A critical role of Rap1b in B-cell trafficking and marginal zone B-cell development

B-cell development is orchestrated by complex signaling networks. Rap1 is a member of the Ras superfamily of small GTP-binding proteins and has 2 isoforms, Rap1a and Rap1b. Although Rap1 has been suggested to have an important role in a variety of cellular processes, no direct evidence demonstrates a role for Rap1 in B-cell biology. In this study, we found that Rap1b was the dominant isoform of Rap1 in B cells. We discovered that Rap1b deficiency in mice barely affected early development of B cells but markedly reduced marginal zone (MZ) B cells in the spleen and mature B cells in peripheral and mucosal lymph nodes. Rap1b-deficient B cells displayed normal survival and proliferation in vivo and in vitro. However, Rap1b-deficient B cells had impaired adhesion and reduced chemotaxis in vitro, and lessened homing to lymph nodes in vivo. Furthermore, we found that Rap1b deficiency had no marked effect on LPS-, BCR-, or SDF-1–induced activation of mitogen-activated protein kinases and AKT but clearly impaired SDF-1–mediated activation of Pyk-2, a key regulator of SDF-1–mediated B-cell migration. Thus, we have discovered a critical and distinct role of Rap1b in mature B-cell trafficking and development of MZ B cells.      

THE VALUE OF PENICILLIN IN THE TREATMENT OF AGRANULOCYTOSIS CAUSED BY THIOURACIL

Thiouracil has been found to be an effective drug in the treatment of hyperthyroidism. Agranulocytosis following its use occurred in nine cases, four of whichterminated fatally. In five others a complete and rapid recovery took place following penicillin therapy. The latter drug is believed to be ideal for all cases of agranulocytosis, and especially those in which chemotherapy has been used and mayhave been responsible for the condition. Thus far we have not seen any report ofany untoward effect on the hemopoietic system from the use of penicillin.

The use of antibacterial agents for the treatment of agranulocytosis was suggested by Dameshek and Wolfson²¹ in 1942. It was believed by these authors thatpatients with agranulocytosis died not of the leukopenia per se but of the sepsiswhich developed secondarily to the lack of granulocytes. Two very severe casesof aminopyrine agranulocytosis treated with sulfathiazole made complete recoveries. For the treatment of sulfonamide agranulocytosis, it was suggested thata preparation differing from that which had already been used be given. Withthe discovery of penicillin, and its complete lack of possible deleterious effect onthe bone marrow, its use was suggested by Dameshek¹⁷ (1944). A report on thebeneficial effects of this medication in a case of sulfonamide agranulocytosis waslater reported by Dameshek and Knowlton¹⁸ and similar cases by Sprague andFerguson¹⁹ and by Meredith and Fink.²⁰

Since sulfonamides may cause further toxic effect on the bone marrow, we feelthat their use should be avoided in the treatment of agranulocytosis, especiallywhere a history of previous use is obtained. We do not agree with others^{21, 22}who continue the use of sulfonamides in the treatment of leukopenia or agranulocytosis where these very drugs may have been responsible for the condition. Itwould seem better judgment to use penicillin, which by combating the bacterialinvasion of the body and the consequent toxemia enables the patient to surviveuntil the bone marrow cells regenerate.

     

Towards medical therapy of calcific aortic stenosis lessons from molecular biology.

Calcific aortic stenosis, with a prevalence of 3–9%, isthe most frequent heart valve disease and the main reason forvalve replacement in patients over 60 years of age.¹ Histopathologically,sclerosis of the aortic valve is defined as fibrous thickeningand calcification of the valve cusps.² For many years, thesechanges were thought to be caused by passive calcium precipitationwithin the valve. However, there is increasing evidence thatthe development and progression of calcific aortic stenosismay be triggered by underlying genetic and acquired risk factors.³Early     

Cell-intrinsic requirement for pRb in erythropoiesis

Retinoblastoma (Rb) and family members have been implicated as key regulators of cell proliferation and differentiation. In particular, accumulated data have suggested that the Rb gene product pRb is an important controller of erythroid differentiation. However, current published data are conflicting as to whether the role of pRb in erythroid cells is cell intrinsic or non–cell intrinsic. Here, we have made use of an in vitro erythroid differentiation culture system to determine the cell-intrinsic requirement for pRb in erythroid differentiation. We demonstrate that the loss of pRb function in primary differentiating erythroid cells results in impaired cell cycle exit and terminal differentiation. Furthermore, we have used coculture experiments to establish that this requirement is cell intrinsic. Together, these data unequivocally demonstrate that pRb is required in a cell-intrinsic manner for erythroid differentiation and provide clarification as to its role in erythropoiesis.      

