A comparison of different definitions of growth response in short prepubertal children treated with growth hormone.

BACKGROUND: How to define poor growth response in the management of short growth hormone (GH)-treated children is controversial. AIM: Assess various criteria of poor response. SUBJECTS AND METHODS: Short GH-treated prepubertal children [n = 456; height (Ht) SD score (SDS) ≤-2] with idiopathic GH deficiency (IGHD, n = 173), idiopathic short stature (ISS, n = 37), small for gestational age (SGA, n = 54), organic GHD (OGHD, n = 40), Turner syndrome (TS, n = 43), skeletal dysplasia (n = 15), other diseases (n = 46) or syndromes (n = 48) were evaluated in this retrospective multicenter study. Median age at GH start was 6.3 years and Ht SDS -3.2. RESULTS: Median [25-75 percentile] first-year gain in Ht SDS was 0.65 (0.40-0.90) and height velocity (HtV) 8.67 (7.51-9.90) cm/year. Almost 50% of IGHD children fulfilled at least one criterion for poor responders. In 28% of IGHD children, Ht SDS gain was <0.5 and they had lower increases in median IGF-I SDS than those with Ht SDS >0.5. Only IGHD patients with peak stimulated growth hormone level <3 µg/l responded better than those with ISS. A higher proportion of children with TS, skeletal dysplasia or born SGA had Ht SDS gain <0.5. CONCLUSION: Many children respond poorly to GH therapy. Recommendations defining a criterion may help in managing short stature patients.

Growth of Belgian and Norwegian children compared to the WHO growth standards: prevalence below -2 and above +2 SD and the effect of breastfeeding.

BACKGROUND: New national growth references have been published in Belgium and Norway. The WHO recommends universal use of their 2006 Child Growth Standards based on data from breastfed children. OBJECTIVE: To compare the growth of Belgian and Norwegian children with the WHO standards. PARTICIPANTS: 6985 children 0-5 years of age from Belgium and Norway. DESIGN: Proportion of children below -2 SD and above +2 SD of the WHO standards was calculated for length/height, weight, body mass index and head circumference. Average SD scores of exclusively breastfed children of non-smoking mothers were compared with national reference data and with the WHO standards. RESULTS: Generally, the number of Belgian and Norwegian children below -2 SD lines of the WHO standards was lower and above +2 SD higher than expected. The largest differences were for head circumference (0.97% Belgian and 0.18% Norwegian children below -2 SD, 6.55% Belgian and 6.40% Norwegian children above +2 SD) and the smallest for length/height (1.25% Belgian and 1.43% Norwegian children below -2 SD, 3.47% Belgian and 2.81% Norwegian children above +2 SD). The growth pattern of breastfed children of non-smoking mothers was in both countries more alike the local national growth references than the WHO standards. CONCLUSIONS: There are significant deviations in the proportion of children outside normal limits (±2 SD) of the WHO standards. This was true for all children, including those who were exclusively breastfed. Hence, adoption of the WHO growth charts could have consequences for clinical decision-making. These findings advocate the use of national references in Belgium and Norway, also for breastfed children.

Nordic consensus on treatment of undescended testes.

AIM: To reach consensus among specialists from the Nordic countries on the present state-of-the-art in treatment of undescended testicles. METHODS: A group of specialists in testicular physiology, paediatric surgery/urology, endocrinology, andrology, pathology and anaesthesiology from all the Nordic countries met for two days. Before the meeting, reviews of the literature had been prepared by the participants. RECOMMENDATIONS: The group came to the following unanimous conclusions: (1) In general, hormonal treatment is not recommended, considering the poor immediate results and the possible long term adverse effects on spermatogenesis. Thus, surgery is to be preferred. (2) Orchiopexy should be done between 6 and 12 months of age, or upon diagnosis, if that occurs later. (3) Orchiopexy before age one year should only be done at centres with both paediatric surgeons/urologists and paediatric anaesthesiologists. (4) If a testis is found to be undescended at any age after 6 months, the patient should be referred for surgery--to paediatric rather than general surgeons/urologists if the boy is less than one year old or if he has bilateral or non-palpable testes, or if he has got relapse of cryptorchidism.

Soluble CD30 and CD23 in cord blood are not related to atopy in early childhood.

