Lentigo maligna (melanoma): A systematic review and meta-analysis on surgical techniques and presurgical mapping by reflectance confocal microscopy.

Because of an increased risk of local recurrence following surgical treatment of lentigo maligna (melanoma) (LM/LMM), the optimal surgical technique is still a matter of debate. We aimed to evaluate the effect of different surgical techniques and reflectance confocal microscopy (RCM) on local recurrence and survival outcomes. We searched MEDLINE, Embase and PubMed databases through 20 May 2022. Randomized and observational studies with ≥10 lesions were eligible for inclusion. Bias assessment was performed using the Methodological Index for Non-Randomized Studies instrument. Meta-analysis was performed for local recurrence, as there were insufficient events for the other clinical outcomes. We included 41 studies with 5059 LM and 1271 LMM. Surgical techniques included wide local excision (WLE) (n = 1355), staged excision (n = 2442) and Mohs' micrographic surgery (MMS) (n = 2909). Six studies included RCM. The guideline-recommended margin was insufficient in 21.6%-44.6% of LM/LMM. Local recurrence rate was lowest for patients treated by MMS combined with immunohistochemistry (<1%; 95% CI, 0.3%-1.9%), and highest for WLE (13%; 95% CI, 7.2%-21.6%). The mean follow-up varied from 27 to 63 months depending on surgical technique with moderate to high heterogeneity for MMS and WLE. Handheld-RCM decreased both the rate of positive histological margins (p < 0.0001) and necessary surgical stages (p < 0.0001). The majority of regional (17/25) and distant (34/43) recurrences occurred in patients treated by WLE. Melanoma-associated mortality was low (1.5%; 32/2107), and more patients died due to unrelated causes (6.7%; 107/1608). This systematic review shows a clear reduction in local recurrences using microscopically controlled surgical techniques over WLE. The use of HH-RCM showed a trend in the reduction in incomplete resections and local recurrences even when used with WLE. Due to selection bias, heterogeneity, low prevalence of stage III/IV disease and limited survival data, it was not possible to determine the effect of the different surgical techniques on survival outcomes.

A systematic review of outcome reporting in laser treatments for dermatological diseases.

The standardization of outcome reporting is crucial for interpretation and comparison of studies related to laser treatment of skin disorders. In collaboration with the Cochrane Skin-Core Outcome Set Initiative (CS-COUSIN), a procedure has been proposed to find consensus on the most important generic outcome domains (what to measure) for implementation in the international Laser TrEAtment in Dermatology (LEAD) registry. As the first step in the development of a generic outcome set for the LEAD registry, we undertook a systematic review to identify outcomes, outcome measurement instruments, methods and definitions reported in recently published literature of laser treatments for skin disorders. A systematic search was conducted and generated a total of 707 papers. We assessed 150 studies including all types of studies involving laser treatments for the skin. Two researchers independently extracted the type, definition and frequency of all outcomes and used outcome measurement instruments. We identified 105 verbatim outcomes that were categorized into eight domains recommended by the COMET framework: appearance, long-term effects, physician and patient-reported physical signs, satisfaction, health-related quality of life, psychological functioning and adverse events. Heterogeneity in outcome reporting (e.g. categories and outcome measurement instruments) was high, and definitions were insufficiently reported. There was a clear under representation of life impact domains, including satisfaction (23%) quality of life (3%) and psychological functioning (1%). Outcome reporting concerning laser treatments for the skin is heterogeneous. Standardized outcomes are needed for improving evidence synthesis. Results of this review will be used in the next step to reach consensus between stakeholders on the outcome domains to be implemented in the LEAD registry.

Correction of a dominant-negative von Willebrand factor multimerization defect by small interfering RNA-mediated allele-specific inhibition of mutant von Willebrand factor.

