M1 and M2 mAChRs activate PDK1 and regulate PKC ßI and ε and the exocytotic apparatus at the NMJ.

Neuromuscular junctions (NMJ) regulate cholinergic exocytosis through the M1 and M2 muscarinic acetylcholine autoreceptors (mAChR), involving the crosstalk between receptors and downstream pathways. Protein kinase C (PKC) regulates neurotransmission but how it associates with the mAChRs remains unknown. Here, we investigate whether mAChRs recruit the classical PKCßI and the novel PKCε isoforms and modulate their priming by PDK1, translocation and activity on neurosecretion targets. We show that each M1 and M2 mAChR activates the master kinase PDK1 and promotes a particular priming of the presynaptic PKCßI and ε isoforms. M1 recruits both primed-PKCs to the membrane and promotes Munc18-1, SNAP-25, and MARCKS phosphorylation. In contrast, M2 downregulates PKCε through a PKA-dependent pathway, which inhibits Munc18-1 synthesis and PKC phosphorylation. In summary, our results discover a co-dependent balance between muscarinic autoreceptors which orchestrates the presynaptic PKC and their action on ACh release SNARE-SM mechanism. Altogether, this molecular signaling explains previous functional studies at the NMJ and guide toward potential therapeutic targets.

Advanced Photonic Thin Films for Solar Irradiation Tuneability Oriented to Greenhouse Applications.

The world population is growing by 1 billion people every 10 years. There will come a time when there will be more people to feed but less land to grow food. Greenhouses can be the solution to this problem because they provide the highest production yield per m2 and also use less water, provide food safety, and offer high quality. Photosynthetic active radiation (PAR) favors vegetable growth with a specific blue and red light ratio. Thus, increasing the amount of red light improves chlorophyll absorption and photosynthetic efficiency. In this article, we present a hybrid system that combines luminescent materials and photonic crystals for better management of the light reaching the greenhouse. The luminescent dyes considered herein are combined ensuring a Förster resonance energy transfer (FRET) nonradiative mechanism to enhance the absorption range. The designed photonic crystal maximizes reflections in the Near-Infrared (NIR) range, and therefore, thermal losses are minimized. Thus, by converting harmful or ineffective radiation for plant growth to the PAR region, we aim to demonstrate growth-condition enhancement for the different vegetables that have been used as a model.

Adenosine receptors and muscarinic receptors cooperate in acetylcholine release modulation in the neuromuscular synapse.

Adenosine receptors (ARs) are present in the motor terminals at the mouse neuromuscular junction. ARs and the presynaptic muscarinic acetylcholine receptors (mAChRs) share the functional control of the neuromuscular junction. We analysed their mutual interaction in transmitter release modulation. In electrophysiological experiments with unaltered synaptic transmission (muscles paralysed by blocking the voltage-dependent sodium channel of the muscle cells with µ-conotoxin GIIIB), we found that: (i) a collaborative action between different AR subtypes reduced synaptic depression at a moderate activity level (40 Hz); (ii) at high activity levels (100 Hz), endogenous adenosine production in the synaptic cleft was sufficient to reduce depression through A1 -type receptors (A1 Rs) and A2 A-type receptors (A2 A Rs); (iii) when the non-metabolizable 2-chloroadenosine (CADO) agonist was used, both the quantal content and depression were reduced; (iv) the protective effect of CADO on depression was mediated by A1 Rs, whereas A2 A Rs seemed to modulate A1 Rs; (v) ARs and mAChRs absolutely depended upon each other for the modulation of evoked and spontaneous acetylcholine release in basal conditions and in experimental conditions with CADO stimulation; (vi) the purinergic and muscarinic mechanisms cooperated in the control of depression by sharing a common pathway although the purinergic control was more powerful than the muscarinic control; and (vii) the imbalance of the ARs created by using subtype-selective and non-selective inhibitory and stimulatory agents uncoupled protein kinase C from evoked transmitter release. In summary, ARs (A1 Rs, A2 A Rs) and mAChRs (M1 , M2 ) cooperated in the control of activity-dependent synaptic depression and may share a common protein kinase C pathway.

Adenosine A1 and A2A receptor-mediated modulation of acetylcholine release in the mice neuromuscular junction.

