Hereditary hemochromatosis screening: effect of mutation penetrance and prevalence on cost-effectiveness of testing algorithms.

Screening for hereditary hemochromatosis, although largely discussed, is not yet implemented in clinical practice. We evaluated the cost-effectiveness of 165 hemochromatosis population-screening algorithms involving two or three of several screening tests by developing a computer program that simulates all possible screening scenarios. Input data comprised government estimates of health services data and costs and a virtual population with user-defined demographic characteristics (including variable HFE mutation frequencies and penetrance values). We show that when C282Y homozygote prevalence is set at 3:1000, population screening appears cost-effective when penetrance of the biochemical phenotype is >0.70. When only hepatocellular carcinoma and cirrhosis are considered as the cost-driving complications, population-based screening is not significantly more cost-efficient than no screening, but life expectancy of individuals identified with hereditary hemochromatosis and treated is still improved by 7 years. Among the 165 screening algorithms tested in 91 different virtual populations of one million individuals, biochemical tests usually perform better as the initial test than genetic testing. Indeed, the genetic testing is most cost-effective as the final confirmatory test. Finally, for most combinations of prevalence and penetrance of HFE, one screening algorithm--unbound iron-binding capacity + transferrin saturation--appeared robust enough to be always within the top 5 most cost-effective strategies.

Potent inhibition of NFAT activation and T cell cytokine production by novel low molecular weight pyrazole compounds.

NFAT (nuclear factor of activated T cell) proteins are expressed in most immune system cells and regulate the transcription of cytokine genes critical for the immune response. The activity of NFAT proteins is tightly regulated by the Ca(2+)/calmodulin-dependent protein phosphatase 2B/calcineurin (CaN). Dephosphorylation of NFAT by CaN is required for NFAT nuclear localization. Current immunosuppressive drugs such as cyclosporin A and FK506 block CaN activity thus inhibiting nuclear translocation of NFAT and consequent cytokine gene transcription. The inhibition of CaN in cells outside of the immune system may contribute to the toxicities associated with cyclosporin A therapy. In a search for safer immunosuppressive drugs, we identified a series of 3,5-bistrifluoromethyl pyrazole (BTP) derivatives that block Th1 and Th2 cytokine gene transcription. The BTP compounds block the activation-dependent nuclear localization of NFAT as determined by electrophoretic mobility shift assays. Confocal microscopy of cells expressing fluorescent-tagged NFAT confirmed that the BTP compounds block calcium-induced movement of NFAT from the cytosol to the nucleus. Inhibition of NFAT was selective because the BTP compounds did not affect the activation of NF-kappaB and AP-1 transcription factors. Treatment of intact T cells with the BTP compounds prior to calcium ionophore-induced activation of CaN caused NFAT to remain in a highly phosphorylated state. However, the BTP compounds did not directly inhibit the dephosphorylation of NFAT by CaN in vitro, nor did the drugs block the dephosphorylation of other CaN substrates including the type II regulatory subunit of protein kinase A and the transcription factor Elk-1. The data suggest that the BTP compounds cause NFAT to be maintained in the cytosol in a phosphorylated state and block the nuclear import of NFAT and, hence, NFAT-dependent cytokine gene transcription by a mechanism other than direct inhibition of CaN phosphatase activity. The novel inhibitors described herein will be useful in better defining the cellular regulation of NFAT activation and may lead to identification of new therapeutic targets for the treatment of autoimmune disease and transplant rejection.

Analysis of two matrix metalloproteinase inhibitors and their metabolites for induction of phospholipidosis in rat and human hepatocytes(1).