Essentials Substitution therapy for von Willebrand (VW) disease leaves mutant VW factor (VWF) unhindered. Presence of mutant VWF may negatively affect phenotypes despite treatment. Inhibition of VWF by allele-specific siRNAs targeting single-nucleotide polymorphisms is effective. Allele-specific inhibition of VWF p.Cys2773Ser improves multimerization. SUMMARY: Background Treatment of the bleeding disorder von Willebrand disease (VWD) focuses on increasing von Willebrand factor (VWF) levels by administration of desmopressin or VWF-containing concentrates. Both therapies leave the production of mutant VWF unhindered, which may have additional consequences, such as thrombocytopenia in patients with VWD type 2B, competition between mutant and normal VWF for platelet receptors, and the potential development of intestinal angiodysplasia. Most cases of VWD are caused by dominant-negative mutations in VWF, and we hypothesize that diminishing expression of mutant VWF positively affects VWD phenotypes. Objectives To investigate allele-specific inhibition of VWF by applying small interfering RNAs (siRNAs) targeting common single-nucleotide polymorphisms (SNPs) in VWF. This approach allows allele-specific knockdown irrespective of the mutations causing VWD. Methods Four SNPs with a high predicted heterozygosity within VWF were selected, and siRNAs were designed against both alleles of the four SNPs. siRNA efficiency, allele specificity and siRNA-mediated phenotypic improvements were determined in VWF-expressing HEK293 cells. Results Twelve siRNAs were able to efficiently inhibit single VWF alleles in HEK293 cells that stably produce VWF. Transient cotransfections of these siRNAs with two VWF alleles resulted in a clear preference for the targeted allele over the untargeted allele for 11 siRNAs. We also demonstrated siRNA-mediated phenotypic improvement of the VWF multimerization pattern of the VWD type 2A mutation VWF p.Cys2773Ser. Conclusions Allele-specific siRNAs are able to distinguish VWF alleles on the basis of one nucleotide variation, and are able to improve a severe multimerization defect caused by VWF p.Cys2773Ser. This holds promise for the therapeutic application of allele-specific siRNAs in dominant-negative VWD.

Variation in the diagnosis and clinical management of lentigo maligna across Europe: a survey study among European Association of Dermatologists and Venereologists members.

BACKGROUND: Lentigo maligna (LM), a form of melanoma in situ, is treated to prevent progression to lentigo maligna melanoma (LMM). Surgical treatment is the gold standard. However, treatment guidelines are based on expert opinion, and comparative studies are lacking. OBJECTIVE: The objective of this study was to assess the diagnostic methods and clinical management of LM patients among European dermatologists and residents. METHODS: A survey consisting of 29 questions about diagnostic methods and treatment options used for LM patients was sent to 3308 members of the European Association of Dermatologists and Venereologists (EADV). RESULTS: Most questions were multiple choice, and multiple answers could be ticked per question. A total of N = 415 (12.5%) completed surveys were included in the analyses. A combination of clinical diagnosis (65.7%), dermatoscopy (83.4%) and histopathology (88.2%) is used by most respondents to diagnose LM. Tissue for histopathological evaluation was collected most often using a single punch biopsy in 61.0%. The most common treatment for LM patients <60 years of age is surgery (97.6%). For LM patients >70 years of age, 66.8% of the respondents preferred surgical treatment. Non-surgical options such as radiotherapy (17.0%), topical imiquimod (30.6%), watchful waiting (19.6%) or cryotherapy (20.4%) were used in this elderly group. Subanalysis showed that respondents who take into account patient preference used topical imiquimod, radiotherapy and watchful waiting more often. CONCLUSION: In conclusion, the results of this survey show that there is a variance in the diagnostic methods and treatment modalities used for LM across Europe. Surgery remains the most utilized option. However, non-surgical options, such as topical imiquimod and radiotherapy, are most often used for elderly patients. We recommend that future studies focus on patient preference and compare surgical to non-surgical therapy.

A systematic review on the role of imiquimod in lentigo maligna and lentigo maligna melanoma: need for standardization of treatment schedule and outcome measures.