Reduced incidence of ischemic stroke in patients with severe factor XI deficiency

Inherited disorders of hemostasis are natural models for investigating mechanisms of thrombosis and development of antithrombotic therapy. Because mice with total factor XI deficiency are protected against ischemic stroke and do not manifest excessive bleeding, we investigated the incidence of ischemic stroke in patients with severe inherited factor XI deficiency. Incidence of ischemic stroke in 115 patients aged 45 years or more with severe factor XI deficiency (activity less than 15 U/dL) was compared with incidence in the Israeli population as estimated from a stroke survey of 1528 patients. Adjustment for major risk factors of stroke (hypertension, diabetes mellitus, hypercholesterolemia, current smoking) was based on comparison of their prevalence in the stroke survey to an Israeli health survey of 9509 subjects. Incidence of myocardial infarction in the factor XI cohort was also recorded. After adjustment for the 4 major risk factors of ischemic stroke, the expected incidence of ischemic stroke was 8.56 compared with one observed (P = .003). The reduced 1:115 incidence of ischemic stroke contrasted with a 19:115 incidence of myocardial infarction, similar to the expected incidence. Thus, severe factor XI deficiency probably is protective against ischemic stroke but not against acute myocardial infarction.      

Efficient marking of human cells with rapid but transient repopulating activity in autografted recipients

Short-term hematopoietic reconstituting cells have been identified in mice, nonhuman primates, and among human cells that engraft xenogeneic hosts. We now present clonal marking data demonstrating a rapid but unsustained contribution of cultured human autografts to the initial phase of hematologic recovery in myeloablated patients. Three patients received transplants of granulocyte colony-stimulating factor–mobilized autologous peripheral blood (PB) cells, of which a portion (8%-25% of the CD34⁺ cells) had been incubated in vitro with growth factors (5 days) and clinical grade LN retrovirus (3-5 days). More than 9% of the clonogenic and long-term culture-initiating cells harvested were transduced. Semiquantitative and linear amplification-mediated polymerase chain reaction analyses of serial PB samples showed that marked white blood cells appeared in all 3 patients within 11 days and transiently constituted up to 0.1% to 1% of those produced in the first month. However, within another 2 to 9 months, marked cells had permanently decreased to very low levels. Analysis of more than 50 vector insertion sites showed none of the clones detected in the first month were active later. Eighty percent of inserts were located within or near genes, 2 near CXCR4. These findings provide direct evidence of cells with rapid but transient repopulating activity in patients and demonstrate their efficient transduction in vitro.      

Differential requirement for DOCK2 in migration of plasmacytoid dendritic cells versus myeloid dendritic cells

The migratory properties of dendritic cells (DCs) are important for their functions. Although several chemokines and their receptors have been implicated in DC migration, the downstream signaling molecules are largely unknown. Here we show that DOCK2, a hematopoietic cell-specific CDM family protein, is indispensable for migration of plasmacytoid DCs (pDCs), but not myeloid DCs (mDCs). Although DOCK2-deficiency did not affect development of pDCs, DOCK2-deficient (DOCK2^–/–) mice exhibited a severe reduction of pDCs in the spleen and lymph nodes. Adoptive transfer experiments revealed that DOCK2^–/– pDCs failed to migrate into the periarteriolar lymphoid sheaths of the spleen. In DOCK2^–/– pDCs, chemokine-induced Rac activation was severely impaired, resulting in the reduction of motility and the loss of polarity during chemotaxis. In contrast, DOCK2^–/– mDCs did not show any defects in Rac activation and migration. These results indicate that pDCs and mDCs use distinct molecules to activate Rac during chemotaxis.     

KNEE FUNCTION AFTER INTRA-ARTICULAR HYDROCORTISONE

The effect of intra-articular hydrocortisone on knees affectedby rheumatoid synovitis has been assessed objectively by theuse of an electrogoniometer. The range of flexion during walkinghas been shown to improve, in some cases into the normal range,and this effect was maintained to a significant degree for eightweeks in a group of 12 patients. Function was shown to improvemore slowly than pain and this may have an important bearingon future assessment of anti-inflammatory drugs. The benefitsof hydrocortisone have been measured against intra-articularsaline in a single-blind study and shown to be significant bothsymptomatically and functionally. ^*Present address: Melbourne, Australia.     