Atopic disease, including atopic dermatitis (AD), is associated with a T-helper 2 (Th2)-dependent immune response. The cytokine receptor CD30 appears to be preferentially expressed on, and its soluble form (sCD30) released by, Th2 cells. Therefore, sCD30 may be a potential marker for atopic disorders. The aim of this study was to test the hypothesis that the sCD30 level in cord blood could be used to predict the development of atopy or AD in early childhood. In a case-control study, levels of sCD30, as well as soluble low-affinity immunoglobulin E (IgE) receptor (sCD23), interleukin-4 (IL-4) and IgE, were measured in cord blood in 35 children who subsequently developed allergic sensitization and AD before the age of three, and the results were compared to those of 35 matched children without a history of atopy. There was no difference in cord blood levels of sCD30 between controls (32.5 U/ml; 19.7-80.1) and children with subsequent atopy and AD (32.2 U/ml; 22-75.9) (median; quartiles). The concentration of sCD30 showed no relation to the levels of total IgE, sCD23 or IL-4. Levels of sCD23 were similar in children with subsequent atopy (60.2 U/ml; 44.5-76.8) and controls (55.2 U/ml; 38.3-72.5), whereas IL-4 was detectable in 10 of the atopic children and in only two of the controls (p <0.05). In conclusion, cord blood levels of sCD30 or sCD23 do not seem to be related to the subsequent development of atopy or AD at the age of three.

Studies of Atlantic salmon (Salmo salar L.) blood, spleen and head kidney leucocytes using specific monoclonal antibodies, immunohistochemistry and flow cytometry.

Monoclonal antibodies (mabs) raised against Atlantic salmon serum IgM (C7G7 and G2H3) and isolated peripheral blood leucocytes (PBL) (E3D9, C4B6 and D8B3) were applied in this study. Using immunoenzymehistochemistry, immunofluorescence and flow cytometry, the distribution of mab+ cells in blood, spleen and head kidney from Atlantic salmon were studied. Immunostaining on cytospin preparations and flow cytometry of isolated PBL showed that the Ig+ cells recognised by C7G7 and G2H3 were mononuclear leucocytes (MNL). The cytospin preparations showed some Ig+ cells with strong cytoplasmic staining, most likely plasma cells. The salmon blood neutrophils were the only E3D9+ cells in cytospin preparations of PBL, and E3D9 recognised about 94% of the defined neutrophil fraction in flow cytometry. The reactivities of C4B6 and D8B3 were to a large degree similar in both immunoenzymehistochemistry and flow cytometry, recognising both MNL and blood neutrophils. Immunofluorescence double staining of PBL with C4B6 and D8B3 showed double staining of all mab+ cells and D8B3 was apparently not able to block the binding of C4B6 to PBL. Immunofluorescence double staining of PBL also revealed more E3D9+ than C4B6+ neutrophils. In immunostaining on cryostat sections of spleen and head kidney, staining of cells was observed with all the mabs, the head kidney generally containing more positive cells than the spleen. Some potential applications for immunological studies using these mabs are suggested.

Insulin-like Growth Factor-1 (IGF-1) is a Costimulator of the Expansion of Lineage Committed Cells Derived from Peripheral Blood Mobilized CD34(+) Cells in Multiple Myeloma Patients.

The effect of insulin-like growth factor-1 (IGF-1) on highly enriched human apheresis CD34(+) progenitor cells was investigated in vitro. The progenitor cells were mobilized by treatment with cyclophosphamide + granulocyte-colony stimulating factor (G-CSF) in patients with multiple myeloma. CD34(+) cells were cultured for 7 days in serumfree medium containing stem cell factor (SCF), granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-3 (IL-3), and this is referred to as cytokine-dependent proliferation. After 7 days of cytokine-dependent proliferation the total number of viable cells increased 1.6-8.2 times, and subsets of cells expressing the granulocyte marker CD15, the myelomonocytic marker CD64 and the erythrocyte phenotype CD71(high)/CD64(-) were detected among the in vitro cultured cells. Addition of G-CSF together with SCF + IL-3 + GM-CSF increased the number of CD15(+) and CD64(+) cells, but without altering the number of erythroid cells. IGF-1 caused a dose-dependent increase in the number of CD15(+), CD64(+) and CD71(high)/CD64(-) cells, and this increase was detected when cells were cultured in both SCF + IL-3 + GM-CSF alone and G-CSF + SCF + IL-3 + GM-CSF. A minor subset of CD34(+) cells could still be detected among in vitro cultured cells and the number of CD34(+) cells was not altered by adding G-CSF and/or IGF-1. Morphologically recognizable mature granulocytes or erythroid cells could not be detected for any of the combinations investigated. We conclude that IGF-1 can enhance the in vitro proliferation of committed progenitor cells derived from apheresis CD34(+) cells.

Concentrations of cytokines in plasma of patients with large burns: their relation to time after injury, burn size, inflammatory variables, infection, and outcome.