Lentigo maligna (LM) is an in situ variant of melanoma. Our objective was to systematically review clinical and histological clearance and recurrence rates of imiquimod treatment of LM with emphasis on progression to lentigo maligna melanoma (LMM). PubMed, EMBASE and the Cochrane library were searched from inception to May 2015. Articles were included if they described histologically proven LM treated with imiquimod 5% monotherapy or combined with another topical therapy. Analysed outcomes were clinical and histological clearance, recurrence rates and number of LMM. The quality was assessed using the GRADE-like checklist, and results were reported according to the PRISMA Statement. Twenty-six case reports, 11 retrospective studies, three prospective studies and one randomized controlled trial were included. One case report of poor quality was excluded. Complete clinical clearance was seen in 369 of 471 patients (78.3%). Histological clearance was present in 285 of 370 (77%) patients. LMM was diagnosed in nine (1.8%) patients 3.9 months (range 0-11 months) post-treatment. Univariate multinominal logistic regression showed that 6-7 applications/week had a 6.47 greater odds (P = 0.017) of resulting in complete clinical clearance compared to 1-4 applications/week. An intensity of 6-7 applications/week showed a 8.85 greater odds (P = 0.003) of resulting in histological clearance compared to 1-4 applications. Applying imiquimod >60 times during a treatment period of 12 weeks (range 4-36) showed a 7.75 greater odds (P = 0.001) of resulting in histological clearance compared to <60 total applications. In conclusion, a treatment schedule using imiquimod 6-7 applications per week, with at least 60 applications, shows the greatest odds of complete clinical and histological clearance of LM. Imiquimod is an option for patients unfit for or not willing to undergo surgery or radiotherapy. Nine cases of LM progressed to LMM shortly after treatment. Our hypothesis is that these LMM may have been present before starting imiquimod.

Effects of acute restraint-induced stress on glucocorticoid receptors and brain-derived neurotrophic factor after mild traumatic brain injury.

We have previously reported that experimental mild traumatic brain injury results in increased sensitivity to stressful events during the first post-injury weeks, as determined by analyzing the hypothalamic-pituitary-adrenal (HPA) axis regulation following restraint-induced stress. This is the same time period when rehabilitative exercise has proven to be ineffective after a mild fluid-percussion injury (FPI). Here we evaluated effects of stress on neuroplasticity. Adult male rats underwent either an FPI or sham injury. Additional rats were only exposed to anesthesia. Rats were exposed to 30 min of restraint stress, followed by tail vein blood collection at post-injury days (PID) 1, 7, and 14. The response to dexamethasone (DEX) was also evaluated. Hippocampal tissue was collected 120 min after stress onset. Brain-derived neurotrophic factor (BDNF) along with glucocorticoid (GR) and mineralocorticoid (MR) receptors was determined by Western blot analysis. Results indicated injury-dependent changes in glucocorticoid and mineralocorticoid receptors that were influenced by the presence of dexamethasone. Control and FPI rats responded differentially to DEX in that GR increases after receiving the lower dose of DEX were longer lasting in the FPI group. A suppression of MR was found at PID 1 in vehicle-treated FPI and Sham groups. Decreases in the precursor form of BDNF were observed in different FPI groups at PIDs 7 and 14. These findings suggest that the increased sensitivity to stressful events during the first post-injury weeks, after a mild FPI, has an impact on hippocampal neuroplasticity.

Heightening of the stress response during the first weeks after a mild traumatic brain injury.

The effects of a mild traumatic brain injury range from white matter disruption to affective disorders. We set out to determine the response to restraint-induced stress after a mild fluid-percussion injury (FPI), an experimental model for brain injury. Hypothalamic-pituitary-adrenal (HPA) axis regulation of corticosterone (CORT) and adrenocorticotropic hormone (ACTH) was determined during the first post-injury weeks, which corresponds to the same time period when rehabilitative exercise has been shown to be ineffective after a mild FPI. Adult male rats underwent either an FPI or sham injury. Additional rats were only exposed to anesthesia. HPA regulation was evaluated by measuring the effects of dexamethasone (DEX) treatment on CORT and ACTH. Tail vein blood was collected following 30-min restraint stress, at post-injury days (PID) 1, 7 and 14, prior to (0 min) and at 30, 60, 90 and 120 min after stress onset. Results from these studies indicate that the stress response was significantly more pronounced after FPI in that CORT and ACTH restraint-induced increases were more pronounced and longer lasting compared to controls. DEX suppression of CORT and ACTH was observed in all groups, suggesting that stress hyper-responsiveness after mild FPI is not attributable to reduced sensitivity of CORT feedback regulation. The increased sensitivity to stressful events in the first two post-injury weeks after a mild FPI may have a negative impact on early rehabilitative therapies.

The glossopharyngeal nerve as a novel pathway in immune-to-brain communication: relevance to neuroimmune surveillance of the oral cavity.