The Philadelphia Chromosome and Galactose-1-Phosphate Uridyl Transferase

Because galactose-1-phosphate uridyl transferase has been reported to beelevated in the blood of patients with mongolism (21 trisomy), assay of thisenzyme in the erythrocytes and leukocytes was performed in patients withthe Philadelphia chromosome. Twenty normal individuals and 16 patients withthe Ph¹ chromosome were studied; 15 of the latter had chronic myelogenousleukemia and 1 had an unusual myeloproliferative disorder. The mean leukocyte enzyme level in the Ph¹ group was not different from that in the normalgroup. The mean erythrocyte enzyme level in the Ph¹ group was higher thanthat in the normal group; this difference might have been due to a youngerpopulation of red cells in the Ph¹ patients.

To interpret the results, three postulates are presented. First, the relationship between chromosome 21 and this enzyme activity may be obscuredby other controlling factors. Second, the long arm of this chromosome mayplay no role in transferase activity. Third, there may be no reduction ofgenetic material in a Ph¹ karyotype.

Submitted on August 12, 1966 Accepted on December 20, 1966     

The role of angiopoietins in the development of endothelial cells from cord blood CD34+ progenitors

Circulating endothelial progenitors contribute to neovascularization at sites of injury and tumorigenesis in postnatal life. Yet, the molecular mechanisms initiating the endothelial developmental program of these precursors remain elusive. Here we provide evidence that endothelial development from progenitors circulating in human cord blood requires angiopoietins, a set of growth factors also involved in vascular branching during embryogenesis. We show that cord blood cells with the potential for endothelial development reside in a CD34⁺CD11b⁺ subset capable of autonomously producing and binding angiopoietins. Functionally, endogenous angiopoietin-1 regulates initial endothelial cell commitment, whereas angiopoietin-2 enhances expansion of the endothelial cell progeny. These findings suggest a role for angiopoietins as regulators of endothelial development from circulating progenitors and imply a function of angiopoietins at distinct developmental steps in postnatal angiogenesis.      

Ca2+ channels, Ca2+ sparks, and regulation of arterial smooth muscle function

Summary In cardiac, skeletal, and arterial muscle, transient, spatially localized elevations in [Ca²⁺]_i, termed "Ca²⁺ sparks", have been observed using confocal laser scanning microscopy. Ca²⁺ sparks are thought to represent "elementary" Ca²⁺ release events, which arise from one or more ryanodine receptor (RyR) channels in the sarcoplasmic reticulum (SR). In striated muscle, Ca²⁺ sparks are thought to be key elements of excitation-contraction coupling. In arterial smooth muscle, Ca²⁺ sparks have been suggested to oppose myogenic vasoconstriction and to influence vasorelaxation. Using a developmental model, we have investigated whether RyRs causing Ca²⁺ sparks and activation of Ca²⁺-activated K⁺ (K_Ca) channels (STOCs) function as "elementary" Ca²⁺ release units that regulate arterial mygenic tone. Whereas increases in the global [Ca²⁺]_i induce sustained constriction of arterial smooth muscle, Ca²⁺ sparks induce vasodilation through the local activation of K_Ca channels. In cerebral arteries, the global bulk [Ca²⁺]_i and a Ca²⁺ spark frequency < 10^-2 Hz/cell do not cause sufficient K_Ca channel activity to regulate membrane potential of smooth muscle cells and myogenic tone. The frequency of Ca²⁺ sparks and STOCs is regulated by agents that modulate protein kinase G and protein kinase A activity. Our findings suggest that "elementary" Ca²⁺ release units may represent novel, important therapeutic targets for regulating function of the intact arterial smooth muscle tissue.     

The Use of FE59 and CR51 for Estimating Red Cell Production and Destruction: An Interpretive Review

In surveying some of the limitations of studies with Cr⁵¹ and Fe⁵⁹ my purposehas been not to deny their usefulness but to put them in perspective. Thesetechnics have made possible many extensions of the fundamental understanding of red cell physiology and iron metabolism. They will continue to be valuable experimental tools. It is hoped that if some of the limitations of isotopetechnics are considered, the non-experimental use of these isotopes will beconfined to situations in which otherwise unavailable information of diagnosticor therapeutic importance can be obtained. Unfortunately isotope technicsare used when more conventional means would be adequate or even preferable. An extreme example is the suggestion²⁴ that repeated Fe⁵⁹ turnoverstudies might be used to determine the total dose of parenteral iron (as Imferonor saccharated iron oxide) to be given in iron deficiency anemia, pointing outthat in so doing the possibility of iatrogenic hemochromatosis could be avoided.The usual calculations for determining dose²⁵ however are not only safer butmore accurate.

