and the amega and TEC patients; normal numbers were reached in 25% of DBA studies. Improved in vitro erythropoiesis with SCF in all types of inherited marrow failure syndromes does not suggest a common defect involving kit or SCF, but implies that SCF may be helpful in the treatment of hematopoietic defects of varied etiologies.

Erythropoiesis is distinct at each stage of ontogeny.

In vitro erythropoiesis from fetuses, newborn infants, and adults was compared in methyl cellulose cultures. Fetal and newborn blood erythroid colony formation tended to be more sensitive to erythropoietin than adult. The day of maximal colony formation was earlier in fetal than in newborn or adult cultures. The number of colonies/100,000 mononuclear cells on d 13 of culture and on the day of peak growth was highest in fetal, intermediate in newborn, and lowest in adult cultures. Burst forming units-erythroid/mL of blood on culture d 13 and the day of peak growth were similar in fetuses and newborns, and both were significantly greater than in adults. The proportional synthesis of gamma-globin in fetal colonies was 2-fold greater than in newborn colonies, and 6-fold greater than in adult colonies. Thus, fetal, newborn, and adult erythroid progenitor cultures are each unique with regard to erythropoietin sensitivity, growth time course, number of erythroid colonies, and the proportion of gamma-globin synthesis.

Sickle and thalassemic erythroid progenitor cells are different from normal.

Blood erythroid progenitors (BFU-E) from patients with sickle and thalassemic syndromes were compared with those from normal individuals. The day of maximal colony formation in methyl cellulose was slightly later in the cultures from the patients with hemoglobinopathies than in the normal cultures. The number of colonies/100,000 mononuclear cells was similar in all cultures on day 13, but was higher in the hemoglobinopathy cultures on the day of maximal growth. The number of BFU-E/mL of blood was significantly higher than normal at all times in both sickle cell anemia and thalassemia. The proportional synthesis of gamma globin was twice normal in all sickle cultures, and 4 times normal in those from beta+-thalassemia. Hemin and interleukin-3 increased the numbers of erythroid colonies in all cultures, but did not consistently alter the globin synthesis patterns. Each progenitor population has a unique pattern in terms of time course, number of BFU-E, and level of gamma globin synthesis. These features indicate distinct types of BFU-E, or differences in accessory cells, or both, which distinguish blood-borne erythropoiesis in normals and those with hemoglobinopathies.

Coryneform group A-4 endophthalmitis. An experimental animal model.

Diphtheroids, members of the coryneform family of bacteria, increasingly have been recognized as the cause of serious ocular diseases. After isolation of coryneform group A-4 from two patients with delayed endophthalmitis after cataract extraction and intraocular lens implantation, 10(7) organisms were injected into the vitreous of seven New Zealand white rabbits, producing endophthalmitis in all eyes inoculated. Coryneform group A-4 subsequently was isolated in six of seven eyes receiving 10(7) organisms, proving Koch's postulates. Five of these seven eyes were treated with a single dose of intravitreal gentamicin, and three eyes remained culture positive. Eyes inoculated with 10(5) or 10(2) coryneform group A-4 organisms had transient anterior chamber and vitreal inflammation; all vitreous cultures were negative. These studies demonstrate that coryneform group A-4 endophthalmitis can be reproduced in an animal model and that gentamicin may not sterilize an eye infected with this organism. Future studies are needed to determine the optimum antibiotic regimen for treatment of this type of endophthalmitis.