Glossopharyngeal afferents may be the neural channel by which immune challenge of the posterior oral cavity conveys information to the brain. If this is the case, then bilateral transection of the glossopharyngeal nerves (GLOx) should disrupt this communication. Injection of lipopolysaccharide (LPS) or interleukin (IL)-1beta into the soft palate (ISP) of sham-operated rats induced a dose-related febrile response. GLOx significantly attenuated the febrile response induced by ISP injection of both LPS and IL-1beta. In contrast, GLOx did not affect the febrile response when LPS or IL-1beta were injected intraperitoneally, indicating that the effect of GLOx is not systemic. These results provide experimental evidence for a novel neural pathway for immune-to-brain communication.

Fetal alcohol exposure attenuates interleukin-1beta-induced fever: neuroimmune mechanisms.

Central mechanisms for the attenuating effects of fetal alcohol exposure (FAE) on interleukin-1beta (IL-1beta)-induced fever were studied in adult male offspring of dams fed a liquid diet supplemented with ethanol (E), in pair-fed (P) control and in normal (N) offspring. Hypothalamic levels of IL-1 were significantly lower in E than in N rats at 2 h, but not at 4 and 6 h, after intraperitoneal administration of lipopolysaccharide. Fever induced by intracerebroventricular (i.c.v.) IL-1 was significantly lower in E than in N and P rats. In contrast, E rats showed a normal febrile response to i.c.v. prostaglandin-E2. Thus, whereas FAE does not affect central thermoregulatory mechanisms, per se, FAE alters the kinetics of hypothalamic IL-1 production/appearance and decreases the responsiveness of central mechanisms which mediate the febrile response to IL-1.

Thymocyte development in male fetal alcohol-exposed rats.

We previously reported altered responses of thymocytes to mitogen stimulation after fetal alcohol exposure (FAE) in prepubertal male Sprague-Dawley rats. The purpose of this study was to examine the effect of FAE on the developmental pattern of thymocyte subsets. In the first experiment, we found that the proportion of double-labeled CD4+CD8+ thymocytes is identical in fetal alcohol-exposed (E) and control (C) animals at 34 and 45 days of age. In the second experiment--at 20, 28, 35, and 48 days of age--we examined the proportion of CD4+ and CD8+ thymocytes that express or are devoid of the maturational markers, the alpha/beta configuration of the T-cell receptor (TcR), and the restriction fragment C of the common leukocyte antigen (CD45RC). We found significant age-dependent effects on the numbers of total double-positive CD4-TcR and CD8-TcR or CD45RC thymocytes, and significantly lower numbers of total CD4+ and CD8+ cells in E than in C rats throughout this period--a finding consistent with the significantly lower total number of thymocytes in E than in C rats. The developmental patterns for both markers were similar in E and C groups, in both the rising (days 20 to 28) and declining (days 35 to 48) phases. However, on day 35, E rats had significantly lower numbers of double-positive CD8-TcR and CD8-CD45RC cells than C rats. It therefore seems that FAE tends to accelerate the decline of double-positive CD8-TcR and CD8-CD45RC cells. The contribution of this phenotypic change to the thymic functional alterations induced by FAE remains to be determined.

Effects of prenatal alcohol and pair feeding on lipopolysaccharide-induced secretion of TNF-alpha and corticosterone.

Fetal alcohol exposure (FAE) produces profound alterations in immunological and neuroendocrine functions. The present study examined the effects of FAE on the secretion of tumor necrosis factor (TNF-alpha) and corticosterone following administration of lipopolysaccharide (LPS) in normal (N) adult rats, in adult offspring of dams fed a liquid diet supplemented with ethanol (E), and in pair-fed control offspring (P). LPS-induced TNF-alpha secretion was not affected by either gender or prenatal treatment. In contrast, LPS-induced corticosterone secretion was significantly greater in female than in male rats, and at 60-min post-LPS was significantly higher in E and P, compared to N females. Ovariectomy significantly inhibited LPS-induced TNF-alpha secretion in E, but not in P and N, rats and chronic replacement with 17-beta-estradiol markedly inhibited TNF-alpha secretion in ovariectomized E and N, but not in P, rats. In contrast, ovariectomy reduced the effects of LPS on corticosterone secretion in all groups, and chronic replacement with 17-beta-estradiol reversed this effect. These findings indicate that LPS-induced secretion of corticosterone, but not TNF-alpha, is affected by prenatal manipulations and by gender. In addition, alterations in the hormonal environment in females modulate LPS-induced corticosterone secretion in all prenatal treatment groups, but differentially influence TNF-alpha secretion in rats exposed to alcohol, restricted feeding, or normal diets in utero.