ABT-770 [(S)-N-[1-[[4'-trifluoromethoxy-[1,1'-biphenyl]-4-yl]oxy]methyl-2-(4,4-dimethyl-2,5-dioxo-1-imidazolidinyl)ethyl]-N-hydroxyformamide], a matrix metalloproteinase inhibitor (MMPI), produced generalized phospholipidosis in rats. Phospholipid accumulation was accompanied by retention of drug-related material and was associated with increased mortality. Generation of a successful drug candidate depended upon understanding the cause of the phospholipidosis and redesigning the chemical structure accordingly. ABT-770 and other MMPIs, plus several metabolites of each, were assayed for their ability to induce phospholipidosis in primary cultured rat and human hepatocytes. Phospholipid accumulation was detected by following the incorporation of a fluorescent phospholipid analogue into intracytoplasmic inclusion bodies characteristic of phospholipid storage disorders. At 24 and 48 hr, none of the parent compounds induced phospholipidosis in vitro in rat or human hepatocytes. Phospholipidosis was associated primarily with an amine metabolite of ABT-770. The amine metabolite of another MMPI, ABT-518 ([S-(R*,R*)]-N-[1-(2,2-dimethyl-1,3-dioxol-4-yl)-2-[[4-[4-(trifluoromethoxy)-phenoxy]phenyl]sulfonyl]ethyl]-N-hydroxyformamide), produced little phospholipidosis in rat and human hepatocytes even at concentrations up to 100 microM. The presence or absence of phospholipidosis in the in vitro assay correlated well with ultrastructural findings and drug accumulation in rat tissues. ABT-770, which produced phospholipidosis associated with its amine metabolite in vitro and in vivo, also generated a higher tissue to plasma distribution of metabolites particularly in tissues where phospholipidosis was observed. ABT-518 and its amine metabolite, however, produced low tissue to plasma ratios and induced little to no phospholipidosis in vitro or in vivo. These results demonstrate that the phospholipidosis observed for ABT-770 could be attributed to a cationic metabolite, and that altering the properties of such a metabolite, by modification of the parent compound, alleviated the disorder.

Efficacy of continuation ECT and antidepressant drugs compared to long-term antidepressants alone in depressed patients.

OBJECTIVE: The purpose of this study was to evaluate the efficacy of continuation ECT in depression. METHOD: The authors used retrospective chart review to identify 29 patients who received continuation ECT plus long-term antidepressant treatment after a positive response to acute treatment with ECT for a depressive episode (continuation ECT group). A retrospective case-controlled approach was used to ascertain a matching group of 29 patients who received long-term antidepressant treatment alone after responding positively to acute ECT (antidepressant-alone group). All 58 patients (46 with unipolar depression, 12 with bipolar disorder) had been chronically depressed before receiving acute ECT. Data from medical records were analyzed by using survival analysis and proportional hazards regression to determine outcome and risk factors. RESULTS: The mean duration of the follow-up period for all patients was 3.9 years (5.4 years for the continuation ECT patients and 2.4 years for the antidepressant-alone patients). Outcome was significantly better in the continuation ECT group. The cumulative probability of surviving without relapse or recurrence at 2 years was 93% for continuation ECT patients and 52% for antidepressant-alone patients. At 5 years, survival declined to 73% for continuation ECT patients, but fell to 18% for antidepressant-alone patients. Mean survival times were 6.9 years for the continuation ECT patients and 2.7 years for the antidepressant-alone patients. CONCLUSIONS: The findings provide strong support for the efficacy of continuation ECT plus long-term antidepressant treatment in preventing relapse and recurrence in chronically depressed patients who have responded to acute treatment with ECT.

Internalization of type-A endothelin receptor.

Endothelins (ETs) are 21 amino acid peptides which bind to ET(A)- and ET(B)-receptors to evoke diverse physiological responses. This report studies the internalization of ET(A)-receptor in Chinese hamster ovary (CHO) cells which were stably transfected with ET(A)-receptor cDNA. ET-1 binding induced ET(A) internalization in a time-dependent manner with 40% of ET(A)-receptors internalized at 37 degrees C after 30 min. To localize internalized ET(A)-receptor, cells were immunostained using a polyclonal antibody against the extracellular loop between IV and V transmembrane segments of the ET(A)-receptor. To examine the fate of internalized ET-1, cells were treated with 10 nM biotinylated ET-1 coupled with Texas Red-labeled streptavidin. In the absence of ET-1, a majority of ET(A) was localized on the surface of cells. After ET-1 treatment for 60 min, internalized ET(A)-receptors were localized in a perinuclear structure. ET-1 remained bound to ET(A)-receptor after internalization for up to 60 min and then dissociated from the receptor. After dissociation, ET-1 possibly became degraded and ET(A) recycled back to the cell surface. Protein kinase inhibitors such as KT5926 and staurosporine partially inhibited ET(A)-receptor internalization. The results of this study may facilitate the understanding of pathways involved in ET-1-induced receptor internalization.

