Haptoglobin and its association with naturally occurring diseases in Holstein heifer calves / [Haptoglobina e sua associação com doenças ocorridas naturalmente em bezerras da raça Holandesa]

The present study evaluated the use of haptoglobin (Hp) as an indicator of health and performance in 166 Holstein heifer calves reared in an intensive production system. Calves were evaluated at D6-9; D10-13; D20-23; D35-38 and D65-68, corresponding to the days of life. The absence or presence of diseases was evaluated by physical examination and classification of scores. The performance parameters evaluated were body weight, height at withers and hind width. Hp was measured by spectrophotometric technique. The highest prevalence of diarrhea (59.4%; 98/165) was observed in D10-13, bovine respiratory disease (BRD) was on D35-38 (25.8%; 42/163), and umbilical inflammations in D6-D9 (7.8%; 13/166). Highest values of Hp were observed in animals with diarrhea (P=0.02), and umbilical inflammation (P=0.057), in comparison with the group of healthy calves. A significant negative correlation was observed between Hp and performance index. This protein presented an important relation with diarrhea and performance of the calves, opening perspectives on its utilization as a biomarker of diseases.(AU)

O presente estudo avaliou o uso da haptoglobina (Hp) como indicadora de sanidade e desempenho em 166 bezerras Holandesas criadas em um sistema de produção intensivo. As bezerras foram avaliadas nos momentos D6-9; D10-13; D20-23; D35-38 e D65-68, sendo estes correspondentes aos dias de vida. A ausência ou a presença de doenças foi avaliada por meio do exame físico e da classificação por escores. Os parâmetros de desempenho avaliados foram peso corporal, altura de cernelha e largura de garupa. A Hp foi mensurada por técnica espectrofotométrica. A maior prevalência de diarreia (59,4%; 98/165) foi observada em D10-13, doença respiratória bovina (DRB) ocorreu em D35-38 (25,8%; 42/163) e inflamações umbilicais em D6-D9 (7,8%; 13/166). O valor de Hp foi maior nos animais que apresentaram diarreia (P=0,02) e inflamações umbilicais (P=0,057), em comparação ao grupo de bezerras saudáveis. Houve correlação negativa significativa entre a Hp e os índices de desempenho. Essa proteína apresentou uma importante relação com a diarreia e com o desempenho das bezerras, abrindo perspectivas sobre a sua utilização como biomarcadora de doenças.(AU)

Comparação entre as abordagens lateral direita e mediana ventral em cadelas submetidas à ovariossalpingo-histerectomia / Comparison between the right lateral and ventral median approach in a bitch undergoing ovariosalpinghysterectomy

A fim de comparar as abordagens abdominais, pela linha mediana ventral e lateral direita em cadelas pré-púberes e adultas submetidas a ovariossalpingo-histerectomia, utilizaram-se 28 cadelas hígidas, distribuídas em dois grupos experimentais de igual número: grupo abordagem mediana ventral (AMV) e grupo abordagem lateral direita (ALD), com sete animais adultos e sete animais pré-púberes em cada grupo. O procedimento cirúrgico foi dividido em nove manobras cirúrgicas distintas, e o tempo para conclusão de cada uma delas, suas facilidades e dificuldades, assim como o tempo cirúrgico total, foram determinados. O tempo médio desde o início da incisão da pele até a entrada na cavidade peritoneal foi menor nas cadelas adultas (P≤0,001) e pré-púberes (P≤0,001) do grupo AMV, mas o tempo médio para identificação uterina foi menor nas cadelas pré-púberes (P≤0,001) do grupo ALD. O tempo cirúrgico total foi menor utilizando-se a abordagem lateral direita (grupo ALD) nas cadelas adultas (P≤0,001) e pré-púberes (P≤0,001). Seu uso não se relacionou com complicações cirúrgicas e facilitou a identificação uterina, possibilitando redução no tempo cirúrgico total. Assim, a abordagem lateral direita demonstrou ser uma alternativa segura em cadelas adultas e pré-púberes submetidas à OSH eletiva.