Fetal alcohol exposure augments the blunting of tumor necrosis factor production in vitro resulting from in vivo priming with lipopolysaccharide in young adult male but not female rats.

We previously reported altered responses of thymocytes and splenocytes to mitogen stimulation in fetal alcohol-exposed (FAE) male Sprague-Dawley rats. We also reported enhanced neuroendocrine responses to stressful stimuli in these animals. The experiments we describe herein aimed at testing whether young adult FAE rats manifest a notable dysregulation in the neuroendocrine-immune response to pathogen administration. We tested the effect of in vivo priming of the animal with a low dose of endotoxin [lipopolysaccharide (LPS), 5 micrograms/kg], considered to be suboptimal from the perspective of mounting detectable levels of circulating monokines several hours after administration, upon the production of immunoreactive tumor necrosis factor (TNF-alpha) in response to a further in vitro challenge of peripheral blood mononuclear cells with 2.5 micrograms/ml of LPS 90 min after priming. We show that the response to the LPS pathogen in vitro after priming is significantly blunted (p < 0.01) in male rats exposed prenatally to alcohol, compared with control male animals. FAE female rats and FAE ovariectomized female rats do not show significant differences in the priming response, compared with control animals. We also show that there is no correspondence between plasma corticosterone levels and TNF-alpha production after priming in any of the groups tested.

Effect of acute dietary restriction on the colonization of MADB106 tumor cells in the rat.

The effects of acute food restriction (i.e. 24-72 h) on (1) the colonization of MADB106 tumor cells; (2) the response of specific T cell subsets in peripheral blood (i.e. CD4+ and CD8+ cells), and (3) natural killer cell activity (NKCA) in the spleen were studied in the Fischer 344 rat. Previous studies have demonstrated that the spread of this tumor cell is enhanced by exposure to an acute stressor within 24 h of tumor inoculation. Consistent with these reports, 72-hour food restricton after tumor inoculation enhanced colonization of tumor cells to the lungs when assessed 4 weeks after inoculation. Food restriction was found to markedly influence the percentage of T cell subsets (i.e. CD4+ and CD8+ cells) and NKCA in the early (24-72 h) postinoculation stage. At 72 h after inoculation, food restriction was associated with a significant reduction in the percentage of CD4+ cells in tumor- or saline-inoculated animals. The percentage of CD8+ cells was significantly increased at 24 and 72 h after tumor inoculation in ad libitum, but not in food-deprived animals. NKCA at 72 h was significantly reduced in saline-treated food-deprived animals compared to animals fed ad libitum. Given that glucocorticoids are typically increased during acute food deprivation and that glucocorticoids are in some instances associated with depressed NKCA, the present study investigated whether there was a relationship between plasma glucocorticoid levels (i.e. corticosterone) and NKCA; however, no significant relationship was found. In conclusion, the present findings demonstrate that 72-hour food deprivation is associated with enhancement of tumor metastasis. This outcome is mediated, at least in part, by the modulatory effect of the physiological response to acute food restriction upon the distribution of circulating T cells and NKCA in the spleen during the early (24-72 h) postinoculation phase.

Effects of fetal alcohol exposure on fever, sickness behavior, and pituitary-adrenal activation induced by interleukin-1 beta in young adult rats.