OBJECTIVE: To monitor longitudinally the concentrations of cytokines in the plasma of patients with severe burns. DESIGN: Prospective open study. SETTING: Burns unit, university hospital, Norway. SUBJECTS: 27 patients (5 women and 22 men, mean age 37 (range 13-82) years). INTERVENTIONS: Measurement of plasma concentrations of interleukin-1beta(IL-1beta), interleukin-1 receptor antagonist (IL-1ra), interferon-7(IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha) were measured by enzyme linked immunosorbent assays (ELISA). MAIN OUTCOME MEASURES: Changes in concentrations, and correlation with morbidity and mortality. RESULTS: The concentration of IL-1beta and IL-1ra were increased in all patients and highest at the time of admission. Initially there was little or no circulating IFN-gamma, but this increased from day 5-10 in all patients. Only 8/15 patients had transient increases in circulating TNF-alpha. Concentrations of IL-1ra correlated with total burn surface area (TBSA) and area of third degree burn, as well as with plasma concentrations of C - reactive protein (CRP). Concentrations of IL-1beta and IL-1ra were higher in patients who developed infective complications than in those who did not (interleukin-8 (IL-8) has previously been shown to follow this pattern as well). Patients who survived had significantly higher IL-1beta concentrations than those who died (13(1) compared with 3 (1) pg/ml, p = 0.005) CONCLUSION: There are significant time-dependent changes in plasma concentrations of IL-1beta, IL-1ra, IFN-gamma and TNF-alpha after serious burns. IL-1ra concentrations may be influenced by size of the burn and the acute phase response; IL-1beta, IL-1ra and IL-8 may have a role in the host's response to infection; and IL-1beta may influence outcome.

Effects of topical nasal steroids on human respiratory mucosa and human granulocytes in vitro.

Human respiratory mucosa and human granulocytes were exposed to topical nasal steroids in vitro. The preparations containing benzalkonium chloride and benzalkonium chloride alone destroyed the mucosa within 10 days. The same preparations also inhibited human neutrophil actin polymerization, degranulation and oxidative burst in vitro in a time and concentration dependent manner. Preparations without benzalkonium chloride, as well as the steroid compounds themselves, did not have these effects. It is concluded that benzalkonium chloride has toxic effects on human respiratory mucosa and human neutrophils in vitro.

Early cell kinetic effects of some drugs on the rat corneal and conjunctival epithelia.

The early cell kinetic response in the rat corneal and conjunctival epithelia was studied after a single topical application of dipivefrine, adrenaline and timolol with and without benzalkonium chloride. Benzalkonium chloride alone in different concentrations, 0.004%, 0.01%, 0.02% and 0.04%, respectively, was also evaluated. The stathmokinetic method and the tritiated thymidine technique were used to estimate the mitotic rate and the labelling index. Dipivefrine (Propine) gave a significant depression of the mitotic rate almost to the same extent as adrenaline in the corneal epithelium, whereas no changes were found in the limbal area and in the conjunctiva. Timolol (Oftan) with or without benzalkonium chloride, and benzalkonium chloride in different concentrations gave no obvious changes of the mitotic rate. No distinct drug effects on the labelling index were observed.

Microbial colonization of large wounds.

This study determines the nature of microbial wound colonization in 28 patients with large burns admitted to the Burn Centre, Haukeland University Hospital, Bergen. Altogether, 748 swabs were taken in 141 sampling procedures. A total of 414 microbial isolates were detected and their resistance patterns to a variety of systemic antimicrobial agents determined. The most frequent isolates were coagulase-negative staphylococci (21.5 per cent) and Staphylococcus aureus (14 per cent), followed by Enterococcus species (11.3 per cent), Pseudomonas aeruginosa (10.9 per cent) and Candida species (9.7 per cent). Forty-one per cent of the enterococci and 36 per cent of the coagulase-negative staphylococci were resistant to the aminoglycosides routinely given in conjunction with surgery in our ward. Only four of the 89 strains of coagulase-negative staphylococci were insensitive to methicillin, and no Staph. aureus were methicillin resistant. The time-related changes of burn wound colonization showed that on admission and during the first week, staphylococci and alpha-haemolytic streptococci were dominant. During the next weeks, these bacteria were gradually superceded by enterococci, gram-negative opportunists (mainly Pseud. aeruginosa, Acinetobacter calcoaceticus and Escherichia coli) and Candida species. The nature of microbial wound colonization and how the flora changes with time should be taken into consideration by those treating thermally injured patients.

Priming of human polymorphonuclear neutrophilic leukocytes by insulin-like growth factor I: increased phagocytic capacity, complement receptor expression, degranulation, and oxidative burst.