Immunocytochemistry shows that purinergic receptors (P1Rs) type A1 and A2A (A1 R and A2 A R, respectively) are present in the nerve endings at the P6 and P30 Levator auris longus (LAL) mouse neuromuscular junctions (NMJs). As described elsewhere, 25 µm adenosine reduces (50%) acetylcholine release in high Mg(2+) or d-tubocurarine paralysed muscle. We hypothesize that in more preserved neurotransmission machinery conditions (blocking the voltage-dependent sodium channel of the muscle cells with µ-conotoxin GIIIB) the physiological role of the P1Rs in the NMJ must be better observed. We found that the presence of a non-selective P1R agonist (adenosine) or antagonist (8-SPT) or selective modulators of A1 R or A2 A R subtypes (CCPA and DPCPX, or CGS-21680 and SCH-58261, respectively) does not result in any changes in the evoked release. However, P1Rs seem to be involved in spontaneous release (miniature endplate potentials MEPPs) because MEPP frequency is increased by non-selective block but decreased by non-selective stimulation, with A1 Rs playing the main role. We assayed the role of P1Rs in presynaptic short-term plasticity during imposed synaptic activity (40 Hz for 2 min of supramaximal stimuli). Depression is reduced by micromolar adenosine but increased by blocking P1Rs with 8-SPT. Synaptic depression is not affected by the presence of selective A1 R and A2 A R modulators, which suggests that both receptors need to collaborate. Thus, A1 R and A2 A R might have no real effect on neuromuscular transmission in resting conditions. However, these receptors can conserve resources by limiting spontaneous quantal leak of acetylcholine and may protect synaptic function by reducing the magnitude of depression during repetitive activity.

Polimialgia reumática: ¿pensamos en ella? / Polymyalgia rheumatica: do we think of it?

La polimialgia reumática (PMR) es una enfermedad inflamatoria crónica que se diagnostica generalmente en las consultas de atención primaria a pacientes mayores de 50 años. El diagnóstico se basa fundamentalmente en el cuadro clínico, pruebas analíticas y la respuesta terapéutica a los corticosteroides. La clínica se caracteriza por dolor simétrico y rigidez de cintura escapular y pelviana y en columna cervical. Existen diferentes tablas de criterios diagnósticos de PMR, aunque se considera que los criterios de Bird son los que poseen mayor sensibilidad. El diagnóstico diferencial se realiza con enfermedades reumatológicas, infecciosas, autoinmunes y traumatológicas. Especial interés presenta la relación entre PMR y arteritis de células gigantes, dado que hay veces que coexisten. El tratamiento debe realizarse con corticosteroides, que proporcionan una pronta mejoría, aunque se desconoce el tiempo de duración de este. También se han realizado estudios con metotrexato e infliximab para disminuir el tiempo y las dosis de corticosteroides (AU)

Polymyalgia rheumatica (PMR) is a chronic inflammatory disease which is generally diagnosed in primary care in patients older than 50 years. The diagnosis is based on clinical findings, analytical tests and the response to corticoids. Clinical findings are symmetrical pain and stiffness affecting shoulders, hips and cervical spine. There are several diagnostic criteria of PMR, with Bird¿s Criteria being the most sensitive. PMR differential diagnosis is made with rheumatic, infectious, autoimmune and osteoarticular diseases. There is a special interest in the relationship between PMR and giant cell arteritis as this association is not uncommon. The treatment of choice for PMR is corticoid therapy, showing a sharp clinical improvement, but its maintenance period is unknown, and should be determined empirically. Some studies show that other immunomodulatory drugs, such as methotrexate and infliximab can be added to reduce the length of the corticoid treatment (AU)

Localization of brain-derived neurotrophic factor, neurotrophin-4, tropomyosin-related kinase b receptor, and p75 NTR receptor by high-resolution immunohistochemistry on the adult mouse neuromuscular junction.

Neurotrophins and their receptors, the trk receptor tyrosine kinases (trks) and p75(NTR), are differentially expressed among the cell types that make up synapses. It is important to determine the precise location of these molecules involved in neurotransmission. Here we use immunostaining and Western blotting to study the localization and expression of neurotrophin brain-derived neurotrophic factor (BDNF) and neurotrophin-4 (NT-4) and the receptors tropomyosin-related kinase b (trkB) and p75(NTR) at the adult neuromuscular junction. Our confocal immunofluorescence results on the whole mounts of the mouse Levator auris longus muscle and on semithin cross-sections showed that BDNF, NT-4, trkB, and p75(NTR) were localized on the three cells in the neuromuscular synapse (motor axons, post-synaptic muscle and Schwann cells).