In order to compare the abdominal approaches, through the ventral midline and right lateral in pre-pubertal adult female dogs undergoing ovariohysterectory we used 28 otherwise healthy dogs, divided into two experimental groups of equal number: Ventral Median Approach Group (VMA) and Right Side Approach Group (RSA), with seven adult animals and seven pre-pubertal animals in each group. The surgical procedure was divided into nine different surgical maneuvers, and the time required for completion of each of them, their strengths and difficulties, as well as the total surgical time were determined. The time from the start of the skin incision to the entrance into the peritoneal cavity was lower in adult female dogs (P≤0.001) and pre-pubertal (P≤0.001) in the VAM group, but the time for uterine identification was lower in pre-pubertal female dogs (P≤ 0.001) in the RSA group. The total surgical time was shorter using the right lateral approach (RSA group) in adult (P≤ 0.001) and pre-pubertal (P≤0.001) female dogs. Thus, the right-side approach has proved to be a safe alternative in adult and pre-pubertal dogs undergoing elective OSH. Its use was not associated with surgical complications, and facilitated uterine identification, allowing a reduction in the total surgical time.

CA15.3, CEA and LDH in dogs with malignant mammary tumors.

BACKGROUND: Presence of tumor markers in serum might be connected to the number of secreting cells and with the stage of the neoplasm. However, there are few studies regarding these markers in veterinary clinical oncology. OBJECTIVES: To determine the serum concentrations of cancer antigen 15.3 (CA 15.3), carcinoembryonic antigen (CEA), and lactate dehydrogenase (LDH) in female dogs with different stages of mammary cancer. ANIMALS: Ninety female dogs, including 30 that were healthy, 40 that had nonmetastatic cancer, 12 with regional metastasis, and 8 with distant lymph node metastasis. METHODS: Prospective case-controlled observational study. Serum samples were collected to measure CA15.3, CEA, and LDH from 60 female dogs with mammary cancer during mastectomy and 30 healthy female dogs during routine check-up. CA15.3 and CEA were determined by chemiluminescent immunoassay and LDH by ultraviolet kinetic method. Western blotting analysis was performed to confirm the specificity and possible cross-reactivity of human CA15.3 and CEA antibodies with canine serum. Group data were compared by ANOVA followed by Student-Newman-Keuls and Tukey's tests. Correlations were investigated by Pearson and Spearman tests. RESULTS: CEA, CA15.3, and LDH were measurable in all groups. Higher serum concentration of CA15.3 and LDH was associated with regional and distant metastases. There was a significantly higher serum CA15.3 concentration in animals with lymph node metastasis when compared with animals without metastasis. There were no significant differences in CEA among groups. Expression of CA15.3 and CEA in canine serum was confirmed by Western blotting. CONCLUSIONS AND CLINICAL IMPORTANCE: Serum CA15.3 can be used to distinguish nonmetastatic from metastatic carcinomas.

Effect of homeopathic medicines on helminth parasitism and resistance of Haemonchus contortus infected sheep.

This study evaluated the effects of homeopathic treatment on control of Haemonchus contortus infection in sheep. Twenty lambs were randomized to three treatments: treated with the homeopathic medicines, Ferrum phosphoricum, Arsenicum album and Calcarea carbonica; treated with a conventional antihelminthic, doramectin, and an untreated control group. Fecal and blood samples were taken from each animal on days 18, 38 and 68 after start of treatment. A significant reduction in number of H. contortus larvae (p<0.01) was observed for animals in the homeopathic treatment group compared to the control group. Fecal egg counts showed negative correlation between haematocrit and haemoglobin concentrations in the homeopathic treatment group (p<0.01); however, the biochemical and immunological parameters showed better correlation, indicating that the homeopathic medicine improved vital functions. Daily weight gain in the homeopathic treatment group was superior to the control and to the antihelminthic groups, 31 and 6.5%, respectively. The cost benefit analysis confirmed that homeopathy group increases economic trend when compared with the other groups.

Chemosensitization effects of XIAP downregulation in K562 leukemia cells.

The effect of downregulation of the expression of the antiapoptotic protein XIAP with antisense oligonucleotides was evaluated in the K562 chronic myeloid leukemia (CML) cell line. This was carried out by studying the effects of downregulation of XIAP expression on cellular viability, cellular apoptosis and on the response to two chemotherapeutical drugs, etoposide and doxorubicin. We document that downregulation of XIAP expression decreased cellular viability, increased cellular apoptosis and enhanced the effects of doxorubicin.

