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* The high rate of proliferation required of the bone marrow renders it highly susceptible to the influence of external factors. * Anaemia is the most common haematological abnormality seen in systemic disorders. * In the anaemia of chronic disease,erythropoietin production is reduced and proliferation of erythroid progenitor cells is also impaired; this anaemia can generally be alleviated by correction of the underlying disease process. * The status of the endocrine system must always be considered in evaluation of a normocytic,normochromic anaemia. * Anaemia in infection can be due to host or parasite factors or to the treatment administered. * Anaemia due to malignant disease responds to erythropoietin therapy in many cases; failure to respond is a poor prognostic sign. View Publication

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Cyclic AMP (cAMP) and cyclic GMP (cGMP) are key second messengers involved in a multitude of cellular events. From the wealth of synthetic analogs of cAMP and cGMP,only a few have been explored with regard to their therapeutic potential. Some of the first-generation cyclic nucleotide analogs were promising enough to be tested as drugs,for instance N(6),O(2)'-dibutyryl-cAMP and 8-chloro-cAMP (currently in clinical Phase II trials as an anticancer agent). Moreover,8-bromo and dibutyryl analogs of cAMP and cGMP have become standard tools for investigations of biochemical and physiological signal transduction pathways. The discovery of the Rp-diastereomers of adenosine 3',5'-cyclic monophosphorothioate and guanosine 3',5'-cyclic monophosphorothioate as competitive inhibitors of cAMP- and cGMP-dependent protein kinases,as well as subsequent development of related analogs,has proven very useful for studying the molecular basis of signal transduction. These analogs exhibit a higher membrane permeability,increased resistance against degradation,and improved target specificity. Furthermore,better understanding of signaling pathways and ligand/protein interactions has led to new therapeutic strategies. For instance,Rp-8-bromo-adenosine 3',5'-cyclic monophosphorothioate is employed against diseases of the immune system. This review will focus mainly on recent developments in cyclic nucleotide-related biochemical and pharmacological research,but also highlights some historical findings in the field. View Publication

The application of multi-parameter flow cytometry for the assessment of T-cell and cytokine functioning has been used by several groups for studying human and mouse samples,although little has been reported for the rat. Here we report the optimisation of immunofluorescent staining for cell surface and intracellular antigens using three-colour flow cytometric analysis to measure the frequency of rat CD3(+)4(+) T-cells that produce IFN-gamma,IL-4 and IL-10. In vitro stimulation of IFN-gamma production required incubation of splenocytes with PMA and ionomycin in the presence of the protein transport inhibitor brefeldin A for 6 h. Three stimulation protocols for IL-4 and IL-10 production were evaluated. In vitro priming of splenic T-cells with antibodies against CD3 and CD28 and recombinant cytokines (IL-2 and IL-4) for 5 days followed by restimulation with PMA and ionomycin was required to stimulate cells to produce either IL-4 or IL-10. Brefeldin A was found to be a more suitable protein transport inhibitor than monensin. This method will be useful for analysing the nature of individual rat cytokine-producing cells in a variety of experimental model systems. View Publication

DYRKs are a new subfamily of dual-specificity kinases that was originally discovered on the basis of homology to Yak1,an inhibitor of cell cycle progression in yeast. At present,mDYRK-3 and mDYRK-2 have been cloned,and mDYRK-3 has been characterized with respect to kinase activity,expression among tissues and hematopoietic cells,and possible function during erythropoiesis. In sequence,mDYRK-3 diverges markedly in noncatalytic domains from mDYRK-2 and mDYRK-1a,but is 91.3% identical overall to hDYRK-3. Catalytically,mDYRK-3 readily phosphorylated myelin basic protein (but not histone 2B) and also appeared to autophosphorylate in vitro. Expression of mDYRK-1a,mDYRK-2,and mDYRK-3 was high in testes,but unlike mDYRK1a and mDYRK 2,mDYRK-3 was not expressed at appreciable levels in other tissues examined. Among hematopoietic cells,however,mDYRK-3 expression was selectively elevated in erythroid cell lines and primary pro-erythroid cells. In developmentally synchronized erythroid progenitor cells,expression peaked sharply following exposure to erythropoietin plus stem cell factor (SCF) (but not SCF alone),and in situ hybridizations of sectioned embryos revealed selective expression of mDYRK-3 in fetal liver. Interestingly,antisense oligonucleotides to mDYRK-3 were shown to significantly and specifically enhance colony-forming unit-erythroid colony formation. Thus,it is proposed that mDYRK-3 kinase functions as a lineage-restricted,stage-specific suppressor of red cell development. (Blood. 2001;97:901-910) View Publication