Neurotrophin-4 couples to locally modulated ACh release at the end of neuromuscular synapse maturation.

We use immunocytochemistry to show that neurotrophin-4 (NT-4) and its receptor proteins (p75(NTR) and tropomyosin-related tyrosine kinase B) are present in neonatal neuromuscular junctions (NMJ) colocalized with several synaptic markers. NT-4 incubation (1h, in the range 2-12 nM) does not change the size of the endplate potential between P6 and P45. However, extended exposure (3h) to a relatively low dose of NT-4 (2 nM) potentiates ACh release (approx. 70%) in adult but not in neonatal muscles. The present results suggest that the developmental mechanism of axonal competition and neonatal elimination of redundant synapses cannot be modulated by added NT-4. However, this neurotrophin was able to modulate synaptic transmission locally in the adult NMJ.

Involvement of brain-derived neurotrophic factor (BDNF) in the functional elimination of synaptic contacts at polyinnervated neuromuscular synapses during development.

We use immunohistochemistry to describe the localization of brain-derived neurotrophic factor (BDNF) and its receptors trkB and p75(NTR) in the neuromuscular synapses of postnatal rats (P6-P7) during the synapse elimination period. The receptor protein p75(NTR) is present in the nerve terminal, muscle cell and glial Schwann cell whereas BDNF and trkB proteins can be detected mainly in the pre- and postsynaptic elements. Exogenously applied BDNF (10 nM for 3 hr or 50 nM for 1 hr) increases ACh release from singly and dually innervated synapses. This effect may be specific for BDNF because the neurotrophin NT-4 (2-8 nM) does not modulate release at P6-P7. Blocking the receptors trkB and p75(NTR) (with K-252a and anti-p75-192-IgG, respectively) completely abolishes the potentiating effect of exogenous BDNF. In addition, exogenous BDNF transiently recruits functionally depressed silent terminals, and this effect seems to be mediated by trkB. Calcium ions, the L-type voltage-dependent calcium channels and protein kinase C are involved in BDNF-mediated nerve ending recruitment. Blocking experiments suggest that endogenous BDNF could operate through p75(NTR) receptors coupled to potentiate ACh release in all nerve terminals because the anti-p75-192-IgG reduces release. However, blocking the trkB receptor (K-252a) or neutralizing endogenous BDNF with the trkB-IgG fusion protein reveals a trkB-mediated release inhibition on almost mature strong endings in dual junctions. Taken together these results suggest that a BDNF-induced p75(NTR)-mediated ACh release potentiating mechanism and a BDNF-induced trkB-mediated release inhibitory mechanism may contribute to developmental synapse disconnection.

Effect of anti-GM2 antibodies on rat sciatic nerve: electrophysiological and morphological study.

We found that a monoclonal human IgM anti-GM2 was fixed in rat sciatic axons and Schwann cells and was able to activate human complement. The passive transfer of IgM and complement in sciatic nerves can induce an acute alteration in nerve conduction. When the transfer of IgM plus complement was repeated for 10 days, the compound action motor potential amplitude was very low and the morphological study showed axons and myelin damage. Without human complement, IgM can only slightly disorganize the myelin by separating some layers, probably by interfering with the functional role of gangliosides in the myelin package.

Presynaptic muscarinic receptors, calcium channels, and protein kinase C modulate the functional disconnection of weak inputs at polyinnervated neonatal neuromuscular synapses.

We studied the relation among calcium inflows, voltage-dependent calcium channels (VDCC), presynaptic muscarinic acetylcholine receptors (mAChRs), and protein kinase C (PKC) activity in the modulation of synapse elimination. We used intracellular recording to determine the synaptic efficacy in dually innervated endplates of the levator auris longus muscle of newborn rats during axonal competition in the postnatal synaptic elimination period. In these dual junctions, the weak nerve terminal was potentiated by partially reducing calcium entry (P/Q-, N-, or L-type VDCC-specific block or 500 muM magnesium ions), M1- or M4-type selective mAChR block, or PKC block. Moreover, reducing calcium entry or blocking PKC or mAChRs results in unmasking functionally silent nerve endings that now recover neurotransmitter release. Our results show interactions between these molecules and indicate that there is a release inhibition mechanism based on an mAChR-PKC-VDCC intracellular cascade. When it is fully active in certain weak motor axons, it can depress ACh release and even disconnect synapses. We suggest that this mechanism plays a central role in the elimination of redundant neonatal synapses, because functional axonal withdrawal can indeed be reversed by mAChRs, VDCCs, or PKC block.