Proteinograma de caprinos da raça Pardo-Alpina infectados naturalmente por parasitos gastrintestinais / Proteinogram of Alpine goat naturally infected by gastrointestinal parasites

The proteinogram of six 12 month-old Alpine goats, intensively raised and naturally infected by gastrointestinal parasites, was evaluated. Blood and feces samples of each animal were monthly collected. Total serum protein and their fractions were determined by agarose gel eletrophoresis, using Tris buffer, pH 9.2. The identified protein fractions were albumin, alfa-globulin, beta1-globulin, beta2-globulin and gama-globulin, whose average and standard deviation (g/dl) were, respectively: 2.35±0.39, 0.69±0.36, 0.70±0.08, 0.48±0.08 and 1.52±0.41. It was not observed significative correlation (P>0.05), according to the Spearman non-parametric test, either between the Strongyloides eggs count per gram of feces or the Haemonchus spp. larval count per gram of feces and the fraction electrophorectly variable.

Weak D type 2 is the most prevalent weak D type in Portugal.

The weak D phenotype is the most common D variant, with a frequency of 0.2-1% in Caucasian individuals. There are several weak D types, with different frequencies in European countries, which may pose serologic problems and have the potential for alloimmunization. Samples from Portuguese individuals were tested for RhD by two or three distinct monoclonal and oligoclonal antisera, in direct agglutination tests. When discrepant results were observed, samples were tested with panels of monoclonal anti-D by LISS-indirect antiglobulin test. Cases that reacted weakly with IgM but positive with IgG anti-D were analysed by PCR-sequence-specific primers and real-time PCR. Ninety-nine samples were referred after being characterized as weak D. This genotype was recognized, with a preponderance of weak D type 2 (63.6%) over type 1 (16.2%) and 3 (14.1%). The high incidence of weak D type 2 in our population is in marked contrast to studies performed in other European populations and might be due to our sample selection criteria or ethnic variation. There are advantages in genotyping serologically depressed D samples to avoid the waste of D-negative RBC units and the use of immunoglobulin in pregnant women, who have no risk of alloimmunization.

Haplotype study of microsatellites flanking the t(15;17) breakpoint in acute promyelocytic leukemia patients from North Portugal.

A higher frequency of acute promyelocytic leukemia (APL) has been noted in countries of Southern Europe and among 'Latino' patients of the United States with acute myeloid leukemia (AML). In order to discover whether there is any genetic predisposition to the disease, we analyzed microsatellites flanking PML and RARalpha genes in 29 t(15;17) APL patients from North Portugal and compared them with a control group of 123 healthy individuals. Fluorescent PCR products were analyzed using an automated capillary electrophoresis system and allele and haplotype frequencies of the two populations were determined. No significant differences were found, suggesting the same genetic origin of patients and healthy individuals. As suggested by the four microsatellites screened, MSI (microsatellite instability) does not explain the increased incidence of t(15;17) APL in this Portuguese population. These results intend to be a new approach to the study of APL, reflecting the particularity of the disease.

The PML-RAR alpha transcript in long-term follow-up of acute promyelocytic leukemia patients.

BACKGROUND AND OBJECTIVES: Detection of PML-RAR alpha transcripts by RT-PCR is now established as a rapid and sensitive method for diagnosis of acute promyelocytic leukemia (APL). Although the majority of patients in long-term clinical remission are negative by consecutive reverse transcription polymerase chain reaction (RT-PCR) assays, negative tests are still observed in patients who ultimately relapse. Conversion from negative to positive PCR has been observed after consolidation and found to be a much stronger predictor of relapse. This study reports on 47 APL patients to determine the correlation between minimal residual disease (MRD) status and clinical outcome in our cohort of patients. DESIGN AND METHODS: The presence of PML-RAR alpha t transcripts was investigated in 47 APL patients (37 adults and 10 children) using a semi-nested reverse transcriptase-polymerase chain reaction to evaluate the prognostic value of RT-PCR tests. RESULTS: All patients achieved complete clinical remission (CCR) following induction treatment with all-trans retinoic acid (ATRA) and chemotherapy (CHT) or ATRA alone. Patients were followed up between 2 and 117.6 months (median: 37 months). Relapses occurred in 11 patients (9 adults and 2 children) between 11.4 and 19 months after diagnosis (median: 15.1 months) while 36 patients (28 adults and 8 children) remained in CCR. Seventy-five percent of patients carried the PML-RAR alpha long isoform (bcr 1/2) which also predominated among the relapsed cases (9 of 11) but did not associate with any adverse outcome (p= 0.37). For the purpose of this analysis, minimal residual disease tests were clustered into four time-intervals: 0-2 months, 3-5 months, 6-9 months and 10-24 months. INTERPRETATION AND CONCLUSIONS: Children showed persisting disease for longer than adults during the first 2 months of treatment. At 2 months, 10 (50%) of 20 patients who remained in CCR and 4 (80%) of 5 patients who subsequently relapsed were positive. Patients who remained in CCR had repeatedly negative results beyond 5.5 months from diagnosis. A positive MRD test preceded relapse in 3 of 4 tested patients. The ability of a negative test to predict CCR (predictive negative value, PNV) was greater after 6 months (>83%), while the ability of a positive test to predict relapse (predictive positive value, PPV) was most valuable only beyond 10 months (100%). This study (i) highlights the prognostic value of RT-PCR monitoring after treatment of APL patients but only from the end of treatment, (ii) shows an association between conversion to a positive test and relapse and (iii) suggests that PCR assessments should be carried out at 3-month intervals to provide a more accurate prediction of hematologic relapses but only after the end of treatment.