The functional characteristics were compared for acute myelogenous leukemia (AML) cells (native blasts and AML cell lines) cultured in three serum-free media (X-vivo 10,X-vivo 15,[Bio-Whitacker,Walkersville,MD] and StemSpan [Stem Cell Technologies,Vancouver,BC,Canada]) and in medium containing 10% inactivated fetal calf serum (FCS). For native AML blasts the following functions were compared: (1) autonomous and cytokine-dependent proliferation; (2) frequency of clonogenic cell; and (3) constitutive cytokine secretion. AML blast proliferation differed between patients independent of the culture medium used,and clonogenic cells were maintained after in vitro culture in all media. In contrast,constitutive cytokine secretion was higher for cells cultured in StemSpan and FCS-containing medium than for cells cultured in the X-vivo media. Native AML blasts incubated in StemSpan also showed a low frequency of apoptotic cells. The three serum-free media could also be used for long-term expansion of well-characterized AML cell lines,but the optimal medium for cell expansion and cytokine secretion differed between cell lines. We conclude that standardized serum-free culture conditions can be used for in vitro studies of native AML blasts and AML cell lines. View Publication

The effect of resiquimod (R-848),an immune-response modifier that is similar to imiquimod,on recurrent herpes simplex virus (HSV) was evaluated using the guinea pig model of genital herpes. Guinea pigs were intravaginally infected with HSV-2 and then were randomized on day 14 to receive nothing or 0.1 mL/kg per dose of subcutaneous resiquimod,given either daily,every other day,or weekly from days 15-35. During a 3-week course of therapy,recurrences in all 3 treated groups were reduced by textgreater80%,compared with the control group. After therapy,recurrences remained significantly (Ptextless.05) decreased in all 3 groups for the next 3 weeks. The group treated weekly developed the fewest recurrences. Significant increases in interleukin-2 levels,produced by incubation of mononuclear cells with HSV-2 antigens,but not in circulating antibody also were detected in the treated groups. Resiquimod treatment may offer significant advantages to present antiviral therapies for the control of recurrent genital herpes. View Publication

A careful prognostic evaluation of patients referred for high-dose therapy (HDT) is warranted to identify those who maximally benefit from HDT as well as those who clearly fail current HDT and are candidates for more innovative treatments. In a series of 110 patients with myeloma who received HDT as first-line therapy,times to event (disease progression and death) were studied through proportional hazard models,in relation to different prognostic factors,including a chromosome 13 fluorescence in situ hybridization (FISH) analysis using a D13S319 probe. Delta13 was detected in 42 patients (38%). Follow-up time among surviving patients and survival time were 48 +/- 3 and 51 +/- 7 months,respectively (median +/- SE). In the univariate analysis,Delta13 was the most powerful adverse prognostic factor for all times to event,especially for the survival time (P textless.0001) and was followed by beta2-microglobulin (beta2m) levels 2.5 mg/L or higher (P =.0001). The comparison of survival prognostic models including beta2m 2.5 mg/L or greater and another factor favored the Delta13/beta2m combination. In 22 patients (20%) with no unfavorable factor,the median survival time was not reached at 111 months. In contrast,among 55 patients (50%) with one unfavorable factor and 33 patients (30%) with 2 unfavorable factors,median survival times were 47.3 +/- 4.6 months and 25.3 +/- 3.2 months,respectively (P textless.0001). We conclude that delta13,adequately detected by FISH analysis,is a very strong factor related to poor survival,especially when associated with a beta2m level of 2.5 mg/L or higher. Routine FISH Delta13 assessment is strongly recommended for patients considered for HDT. View Publication

High expression of the Breast Cancer Resistance Protein (BCRP) gene has been shown to be involved in resistance to chemotherapeutic drugs. Knowledge of the localization of BCRP protein in normal tissues may help unravel the normal function of this protein. Therefore,we characterized the tissue distribution and cellular localization of BCRP in frozen sections of normal human tissues. For this purpose,we used the recently described monoclonal antibody BXP-34 and another independently developed monoclonal antibody directed against BCRP,BXP-21. Both monoclonal antibodies show specific BCRP plasma membrane staining on cytospins obtained from topotecan- or mitoxantrone-selected cell lines,as well as from BCRP-transfected cell lines. Immunoprecipitation experiments using either BXP-21 or BXP-34 yielded a clear M(r) 72,000 BCRP band from BCRP-overexpressing tumor cells. In the topotecan-selected T8 and mitoxantrone-selected MX3 tumor cell lines,BCRP turned out to be differentially glycosylated. In contrast to BXP-34,BXP-21 is able to detect the M(r) 72,000 BCRP protein on immunoblots and is reactive with BCRP in formalin-fixed,paraffin-embedded tissues. Using BXP-21 and BXP-34,prominent staining of BCRP was observed in placental syncytiotrophoblasts,in the epithelium of the small intestine and colon,in the liver canalicular membrane,and in ducts and lobules of the breast. Furthermore,BCRP was present in veinous and capillary endothelium,but not in arterial endothelium in all of the tissues investigated. In the tissues studied,the mRNA levels of BCRP were assessed using reverse transcription-PCR,and these corresponded with the levels of BCRP protein estimated from immunohistochemical staining. The presence of BCRP at the placental syncytiotrophoblasts is consistent with the hypothesis of a protective role of BCRP for the fetus. The apical localization in the epithelium of the small intestine and colon indicates a possible role of BCRP in the regulation of the uptake of p.o. administered BCRP substrates by back-transport of substrate drugs entering from the gut lumen. Therefore,it may be useful to attempt to modulate the uptake of p.o. delivered BCRP substrates,e.g.,topotecan or irinotecan,by using a BCRP inhibitor. Clinical trials testing this hypothesis have been initiated in our institute. View Publication