Interaction between protein kinase C and protein kinase A can modulate transmitter release at the rat neuromuscular synapse.

We used intracellular recording to investigate the functional interaction between protein kinase C (PKC) and protein kinase A (PKA) signal transduction cascades in the control of transmitter release in the neuromuscular synapses from adult rats. Our results indicate that: 1) PKA and PKC are independently involved in asynchronous release. 2) Evoked acetylcholine (ACh) release is enhanced with the PKA agonist Sp-8-BrcAMP and the PKC agonist phorbol ester (PMA). 3) PKA has a constitutive role in promoting a component of normal evoked transmitter release because, when the kinase is inhibited with H-89, the release diminishes. However, the PKC inhibitor calphostin C (CaC) does not affect ACh release. 4) PKA regulates neurotransmission without PKC involvement because, after PMA or CaC modulation of the PKC activity, coupling to the ACh release of PKA can normally be stimulated with Sp-8-BrcAMP or inhibited with H-89. 5) After PKA inhibition with H-89, PKC stimulation with PMA (or inhibition with CaC) does not lead to any change in evoked ACh release. However, in PKA-stimulated preparations with Sp-8-BrcAMP, PKC becomes tonically active, thus potentiating a component of release that can now be blocked with CaC. In normal conditions, therefore, PKA was able to modulate ACh release independently of PKC activity, whereas PKA stimulation caused the PKC coupling to evoked release. In contrast, PKA inhibition prevent PKC stimulation (with the phorbol ester) and coupling to ACh output. There was therefore some dependence of PKC on PKA activity in the fine control of the neuromuscular synaptic functionalism and ACh release.

Anti-GM2 gangliosides IgM paraprotein induces neuromuscular block without neuromuscular damage.

We analyzed the effect on the mouse neuromuscular synapses of a human monoclonal IgM, which binds specifically to gangliosides with the common epitope [GalNAc beta 1-4Gal(3-2 alpha NeuAc)beta 1-]. We focused on the role of the complement. Evoked neurotransmission was partially blocked by IgM both acutely (1 h) and chronically (10 days). Transmission electron microscopy shows important nerve terminal growth and retraction remodelling though axonal injury can be ruled out. Synapses did not show mouse C5b-9 immunofluorescence and were only immunolabelled when human complement was added. Therefore, the IgM-induced synaptic changes occur without complement-mediated membrane attack.

Coupling of presynaptic muscarinic autoreceptors to serine kinases in low and high release conditions on the rat motor nerve terminal.

We used intracellular recording to investigate how muscarinic acetylcholine receptors and the serine kinase signal transduction cascade are involved in regulating transmitter release in the neuromuscular synapses of the levator auris longus muscle from adult rats. Experiments with M1 and M2 selective blockers show that these subtypes of muscarinic receptors were involved in enhancing and inhibiting acetylcholine (ACh) release, respectively. Because the unselective muscarinic blocker atropine considerably increased release, the overall presynaptic muscarinic mechanism seemed to moderate ACh secretion in normal conditions. This muscarinic function did not change when more ACh was released (high external Ca2+) or when there was more ACh in the cleft (fasciculin II). However, when release was low (high external Mg2+ or low external Ca2+) or when there was less ACh in the cleft (when acetylcholinesterase was added, AChE), the response of M1 and M2 receptors to endogenously released ACh shifted to optimize release, thus producing a net potentiation of the Mg2+-depressed level. Protein kinase A (PKA) (but not protein kinase C, PKC) has a constitutive role in promoting a component of normal release because when it is inhibited with N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, 2 HCl, release diminishes. The imbalance of the muscarinic acetylcholine receptors (mAChRs) (with the selective block of M1 or M2) inverts the kinase function. PKC can then tonically stimulate transmitter release, whereas PKA is uncoupled. The muscarinic function can be explained by an increased M1-mediated PKC activity-dependent release and a decreased M2-mediated PKA activity-dependent release. In the presence of high external Mg2+ or low Ca2+, or when AChE is added, both mAChRs may potentiate release through an M2-mediated PKC mechanism and an M1-mediated mechanism downstream of the PKC.