Selective antagonism of the ET(A) receptor reduces neointimal hyperplasia after balloon-induced vascular injury in pigs.

Balloon angioplasty has become an important intervention in clinical cardiology; however, the technique is associated with a high incidence of restenosis, requiring repeated procedures. Endothelin-1 (ET-1), specifically through its action on ET(A) receptors, has been implicated in the cell proliferation and subsequent neointimal formation that leads to restenosis. Therefore we examined a potent antagonist of the ET(A) receptor, A127722.5, in a pig model of balloon angioplasty in iliac and carotid arteries. Ten pigs received A-127722.5 (7.5 mg/kg b.i.d.) orally, starting 3 days before angioplasty and continuing for 4 weeks; 10 additional pigs were treated with the same dosing regimen of the angiotensin-converting enzyme (ACE) inhibitor captopril (3.0 mg/kg b.i.d.), while a third group of 10 animals received placebo. At 2 and 4 weeks after the start of treatment, these doses of the ET(A) receptor antagonist and ACE inhibitor blocked the presser responses induced by big ET-1 and angiotensin I, respectively. In the iliac arteries, neointimal formation, neointimal/medial ratio, and maximal neointimal thickness were all significantly reduced, and the residual lumen area was significantly increased in pigs treated with the ET(A) receptor antagonist compared with placebo and captopril-treated groups. Medial collagen content, collagen deposition, and medial growth also were significantly reduced relative to the placebo group. Beneficial effects also were observed in the carotid arteries, although the results were less striking. Captopril was ineffective in protecting against the effects of balloon angioplasty in both vessels. Our results indicate that an orally active and potent antagonist of the ET(A) receptor inhibits cell proliferation and synthesis of extracellular matrix in pigs and may provide an important therapeutic approach to the prevention of restenosis.

Nuclear localization of the antigen detected by ulcerative colitis-associated perinuclear antineutrophil cytoplasmic antibodies.

Antineutrophil cytoplasmic antibodies (ANCAs) identified in the serum of 50 to 80% of ulcerative colitis (UC) patients yield a perinuclear staining pattern (pANCA) with alcohol-fixed neutrophils. The ANCAs of UC are distinguishable from those described for Wegener's granulomatosis and other vasculitidies. These various non-UC ANCAs recognize neutrophil granule constituents, but the antigenic moiety specific for the UC pANCA remains unknown. Although the perinuclear nature of some ANCA reactions is an artifact of the alcohol fixation of neutrophils, which causes cytoplasmic granules to redistribute around the nucleus, the UC pANCA reaction has been found not to be similarly affected. We postulated a nuclear localization for the UC-associated pANCA antigen and used both confocal laser microscopy and immunoelectron microscopy to examine the neutrophil reaction of UC-associated pANCA-containing sera. Confocal microscopy revealed a nuclear reaction for 88% (22/25) of the sera with 72% (18/25) showing the reaction localizing to the inner side of the nuclear (membrane) periphery. Immunoelectron microscopy showed that the UC-associated pANCA reaction localized primarily over chromatin concentrated toward the nuclear periphery, although the sera did not recognize double-stranded DNA. These results confirm the nuclear localization of the UC-associated pANCA antigen.

Transmission maximum-likelihood reconstruction with ordered subsets for cone beam CT.

An iterative algorithm is presented for accelerated reconstruction of cone beam transmission CT data (CBCT). CBCT supplies an attenuation map for SPECT attenuation compensation and anatomical correlation. Iterative algorithms are necessary to reduce truncation artifacts and 3D reconstruction artifacts. An existing transmission maximum-likelihood algorithm (TRML) is accurate but the reconstruction time is too long. The new algorithm is a modified EM algorithm, based on ordered subsets (OSEM). OSEM was evaluated in comparison to TRML using a thorax phantom and a 3D Defrise phantom. A wide range of image measures were evaluated, including spatial resolution, noise, log likelihood, region quantification, truncation artifact removal, and 3D artifact removal. For appropriate subset size, OSEM produced essentially the same image as TRML, but required only one-tenth as many iterations. Thus, adequate images were available in two to four iterations (20-30 min on a SPARC 2 workstation). Further, OSEM still approximately maximizes likelihood: divergence occurs only for very high (and clinically irrelevant) iterations. Ordered subsets are likely to be useful in other geometries (fan and parallel) and for emission CT as well. Therefore, with ordered subsets, high-quality iterative reconstruction is now available in clinically practical reconstructions times.