The use of Fe⁵⁹ and Cr⁵¹ entails some risk, the main hazards being hepatitis,with the use of donor plasma or cells, and the possibility of untoward effectsfrom radiation. An estimate of the risk of hepatitis can be gained from itsincidence after transfusion. The radiation hazard is more difficult to assess.Leukemia has occurred after large doses of radiation but the extent of the radiation hazard is unknown from the much smaller doses of radiation employedin the usual isotopes studies. Certainly the risk is not such as to preclude theuse of isotopes to obtain information essential for diagnosis. However, whensuch information can be obtained by other means or when results cannot beadequately interpreted, the use of isotopes in clinical medicine appears unwarranted. In considering the use of isotopes in the doubtful case, the dose ofradiation to be delivered should not be thought of as an isolated event butrather as adding to a total radiation dose, which as shown by the British survey²⁸ may be appreciable.

Submitted on December 6, 1960 Accepted on April 3, 1961     

Strategies to promote HDL-C: an emerging therapeutic target.

Hydroxy-methyl glutaryl coenzyme A reductase inhibitors (statins)have accumulated an extraordinary record of successful randomizedclinical trials, transforming the practice of preventive cardiology.However, most placebo-controlled statin trials have reportednot more than 25–35% reduction in morbidity/mortalityduring 5 year follow-up. Thus, despite their established benefitsin primary and secondary prevention, statin drugs fail to preventthe majority of clinical events. In acute coronary syndromes(ACS), despite aggressive use of statins, the prognosis is evenless favourable. In a recent trial of lipid lowering in ACS,the 2 year event rate was still 22% in the most aggressivelytreated patients.¹ These findings highlight the need to developnew strategies to compliment statins in reducing cardiovascularrisk. Emerging therapeutic targets include C-reactive protein,recently shown to have a strong link to outcomes during statinadministration.² However, few therapies other than statins     

Oral chelators deferasirox and deferiprone for transfusional iron overload in thalassemia major: new data, new questions

For nearly 30 years, patients with transfusional iron overload have depended on nightly deferoxamine infusions for iron chelation. Despite dramatic gains in life expectancy in the deferoxamine era for patients with transfusion-dependent anemias, the leading cause of death for young adults with thalassemia major and related disorders has been cardiac disease from myocardial iron deposition. Strategies to reduce cardiac disease by improving chelation regimens have been of the highest priority. These strategies have included development of novel oral iron chelators to improve compliance, improved assessment of cardiac iron status, and careful epidemiologic assessment of European outcomes with deferiprone, an oral alternative chelator available for about a decade. Each of these strategies is now bearing fruit. The novel oral chelator deferasirox was recently approved by the Food and Drug Administration (FDA); a randomized clinical trial demonstrates that deferasirox at 20 to 30 mg/kg/d can maintain or improve hepatic iron in thalassemia as well as deferoxamine. A randomized trial based on cardiac T2* magnetic resonance imaging (MRI) suggests that deferiprone can unload myocardial iron faster than deferoxamine. Retrospective epidemiologic data suggest dramatic reductions in cardiac events and mortality in Italian subjects exposed to deferiprone compared with deferoxamine. These developments herald a new era for iron chelation, but many unanswered questions remain.      

Chemokine-mediated rapid turnover of myeloid-derived suppressor cells in tumor-bearing mice

Tumor growth is associated with aberrant myelopoiesis, including the accumulation of CD11b⁺Gr-1⁺ myeloid-derived suppressor cells (MDSCs) that have the potential to promote tumor growth. However, the identity, growth, and migration of tumor-associated MDSCs remain undefined. We demonstrate herein that MDSCs at tumor site were composed primarily of bone marrow-derived CD11b⁺Gr-1^hiLy-6C^int neutrophils and CD11b⁺Gr-1^int/dullLy-6C^hi macrophages. Unexpectedly, in vivo bromodeoxyuridine (BrdU) labeling and parabiosis experiments revealed that tumor-infiltrating macrophages were replenished more rapidly than neutrophils. CCR2 deficiency caused striking conversion of infiltrating cellular dominance from macrophages to neutrophils in the tumor with the excessive production of CXCR2 ligands and granulocyte-colony stimulating factor in the tumor without affecting tumor growth. Overall, our data established the identity and dynamics of MDSCs in a tumor-bearing host mediated by chemokines and elucidated unexpected effects of the paucity of macrophages on tumor development.     