Differences in phenotype, growth factor requirements, pattern of expression of adhesion molecules and rate of apoptosis displayed by three new myeloid sister leukemic cell lines.

We established three new human myeloid cell lines from one patient, in the presence of granulocyte-macrophage colony-stimulating factor (UPM1-GM), interleukin-3 (UMP1-IL-3) or without exogenous growth factors (UPM1). The 3 lines were characterized by phenotypic, genotypic and functional studies. These cell lines may provide useful tools to study different aspects of leukemic cell biology

Limited synergistic effect of antisense oligonucleotides against bcr-abl and transferrin receptor mRNA in leukemic cells in culture.

The synergistic use of antisense oligonucleotides (ASOs) towards the bcr-abl and the transferrin receptor (TfR) mRNA was studied in a chronic myeloid leukemia (CML) cell line, aiming to improve the efficiency of individual ASO treatment. At 20 microM concentration, bcr-abl ASOs reduced cell growth by 40% and was specific for cells that have the translocation: there was a 34% reduction of BCR-ABL protein. The TfR ASO reduced cell growth by 20% and decreased TfR protein by 24%. The ASOs were more potent at reducing cell growth when used in combination (respectively, -20 and -17% than bcr-abl ASO and TfR ASO when used individually at the 10 microM concentration), thus we postulate that there is synergism of action. Cell cycle analysis also revealed that the sub-G1 peak was bigger in the synergistic treatment.

Critical evaluation of techniques to detect and measure cell death--study in a model of UV radiation of the leukaemic cell line HL60.

The reliability of eight distinct methods (Giemsa staining, trypan blue exclusion, acridine orange/ethidium bromide (AO/EB) double staining for fluorescence microscopy and flow cytometry, propidium iodide (PI) staining, annexin V assay, TUNEL assay and DNA ladder) for detection and quantification of cell death (apoptosis and necrosis) was evaluated and compared. Each of these methods detects different morphological or biochemical features of these two processes. The comparative analysis of the 8 techniques revealed that AO/EB (read in fluorescence microscopy) provides a reliable method to measure cells in different compartments (or pathways) of cell death though it is very time consuming. PI staining and TUNEL assay were also sensitive in detecting very early signs of apoptosis, but do not allow precise quantification of apoptotic cells. These three methods were concordant in relation to induction of apoptosis and necrosis in HL60 cells with the various UV irradiation time periods tested. Both AO/EB (read by flow cytometry) and annexin V-FITC/PI failed to detect the same number of early apoptotic cells as the other three methods. Trypan blue is valueless for this purpose. Giemsa and DNA ladder might be useful as confirmatory tests in some situations.

Leptospira interrogans sorotipo pomona em caprinos: determinaçäo do fibrinogênio, proteína total, transaminases e bilirrubinas, durante infecçäo experimental / Leptospira interrogans serovar pomona in experimental infection of goats: determination of fibrinogen, total protein, transaminases and bilirrubins

Com o objetivos de estudar os aspectos laboratoriais, através da determinaçäo do fibrinogênio plasmático, proteína total plasmática, aspartato e alanina aminostransferases séricas, e bilirrubinas séricas total, direta e indireta, utilizaram-se 20 caprinos mestiços, clinicamente sadios, de ambos os sexos, com dez meses de idade e, com peso vivo médio de oito quilogramas. Os animais foram divididos aleatoriamente em dois grupos de dez: grupo "A", controle e grupo "B", experimental. Nos animais deste último grupo foram inoculados cinco mililitros, via intraperitoneal, de cultura de Leptospira interrogans sorotipo pomona (estirpe M7/87), previamente preparada. Inicialmente, as amostras sanguíneas foram colhidas a partir do 3§ dia após inoculaçäo, em intervalos de quatro dias, entre o 3§ e 15§ dia, passando para seis dias do 16§ ao 44§ dia, e finalmente para sete dias entre o 45§ e 93§ dia. A análise estatística revelou significância a nível de 5 por cento para a bilirrubina total e direta, enquanto para as demais variáveis näo houve diferenças significativas entre os tratamentos