Protein kinase C activity affects neurotransmitter release at polyinnervated neuromuscular synapses.

By using intracellular recording, we studied how protein kinase C (PKC) activity affected transmitter release in singly and dually innervated endplates of the Levator auris longus muscle of 5-6-day-old rats during axonal competition in the postnatal synaptic elimination period. In dually innervated fibers, a second endplate potential (EPP) may appear after the first one when the stimulation intensity is increased. The nerve terminals that generate the lowest and the highest EPP amplitudes are designated "small-EPP generating ending" (SEGE) and "large-EPP generating ending" (LEGE), respectively. Blocking PKC with calphostin C, staurosporine, or chelerythrine results in an increased release from SEGE ( approximately 80%), whereas release from LEGE and from endings generating only one EPP (OEGE) is not significantly affected. Blocking PKC also leads to the recruitment of silent synapses (acetylcholine cannot be released before PKC inhibition). The mean number of functional axon terminals per synapse increases by approximately 47%, and these are now designated the "recruited-EPP generating endings" (REGE). This suggests that axonal PKC can modulate postnatal synaptic elimination by favoring the nerve terminal disconnection of certain weak axonal endings (REGE and SEGE). We conclude that a PKC-mediated mechanism should occupy a pivotal place in neonatal synapse elimination, because functional axonal withdrawal can indeed be turned back by PKC block.

Muscarinic autoreceptors modulate transmitter release through protein kinase C and protein kinase A in the rat motor nerve terminal.

We have used intracellular recording to investigate the existence of a functional link between muscarinic presynaptic acetylcholine (ACh) autoreceptors, the intracellular serine-threonine kinases-mediated transduction pathways and transmitter release in the motor nerve terminals of adult rats. We found the following. (1) Transmitter release was reduced by the M1 muscarinic acetylcholine receptor (mAChR) blocker pirenzepine and enhanced by the M2 blocker methoctramine. The unselective mAChR blocker atropine increased ACh release, which suggests the unmasking of another parallel release-potentiating mechanism. There are therefore two opposite, though finely balanced, M1-M2 mAChR-operated mechanisms that tonically modulate transmitter release. (2) Both M1 and M2 mechanisms were altered when protein kinase C (PKC), protein kinase A (PKA) or the P/Q-type calcium channel were blocked. (3) Both PKC and PKA potentiated release when they were specifically stimulated [with phorbol 12-myristate 13-acetate (PMA) and Sp-8-Br cAMPs, respectively], and both needed the P/Q channel. (4) In normal conditions PKC seemed not to be directly involved in transmitter release (the PKC blocker calphostin C did not reduce release), whereas PKA was coupled to potentiate release (the PKA blocker H-89 reduced release). However, when an imbalance of the M1-M2 mAChRs function was experimentally produced with selective blockers, an inversion of the kinase function occurred and PKC could then stimulate transmitter release, whereas PKA was uncoupled. (5) The muscarinic function may be explained by the existence of an M1-mediated increased PKC activity-dependent potentiation of release and an M2-mediated PKA decreased activity-dependent release reduction. These findings show that there is a precise interrelation pattern of the mAChRs, PKC and PKA in the control of the neurotransmitter release.

Our nine-year experience with the self-locating catheter: comparison of malfunction rate with other Tenckhoff catheter variants.

OBJECTIVE: To evaluate catheter-related complications among self-locating and other variants of Tenckhoff catheters. PATIENTS AND METHODS: We studied all self-locating catheters implanted in our hospitals from May/97 to Dec/05. We extended the follow-up to Jun/05. We collected demographic data, causes of catheter withdrawal and dropping of PD. We studied data about technique and catheter related complications. RESULTS: We included 328 catheters in 285 patients. There were 258 self-locating, 55 straight and 15 curled. There was a higher rate of catheter withdrawal due to malfunction in patients with straight or pig-tail catheters than in self-locating ones (log-rank, p < 0.001). CONCLUSION: In our patients, self-locating catheters have a lower malfunction rate than other variants of Tenckhoff catheters.