The transition from technegas to pertechnegas.

UNLABELLED: We set out to determine the oxygen concentration of the argon-oxygen mixture in the technegas generator where technegas-like behavior changes to pertechnegas-like behavior which allows [99mTc]pertechnetate to enter the solution. METHODS: We prepared radioaerosols analogous to technegas and pertechnegas, with oxygen concentrations between 0% and 5%. They then were examined by thin-layer chromatography using saline as a solvent to measure the amount of [99mTc]pertechnetate moving with the solvent front as a function of oxygen concentration. RESULTS: The amount of mobile pertechnetate markedly increased when the radioaerosols were created in an atmosphere containing between 0.1% and 0.2% oxygen. The transition from technegas-like to pertechnegas-like behavior occurs when the oxygen-argon gas mixture contains lower concentrations of oxygen than those used in the preparation of pertechnegas. CONCLUSION: Argon intended for the preparation of technegas should contain no more than 0.1% oxygen.

Characterization of tumor-infiltrating lymphocytes from murine mammary adenocarcinomas.

The aim of this investigation was to assess the in vitro functional and phenotypic characteristics of lymphocytes isolated from C3H mouse mammary adenocarcinomas. A protocol was developed for the expansion of TILs in long-term culture. The homing pattern of TILs prepared and grown in this manner was studied. Cells that had been in culture for up to 96 days accumulated at higher levels in mammary tumors than in corresponding normal mammary tissue 24 hr after adoptive transfer. The ability of cultured TILs to lyse YAC-1 cells was determined. Peak activity was demonstrated by lymphocytes that had been in culture for three days. By two weeks in culture the level of cytotoxicity returned to that of fresh TILs, and after 45 days it was negligible. T cells were the major constituents in all preparations. The relative frequency of CD8+ cells remained fairly constant over time in culture, but that of CD4+ cells declined. At all time points the CD4:CD8 ratio for TILs was less than 1. The percentage of ASGM1+ bright cells among fresh TILs was low. It increased dramatically within 3 days, remained high for about 7 weeks, and then declined rapidly to pre-culture levels. An unusual large cell characterized by the presence of an intensely PAS positive peripheral region was observed.

Nitric oxide synthase in bovine superior cervical ganglion.

We investigated the mechanism of increases in cyclic GMP levels in bovine superior cervical ganglion (SCG) in response to muscarinic receptor stimulation. Acetylcholine increased cyclic GMP levels in SCG. This increase was inhibited by NG-methyl-L-arginine (NMA), and the inhibition was reversed by L-arginine. Soluble nitric oxide (NO) synthase was partially purified from bovine SCG using 2',5'-ADP Sepharose affinity chromatography. The resulting enzyme activity was Ca2+/calmodulin dependent and required NADPH and tetrahydrobiopterin as cofactors. Superoxide dismutase protected and oxyhemoglobin blocked the effect of NO formed by the enzyme. NMA inhibited the activity of the NO synthase. In western blots, an antibody generated against rat brain NO synthase specifically recognized the NO synthase from SCG as a 155-kDa protein band. Immunohistochemistry using the same antibody demonstrated that NO synthase was localized in postganglionic neuronal cell bodies of the SCG. Immunofluorescent labeling showed that some of the cells staining positive for dopamine-beta-hydroxylase also contained NO synthase. Thus, NO is synthesized in specific cells within bovine SCG, including sympathetic neurons, and mediates the acetylcholine-induced stimulation of soluble guanylyl cyclase.

[Mouth diseases in a prehistoric agricultural population of northeastern North America]. / Pathologies buccales d'une population agricole préhistorique du nord-est américain.