Calmodulin-binding sites on target proteins show considerable variation in primary sequence; hence compounds that block the access of calmodulin to these binding sites may be more selective than compounds that inactivate calmodulin. Suramin and its analogue NF307 inhibit the interaction of calmodulin with the ryanodine receptor. We have investigated whether inhibition of calmodulin binding to target proteins is a general property of these compounds. Suramin inhibited binding of [(125)I]calmodulin to porcine brain membranes and to sarcoplasmic reticulum from skeletal muscle (IC(50)=4.9+/-1.2 microM and 19.9+/-1.8 microM,respectively) and blocked the cross-linking of [(125)I]calmodulin to some,but not all,target proteins in brain membranes by [(125)I]calmodulin. Four calmodulin-binding proteins were purified [ryanodine receptor-1 (RyR1) from rabbit skeletal muscle,neuronal NO synthase (nNOS) from Sf9 cells,G-protein betagamma dimers (Gbetagamma) from porcine brain and a glutathione S-transferase-fusion protein comprising the C-terminal calmodulin-binding domain of the metabotropic glutamate receptor 7A (GST-CmGluR7A) from bacterial lysates]. Three of the proteins employed (Gbetagamma,GST-CmGluR7A and RyR1) display a comparable affinity for calmodulin (in the range of 50-70 nM). Nevertheless,suramin and NF307 only blocked the binding of Gbetagamma and RyR1 to calmodulin-Sepharose. In contrast,the association of GST-CmGluR7A and nNOS was not impaired,whereas excess calmodulin uniformly displaced all proteins from the matrix. Thus suramin and NF307 are prototypes of a new class of calmodulin antagonists that do not interact directly with calmodulin but with calmodulin-recognition sites. In addition,these compounds discriminate among calmodulin-binding motifs. View Publication

Although the source of embryonic stem (ES) cells presents ethical concerns,their use may lead to many clinical benefits if differentiated cell types can be derived from them and used to assemble functional organs. In pancreas,insulin is produced and secreted by specialized structures,islets of Langerhans. Diabetes,which affects 16 million people in the United States,results from abnormal function of pancreatic islets. We have generated cells expressing insulin and other pancreatic endocrine hormones from mouse ES cells. The cells self-assemble to form three-dimensional clusters similar in topology to normal pancreatic islets where pancreatic cell types are in close association with neurons. Glucose triggers insulin release from these cell clusters by mechanisms similar to those employed in vivo. When injected into diabetic mice,the insulin-producing cells undergo rapid vascularization and maintain a clustered,islet-like organization. View Publication

Valproic acid is widely used to treat epilepsy and bipolar disorder and is also a potent teratogen,but its mechanisms of action in any of these settings are unknown. We report that valproic acid activates Wntdependent gene expression,similar to lithium,the mainstay of therapy for bipolar disorder. Valproic acid,however,acts through a distinct pathway that involves direct inhibition of histone deacetylase (IC(50) for HDAC1 = 0.4 mm). At therapeutic levels,valproic acid mimics the histone deacetylase inhibitor trichostatin A,causing hyperacetylation of histones in cultured cells. Valproic acid,like trichostatin A,also activates transcription from diverse exogenous and endogenous promoters. Furthermore,valproic acid and trichostatin A have remarkably similar teratogenic effects in vertebrate embryos,while non-teratogenic analogues of valproic acid do not inhibit histone deacetylase and do not activate transcription. Based on these observations,we propose that inhibition of histone deacetylase provides a mechanism for valproic acid-induced birth defects and could also explain the efficacy of valproic acid in the treatment of bipolar disorder. View Publication