POSTMENOPAUSAL SCREENING FOR OSTEOPENIA

Dual energy X-ray absorptiometry (DXA) measurements of bonemineral density (BMD) in the lumbar spine and femoral neck havebeen performed in 1000 consecutive women aged between 40 and60 years referred for screening for osteopenia. A detailed historywas taken from each woman that included relevant lifestyle parametersand known risk factors for osteoporosis. After exclusions, e.g.because of fractures, corticosteroid or prolonged HRT use, 627women (mean age 53 years) were considered suitable for furtheranalysis. The mean BMD in the lumbar spine (L1–L4) was0.946 g/cm² and in the femoral neck was 0.767 g/cm². Significantcorrelations were found between BMD and years after the menopauseand weight (range r = 0.20–0.24). However, these parametersare not reliable enough predictors of BMD to be of value inclinical practice. If osteopenia is to be the basis for initiatingprophylactic measures against bone loss, then a threshold BMDmust be chosen below which treatment will be advised. Sincethe correlation coefficient between spinal and femoral neckBMD measurements was only 0.64, assessment of any individualrequires consideration of both sites. There is as yet no consensusas to the number of women who may require to be treated andwe have provided BMD values that identify a range of populationsizes (the lowest 20, 30, 40 and 50 percentiles). It shouldbe noted that there was a 16% loss of BMD in the spine and 14%in the femoral neck during the first 5 years after the menopause.If prophylactic treatment for bone loss is to be used, thereis an advantage in initiating treatment early after the menopauseto maintain bone mass at the highest possible level. KEY WORDS: Osteopenia, Osteoporosis, Dual energy X-ray absorptiometry, Bone density     

Screening for cardiovascular disease.

Sir, The paper by Empana et al.¹ reviewing the utility of the Framinghamand PROCAM cardiovascular risk calculation algorithms in a validationstudy against the PRIME cohort raises many issues. Firstly definitionsare of critical importance for defining categorical variables.²The definitions of diabetes and family history have either changed     

New trends in umbilical cord blood transplantation

Since the first report of a successful umbilical cord blood transplantation in 1988, there has been great interest in the use of cord blood as an alternative stem cell source to treat cancer and genetic diseases. More than 4000 cord blood transplantations have been performed worldwide. In this review, the scientific rationale for this therapy, as well as related preclinical studies, cord blood banking issues, and ethical concerns, will be addressed. Results of studies in both pediatric and adult transplantation will be discussed. Finally, new indications for cord blood use and emerging technologies will be addressed.      

The TNF (-308A) polymorphism is associated with microchimerism in transfused trauma patients

Microchimerism (MC), defined as the persistence of allogeneic cells at low concentrations, is well documented in transfused trauma patients. We hypothesized that genetic polymorphisms linked to cytokine production could contribute to trauma-induced immune modulation and development of microchimerism after transfusion of trauma patients. We used high-throughput SYBR-green-based genotyping of single nucleotide polymorphisms (SNPs) to characterize 59 transfused trauma patients, with MC (n = 30) and without MC (n = 29), for 4 functionally significant SNPs: TNF (–308), IL 10 (–1082), IFNG (+874), and TGFB1 (+915). We then compared likelihood for development of MC and the magnitude of immune suppression among subjects with and without these selected immune response SNPs. We identified a significant association between TNF (–308A) SNP and both development of MC and diminished immune responsiveness. Hence predisposing genetic factors may explain, in part, why only a subset of trauma patients develops transfusion-associated microchimerism.      

The whole is greater than its parts.

Many psychosocial factors have been identified as potentialrisk factors for ischaemic heart disease, and strong evidencehas been put forward for several personality factors such asType A behavioural pattern, hostility, depression, or socialfactors such as low socioeconomic status and lack of socialsupport.^1–9 (Table 1) Psychosocial stressors perse increase cardiac vulnerability by altering autonomic tone,and induce increased catecholamines spillover, baroreceptorreflexes inhibition, clotting formation, and angiotensin-IIstimulation. All these responses have detrimental effects onthe cardiovascular system and may explain the high incidenceof adverse cardiac events during follow-up. Associations betweendepressive symptoms and mortality have been reported in patientswithout known coronary artery disease, but these reported associationswere, in general, weaker than those