There is no longer any question that the consumption of refined sugar is a factor in the development of dental caries. In fact, researchers now believe that the production of refined sugar and, particularly, its great availability and use in post-industrial populations has led to a virtual revolution in both the food industry and buccopathology. Clinical and epidemiological studies on the relationship between caries and sugar consumption have been conducted for more than 40 years, and the harmful effects of sugar consumption on the development of dental caries are now well known. Some authors have also demonstrated a historical relationship between caries and sugar over the last three centuries. Another food revolution that had an equally great impact on oral health occurred with the introduction of agriculture. This innovation is discussed from both a technical and food perspective. Agriculture modified the diet of ancient populations by providing new foods that were rich in carbohydrates and by introducing new cooking methods (food was now often boiled instead of being roasted). These two factors alone contributed to an increased rate of dental caries, but at the same time reduced the abrasion of occlusal surfaces and dental crowns. This paper documents the relationship between dental caries, occlusal abrasion, fractures and periodontal disorders, as well as the agriculture practises of an Amerindian population that lived between 1000 and 1500 A.D. in parts of what are now New York State, Quebec and Ontario. The author's findings confirm those of many other researchers who have investigated agricultural populations.(ABSTRACT TRUNCATED AT 250 WORDS)

Phantom study of breast tissue attenuation in myocardial imaging.

The attenuation of photons by the breasts and other soft tissue overlying the chest may decrease the diagnostic accuracy of SPECT myocardial imaging. In this experiment, we measured the attenuation distortion of myocardial polar maps using a thorax phantom with a cardiac insert and added breast tissue. The distortion was measured using a regional semiquantitative analysis. Attenuation compensation was performed using a conebeam radionuclide CT attenuation map. Breast tissue attenuation created apparent "defects" in the polar map, where the intensity was reduced by up to 35% relative to the most intense region. However, the size, location and severity of the reduction depended on cardiac insert orientation and breast placement. For the geometries studied, apparent "defects" were observed in the anterior wall, the apex, the inferior wall and basal regions. These results suggest that attenuation artifacts may occur in almost any location. However, the attenuation compensation nearly eliminated the apparent defects and improved polar map symmetry. After compensation, the variations between regions were generally 5% or less. Therefore, we expect that attenuation compensation will improve diagnostic accuracy in myocardial imaging in female patients and in males with excessive musculature or soft tissue. Without such compensation, diagnosis may be compromised.

Experimentally produced pulmonary arterial occlusion in the dog: identification with pertechnegas inhalation.

Pertechnegas, a variant of technegas, is a fine, dry aerosol formed when technetium-99m pertechnetate is vaporized in a technegas generator under appropriate conditions. After inhalation, the aerosol is deposited almost entirely on the surfaces of terminal respiratory structures. Unlike technegas, however, pertechnegas crosses the alveolar-capillary membrane and leaves the lung through the pulmonary circulation. To demonstrate prolonged retention of pertechnegas in regions of lung supplied by an occluded pulmonary artery, 12 pulmonary arterial occlusions were experimentally produced in two dogs. After inhalation of pertechnegas, a transient focus of increased pulmonary radioactivity was seen in 11 occlusions; 10 of these 11 foci were easily confirmed by means of subsequent perfusion scintigraphy. All 11 foci of retained pertechnegas corresponded to the location of the non-perfused lung. Thus, it is possible to identify ischemic lung with focal retention of pertechnegas (a finding that indicates preserved ventilation but diminished perfusion) by use of a single examination.

Mapping of neural nitric oxide synthase in the rat suggests frequent co-localization with NADPH diaphorase but not with soluble guanylyl cyclase, and novel paraneural functions for nitrinergic signal transduction.

Nitric oxide synthases (NOS Types I-III) generate nitric oxide (NO), which in turn activates soluble guanylyl cyclase (GC-S). The distribution of this NO-mediated (nitrinergic) signal transduction pathway in the body is unclear. A polyclonal monospecific antibody to rat cerebellum NOS-I and a monoclonal antibody to rat lung GC-S were employed to localize the protein components of this pathway in different rat organs and tissues. We confirmed the localization of NOS-I in neurons of the central and peripheral nervous system, where NO may regulate cerebral blood flow and mediate long-term potentiation. GC-S was located in NOS-negative neurons, indicating that NO acts as an intercellular signal molecule or neurotransmitter. However, NOS-I was not confined to neurons but was widely distributed over several non-neural cell types and tissues. These included glia cells, macula densa of kidney, epithelial cells of lung, uterus, and stomach, and islets of Langerhans. Our findings suggest that NOS-I is the most widely distributed isoform of NOS and, in addition to its neural functions, regulates secretion and non-vascular smooth muscle function. With the exception of bone tissue, NADPH-diaphorase (NADPH-d) activity was generally co-localized with NOS-I immunoreactivity in both neural and non-neural cells, and is a suitable histochemical marker for NOS-I but not a selective neuronal marker.