Configuration of immunoglobulin and T cell receptor beta and gamma genes in acute myeloid leukaemia: pitfalls in the analysis of 40 cases.

AIMS: To evaluate the overall incidence of immunoglobulin (Ig) and T cell receptor (TCR) beta and gamma gene rearrangements in a series of 40 cases of acute myeloid leukaemia (AML) and to determine whether structural modifications of these genes could be correlated with the abnormal expression of lymphoid markers in malignant cells. METHODS: All cases were classified according to the criteria of the FAB group and immunophenotyped with a panel of monoclonal antibodies reactive with myeloid and lymphoid differentiation antigens. DNA analysis was performed by the method of Southern using probes for the Ig JH, TCR-C beta 1, and TCR-J tau 1 regions. RESULTS: Phenotypic analysis showed that in addition to myeloid markers, 10 cases expressed lymphoid antigens: CD7 in seven (of which three were TdT positive, one CD2 positive, and one CD19 positive) and CD19 in three. Southern blot analysis showed that bands with sizes different from the germ line control were present in the TCR beta genes in 11 cases: in six of 30 with pure myeloid phenotype and in five of 10 of those expressing lymphoid markers. A close observation of the size and patterns of those bands, however, showed that they could be artefactual. Indeed, further analysis showed that they were either due to resistant Eco RI/Hind III sites at the beta locus or to plasmid contamination. Rearranged genes were eventually found in only two of the 40 cases: at the Ig JH region in one of the 30 with pure myeloid phenotype (3.3%) and at the TCR gamma genes in one of 10 with lymphoid markers (10%). CONCLUSIONS: These observations showed that Ig/TCR gene rearrangements were rare in this AML series (overall incidence of 5%) and that they were not significantly more common in cases with aberrant expression of lymphoid markers. The size and pattern of the potential non-germline bands that can be found in these loci must be carefully evaluated.

[Molluscacide activity of a mixture of 6-n-alkyl salicylic acids (anacardic acid) and 2 of its complexes with copper (II) and lead (II)]. / Atividade moluscicida da mistura de ácidos 6-n-alquil salicílicos (ácido anacárdico) e dos seus complexos com cobre (II) e chumbo (II).

The molluscicide activity of hexanic extract from Anacardium occidentale L. (cashew) nut shell, of copper (II) complex, of lead (II) complex and anacardic acid has been compared in the laboratory in an attempt to obtain better stability than anacardic acid. This was obtained from the hexanic extract of the cashew nut shell by precipitation with lead (II) hydroxide or cupric sulfate plus sodium hydroxide or (II) cupric hydroxide followed by treatment of lead (II) complex with a diluted solution of sulfuric acid. Ten products of the mixture obtained were tested on adults snails of Biomphalaria glabrata at 1 to 10 ppm. The most active products were copper (II) complex, obtained by cupric sulfate plus sodium hydroxide, and anacardic acid (sodium hydroxide) which presented activity at 4 ppm. The anacardic acid's lead content was above the limits accepted by the United States standards.

Acidic isoferritin stimulates differentiation of normal granulomonocytic progenitors.

Acidic isoferritin (AIF) has been shown to be released by cells from patients with leukemia and to have an inhibitory effect on the growth of normal granulomonocytic (GM) progenitors (leukemia-inhibitory activity) during the S phase of the cell cycle. AIF is also produced by normal mature cells of the monocyte-macrophage lineage. We studied the effects of AIF on the differentiation of normal GM progenitors and found an increase in the number of mature cells in AIF-exposed cultures. This increase did not occur when AIF was pretreated with anti-heart ferritin antiserum or when basic isoferritin was used in the place of AIF. The influence of AIF was not mimicked by removing S phase cells by pretreatment with a pulse of high specific activity tritiated thymidine. Thus, the apparent differentiation-stimulating effect of AIF is not likely to be due to selective removal of immature dividing cells. The results suggest that AIF-inhibitory activity on the proliferation of GM progenitors might at least in part be mediated by a stimulus for differentiation of the target cells, thus regulating the number of mature cells which might be formed by a single progenitor cell.