Exposure to alcohol in utero can lead to long-lasting impairments of immune functions and to decreased resistance to infectious agents. We have previously reported that fetal alcohol-exposed rats show markedly decreased lipopolysaccharide-induced fever and suggested that fetal alcohol exposure (FAE) impairs the communication between the immune and the nervous systems. The present study examined the effects of interleukin-1 beta (IL-1) on body temperature, motor activity, ingestive behavior, and pituitary-adrenal activation in fetal alcohol-exposed and control rats. Transmitters for continuous biotelemetric recording of body temperature and motor activity were implanted i.p. in normal (N) adult rats, offspring of dams fed a liquid diet supplemented with ethanol (E), and pair-fed control offspring (P). In one experiment, rats were injected with either IL-1 (2 micrograms/kg, i.p.) or saline at the beginning of the light period. IL-1 produced a marked increase in body temperature, which was significantly lower in E rats than in N and P rats. In a second experiment, rats were administered either IL-1 (10 micrograms/kg, i.p.) or saline at the beginning of the dark period. IL-1 produced an initial transient hypothermia followed by a longer-lasting hyperthermia. During the hyperthermic phase, fever in the E rats was lower than in the P rats, but comparable to fever in the N rats. IL-1 significantly reduced motor activity, during both the hypothermic and hyperthermic phases. This effect was similar in all prenatal treatment groups. IL-1 also suppressed 24-h food consumption in N and P rats and water consumption in P rats, but it did not produce significant anorexia and adypsia in E rats. A third experiment demonstrated that IL-1 (2 micrograms/ kg, ip) significantly increased ACTH and corticosterone release in all prenatal treatment groups. IL-1-induced corticosterone secretion was attenuated in P offspring, compared to both E and N rats. Together, these findings indicate that exposure to ethanol in utero produces impairments in mechanisms that mediate the effects of IL-1 on body temperature (particularly during the light period) and ingestive behavior, but not on motor activity and pituitary-adrenal activation. In view of the adaptive role of IL-1-induced fever and anorexia, these impairments may contribute to the decreased resistance to infections observed in animals and humans following FAE.

Fetal alcohol and thymocyte phenotypes in offspring: response to food deprivation.

Restriction of food availability is a reliable stimulus that leads to significant hypothalamo-pituitary-adrenal (HPA) activation to which rats do not habituate. Based on our previous data that indicated that the HPA response to some, but not all, stressful stimuli is significantly greater in adult offspring of Sprague-Dawley dams exposed to 35% alcohol during the last 2 weeks of gestation than that of control rats and on the mounting neuroendocrine-immune literature that describes the role of pituitary-adrenal products in modulating cellular immunity, we hypothesized that the outcomes of food restriction would be significantly more marked in fetal alcohol-exposed (FAE) offspring, compared with control rats. Data we report herein show that--whereas food restriction at 30-35 days of age produced significant changes in body weight, thymus weight-to-body weight ratio, adrenal weight-to-body weight ratio, plasma corticosterone levels, and in thymocyte number, as well as in the percentage and absolute number of CD4+ and CD8+ thymocytes that express CD45RC-FAE and control rats were equally affected. We conclude that food restriction is another example of a stressful stimulus that fails to distinguish satisfactorily between FAE and control rats of prepubertal age.

Adrenalectomy but not adrenal demedullation during pregnancy prevents the growth-retarding effects of fetal alcohol exposure.

Growth retardation, both in the prenatal and the early neonatal period, is a consistent feature of fetal alcohol exposure, but the mechanism by which alcohol affects growth has not been elucidated. Because other stressors--such as maternal restraint and neonatal glucocorticoid treatment--can also affect growth, we examined the effect of ethanol on pup birthweight under two experimental conditions that altered maternal adrenal function. In the first study when dams were adrenalectomized and given low replacement doses of dexamethasone, the ethanol-exposed offspring of the adrenalectomized dams had birthweights similar to those of dams maintained on regular lab chow diets. In a second study, we found that maternal adrenal demedullation did not alter the reduction in birthweight produced by fetal ethanol exposure. The results suggest that the effects of ethanol on fetal growth may be mediated in part through ethanol-induced changes in the function of the maternal adrenal cortex.

Selective effects of fetal alcohol exposure on rat thymocyte development.

The thymoproliferative response to concanavalin A (ConA) following fetal alcohol exposure (FAE) is higher than control (149%) on day 44, is lower than control (64%) by day 51, and normalizes by day 69 (88% of controls). The ontogeny of HLA-Dr and transferrin receptor (CD71) expression in response to anti-CD3 stimulation is similar among the groups, but is distinct from that of ConA proliferation. The ontogeny of glucocorticoid cytoplasmic receptor (GCCR) sites per thymocyte is also different from the ontogeny of the ConA response. The number of GCCR sites rises sharply (2.5-fold) in control rat thymocytes between days 30 and 44, and remains at that level at later time points. By contrast, the number of GCCR sites per FAE thymocytes rises nearly linearly and normalizes by day 72. Our data support the notion that prenatal alcohol exposure significantly alters thymic development and indicates that the relationship between the development of thymocyte functional responses and that of GCCR is more complex than initially hypothesized.