The early genetic response to light in the green unicellular alga Chlamydomonas eugametos grown under light/dark cycles involves genes that represent direct responses to light and photosynthesis.

In the green unicellular alga Chlamydomonas eugametos, cellular division is readily synchronized by light/dark cycles. Under these conditions, light initiates photosynthetic growth in daughter cells and begins the G1 phase. Genes whose expression is regulated upon illumination are likely to be important mechanisms controlling cell proliferation. To identify some of those genes, two cDNA libraries were prepared with poly(A)+ extracted from cells either stimulated with light for 1 h or held in darkness (quiescent cells) during the same period. To restrict our analysis to those genes that are part of the primary response, cells were incubated in presence of cycloheximide. Differential screening of approximately 40,000 clones in each library revealed 44 clones which hybridize preferentially with a [32P] cDNA probe derived from RNA of light-stimulated cells and 15 clones which react selectively with a [32P] cDNA probe synthesized from poly(A)+ RNA of quiescent cells. Cross-hybridization of these clones identified 4 independent sequences in the light-induced (LI) collection and 2 in the uninduced (LR) library. Four of these cDNAs correspond to mRNAs that are positively or negatively regulated upon activation of photosynthesis. One clone represents a mRNA that accumulates transitorily at both transitions. Finally, LI818 cDNA identifies a new chlorophyll a/b-binding (cab) gene family whose mRNA accumulation is controlled by light and a circadian oscillator. The endogenous timing system controls LI818 mRNA accumulation so that it precedes the onset of illumination by a few hours. On the other hand, light affects LI818 mRNA levels independently of active photosynthesis.

The localization of indium-111-leukocytes, gallium-67-polyclonal IgG and other radioactive agents in acute focal inflammatory lesions.

A variety of radioactive agents, injected directly intravenously have demonstrated foci of inflammation by gamma camera imaging, avoiding the in vitro preparation of labeled leukocytes. This study sought to find out if any of these agents mimicked the biodistribution in abscesses and non-target organs of labeled mixed leukocyte suspensions. Eight different agents were compared with 111In-oxine labeled leukocytes in an acute soft tissue E. coli abscess and an acute arthritic lesion in 24 dogs one day after intravenous administration. These included 67Ga-citrate, human and canine polyclonal immunoglobulin (IgG), rabbit anti-dog polyclonal IgG, serum albumin, monoclonal antibody TNT-1 F(ab')2 against nuclear antigens, 57Co-porphyrin and serum albumin nanocolloid. None of these agents achieved abscess concentrations approaching those obtained with labeled leukocytes, and their abscess/blood and abscess/muscle concentration ratios were considerably lower. No statistically significant differences were found between the different radiolabeled proteins evaluated. The abscess concentration of 99mTc-nanocolloid was much lower than that of other agents, and the results with the oldest agent, 67Ga-citrate, were disappointing in these acute experiments.

Thrombus imaging: a comparison of radiolabeled GC4 and T2G1s fibrin-specific monoclonal antibodies.

Radioimmunoimaging of experimentally-induced canine thrombi has previously been achieved with iodine-131- and indium-111-labeled (131I and 111In) anti-fibrin T2G1s monoclonal antibody (MAb). We now compare T2G1s to another anti-fibrin MAb, designated GC4, for imaging fresh and aged canine thrombi. GC4 is specific for a neoepitope exposed on fibrin later in the thrombolytic process after plasmin digestion. Femoral venous thrombi were induced in six groups of dogs, each containing three dogs. In two groups, the MAbs were compared when the thrombi were 3-hr or 3-days old at the time of injection, and the dogs were killed at 48 hr. In thrombi 3-hr-old, the GC4/T2G1s concentration ratio averaged 0.53 compared to 1.9 in 3-day-old thrombi. Two groups of dogs with thrombi 1- or 3-days-old were heparinized before MAb injection and were killed at 24 hr. The heparinized dogs with thrombi 1- or 3-days-old had GC4/T2G1s mean ratios of 2.3 and 2.9, respectively. In the unheparinized groups, the corresponding ratios were 1.1 and 1.9. GC4 may be more useful for clinical thrombus imaging than T2G1s because spontaneous venous thrombi are usually several days old at the time of presentation and patients are often heparinized immediately.