Differentiation induction in myelodysplasia and acute myeloid leukaemia: use of synergistic drug combinations.

DNA synthesis inhibitors and vincristine greatly enhance the response of leukaemic and dysplastic cells to differentiation inducing agents such as retinoic acid (RET). Differentiation induction therapy is an attractive therapeutic approach in myelodysplasia (MDS) and in acute myeloid leukaemia (AML) in the elderly, since it should be possible to increase the production of mature cells, at the expense of precursor cells, without incurring the complications of intensive cytotoxic therapy. Single agent differentiation therapy has, however, not been highly successful. We have therefore investigated the use of synergistic combinations of agents. We treated nine patients (6 with MDS, 3 with AML) with 13-cis-retinoic acid (up to 100 mg/m2/day) in combination with either 6-thioguanine (20-40 mg/day in 14-57 day courses) or with vincristine (1-2 mg as a single injection during a four-day course of RET). Seven patients responded with an increase in the mature cells of at least one haemopoietic lineage. A concomitant decrease in marrow blasts was observed in 3/4 responding patients. The retention of dysplastic and karyotypic abnormalities and lack of a hypoplastic phase all suggested that differentiation induction was occurring in vivo. Prior failure to respond to therapy with single agents (RET in two and cytosine arabinoside in five patients) suggests that the synergy observed in vitro operates in vivo. In-vitro studies on marrow cells from seven patients demonstrated synergistic differentiation induction in 6/7 samples. The seventh patient was one of the two who did not respond clinically. The second of these clinically unresponsive patients had cells which were relatively refractory to RET in vitro, suggesting that in-vivo and in-vitro responses may be related.

Differentiation of human acute myeloid leukaemia cells in response to exogenous and endogenous stimuli: different patterns of response suggested by a bioassay system.

The object of this study was to devise quantitative bioassay systems suitable for the analysis of differentiation in acute myeloid leukaemia (AML) in response to both endogenous (bone marrow) and exogenous stimuli. Dose response analyses of the relationship between exogenous differentiation stimulus (supplied by peripheral blood mononuclear leucocytes) and clone cell maturity in double layer semi-solid agar cultures, were used to identify a measure of response with a linear relationship to differentiation stimulus on which bioassay systems could be based. All 7 AML samples tested at presentation and 1 sample from a patient during the regenerative phase after therapy, exhibited some response to exogenous differentiation stimulus. Only in the latter was there no significant difference in the response of test and reference cells. The other 7 showed marked variation in the pattern of the response in vitro. There was no close correlation between differentiation capacity in vitro and extent of differentiation observed in vivo. This discrepancy might be related to varying availability of endogenous differentiation stimulus in different patients. The technique for assaying differentiation response was adapted to demonstrate and quantify the endogenous differentiation stimulus (the stimulus provided by the leukaemic patient's marrow cells). The results suggest that in some patients the level of this stimulus may be a major determinant in the level of differentiation achieved in vivo.

Synergistic interaction between differentiation inducers and DNA synthesis inhibitors: a new approach to differentiation induction in myelodysplasia and acute myeloid leukaemia.

Numerous agents induce differentiation and maturation of neoplastic and dysplastic myeloid cells in vitro and some of these agents have been used with limited success in the treatment of patients with myelodysplastic syndromes (MDS) and myeloid leukaemias. We recently proposed that physiological and pharmacological agents which enhance differentiation and maturation in vitro act by two fundamentally different routes: (1) by hastening the progression through various differentiation/maturation steps; (2) by slowing proliferation (usually by inhibition of DNA synthesis). In order to test this thesis we looked for synergistic effects on differentiation using pairs of agents from the two groups in cultures of cells from myelodysplastic and acute myeloid leukaemia (AML) patients and from normal marrow donors. The results with three MDS, two AML and three normal samples show that combinations of differentiation inducing agents (retinoic acid, N-methylformamide) with DNA synthesis inhibitors (6-mercaptopurine, cytosine arabinoside and aphidicolin) produce a differentiation inducing effect equivalent to that of 10-100, or even 1000 fold higher concentrations of single agents. Myelotoxic effects in vitro were not synergistic. The use of these synergistic combinations should greatly enhance the usefulness of differentiation inducers in the therapy of MDS and myeloid leukaemia.