Expression of muscarinic acetylcholine receptors (M1-, M2-, M3- and M4-type) in the neuromuscular junction of the newborn and adult rat.

Using intracellular recording and immunohistochemistry, we studied the presynaptic muscarinic autoreceptor subtypes controlling ACh release in the neuromuscular junctions of the newborn (3-6 days postnatal) and adult (30-40 days) rat. In the Levator auris longus muscles of both newborn and adult rats, acetylcholine release was modified by the M1-receptor selective antagonists pirenzepine (10 microM) and MT-7 (100 nM) and by the M2-receptor selective antagonists methoctramine (1 microM) and AF-DX 116 (10 microM). The M4-receptor selective antagonists tropicamide (1 microM) and MT-3 (100 nM) can also modify the neurotransmitter release in certain synapses of the newborn muscles. The neurotransmitter release was not altered by the M3-receptor selective antagonist 4-DAMP (1 microM) in the adult or newborn rats. However, we directly demonstrate by immunocytochemistry the presence of these receptors in the motor endplates and conclude that M1-, M2-, M3- and M4-type muscarinic receptors are present in all the neuromuscular junctions of the rat muscle both in newborn and adult animals. These receptors may be located in the perisynaptic glial cell as well as at the nerve terminals.

Calcium inflow-dependent protein kinase C activity is involved in the modulation of transmitter release in the neuromuscular junction of the adult rat.

Using intracellular recording, we studied how protein kinase C activity affected miniature endplate potentials (MEPPs) and evoked endplate potentials (EPPs) in the neuromuscular junctions of the levator auris longus muscle from adult rats. The protein kinase C activator phorbol 12-myristate 13-acetate (PMA, 10 nM) increased the quantal content by approximately 150% (P<0.05). On the other hand, the quantal content decreased by approximately 40% (P<0.05) for all the protein kinase C inhibitors tested (Calphostin-C, 10 microM; Chelerythrine, 1 microM; Staurosporine, 200 nM). These changes in acetylcholine release were maintained at plateau for 1 to 7 h. Moreover, none of the protein kinase C activators or inhibitors used could modify the spontaneous MEPP mean size (P>0.05). We reduced the calcium influx in nerve terminals using the P/Q-type channel blocker omega-Aga-IVA(100 nM) or with 5 mM magnesium in physiological solution. In neither situation was the quantal content modified by PMA or by CaC. However, when high Ca2+ (5 mM) was added to a preparation that was previously blocked with omega-Aga-IVA, PMA and CaC had their full effect. We conclude that under physiological conditions PKC is dependent on the calcium inflow through the P/Q-type voltage-dependent calcium channels during evoked activity and works near the maximum rate at normal external calcium concentration.

Iron deficiency--an underrecognized problem in nonanemic and erythrocytic kidney transplant recipients: risks and effects of ACEI and of iron treatment.

The state of iron deposits in long-term kidney graft recipients is not well-known. Angiotensin enzyme inhibitors (ACEIs) reduce hematocrit levels in patients with posttransplant erythrocytosis (PTE), but their action on iron deposits has not been sufficiently evaluated. We designed this study to investigate the prevalence of iron deficiency among patients without anemia, the efficacy of ACEI treatment and its influence on iron deposits, and the risks of iron treatment in patients with symptomatic iron deficiency but no anemia. One hundred thirty eight patients were included if they had a kidney transplant for more than a year, with good renal function, with no anemia, and with neither iron nor rHuEpo, ARA, or ACEI treatment. One hundred seventeen had a normal Ht (group 1) and 21 had PTE (group 2). Iron deficiency was found in 73 (62.4%) group 1 patients and in 10 (47%) group 2 patients. Two group 1 patients with symptoms of iron deficiency were treated with oral iron. Their symptoms disappeared, but one developed PTE. Enalapril treatment decreased Ht levels in PTE but not in control patients. Furthermore, this drug increased iron deposits in PTE and controls with a baseline iron deficiency. We conclude that there is a high prevalence of iron deficiency in long-term transplanted patients without anemia. Furthermore, iron treatment must be carefully administered because of the risk of PTE. Enalapril treatment decreased Ht levels in PTE but not in control patients and increased iron deposits in patients with baseline iron deficiency.