Insulin-like growth factor I (IGF-I) is a GH-dependent peptide regulating mammalian growth that seems to be of importance for the normal development and function of the immune system. Polymorphonuclear neutrophilic leukocytes (PMNLs) are terminally differentiated phagocytes essential for host defense, and in the present study, recombinant human IGF-I was shown to be a powerful primer of mature human PMNLs. IGF-I augmented the PMNL phagocytosis of both immunoglobulin G-opsonized Staphylococcus aureus and complement-opsonized Candida albicans. In addition, the growth factor increased PMNL complement receptor expression [complement receptors 1 (CD35) and 3 (CD11b)] and primed the cells to stronger f-met-leu-phe-induced degranulation of both specific and azurophilic granules [markers: CD11b, CD35 and CD67 (specific granules); CD63 (azurophilic granules)]. In contrast, IGF-I did not alter the PMNL surface expression of Fc gamma RI (CD64), Fc gamma RII (CDw32), or Fc gamma RIII (CD16). PMNLs exposed to IGF-I increased their f-met-leu-phe and phorbol myristate acetate-induced oxidative burst, as evaluated by hydrogen peroxide production, whereas IGF-I did not influence PMNL actin polymerization. The priming of PMNLs by IGF-I was dependent on time and concentration, and saturating amounts of a monoclonal antibody to the IGF-I receptor blocked the priming of PMNLs by this peptide. These experiments demonstrate that IGF-I can selectively stimulate mature PMNL functions, providing further evidence for the interaction between the immune and the endocrine systems.

The frequency of bacteremia and fungemia following wound cleaning and excision in patients with large burns.

Twenty-eight patients with large burn injuries (mean total body surface area burned, 47.1%) who underwent 112 wound cleaning and staged early excision procedures were studied prospectively for bacteremia and fungemia induced by wound manipulation. The patients were given an aminoglycoside preoperatively, perioperatively, and postoperatively. Blood samples were obtained immediately before removal of dressings, after wound cleaning, after 30 minutes of surgery, at the end of each procedure, and 1 hour after surgery was completed. In a group of control patients blood samples were obtained immediately before the dressings were removed, after wound cleaning was completed, and 1 hour afterward. In a second group of control patients blood samples were obtained serially while the burn wounds remained undisturbed. None of the control patients received prophylactic aminoglycosides. Induced bacteremia or fungemia was documented in 50 instances of burn wound cleaning and excision; 31 cases of bacteremia or fungemia occurred after wound cleaning alone. Spontaneous bacteremia, i.e., that occurring in the absence of burn wound manipulation, was demonstrated in 3 of 18 blood culture series, whereas induced bacteremia was observed after 11 of the 17 burn wound cleaning procedures alone. The frequency of bacteremia tended to be higher for patients with inhalation injury than for those with no lung involvement. However, lung infection did not seem to account for many of the positive results of blood culture in this study. In contrast, burn wound infection contributed significantly to both spontaneous and induced bacteremia or fungemia. The micro-organisms most frequently isolated were staphylococci (50%) and enterococci (15.1%).(ABSTRACT TRUNCATED AT 250 WORDS)

Early cell kinetic effects of a single dose of narrow-banded ultraviolet B irradiation on the rat corneal epithelium.

The right eyes of 40 rats were exposed to a single erythemogenic dose of ultraviolet B irradiation (UVB) at 297 nm. The irradiation was directed perpendicular to the center of the cornea. The left eyes served as controls. The animals were randomly assigned into 10 groups. The labeling index (LI) after pulse labeling with tritiated thymidine and the mitotic rate (MR) after Colcemid administration were registered in the corneal epithelium at predetermined intervals up to 96 h after the irradiation. A mathematical method was used to correlate corresponding corneal areas from the different animals. In the central cornea the LI was considerably reduced up to 36 h after the irradiation. The LI increased toward the peripheral cornea and reached normal values at the limbal area. The MR was also reduced up to 36 h. However, this reduction was over the entire epithelium. The block in cell proliferation was followed by increased proliferation.

Cell kinetics of normal and healing rat corneal epithelium during organ culture.

Local epithelial proliferation in corneal, limbal, and conjunctival rat epithelium was investigated during organ culture of corneo-scleral shells, by relating the number of tritium labelled cells to the total number of cells (labelling index). The changes in labelling index after a mechanically induced central corneal abrasion were also studied. Local labelling indices were measured 2 h, 1, 2, 4 and 7 days after incubation. The average labelling index increased in un-abraded specimens 4-fold after 4 days of incubation, while the total number of cells only decreased 30-50%. In eyes with a central corneal abrasion the labelling index increased 10-fold in the surrounding epithelial cells after 1 day. The labelling index of the limbal and conjunctival epithelial cells increased to the same extent after 2 days of incubation. The finding of a reduced total number of cells and an increased cell proliferation in un-abraded eyes may be explained by increased loss of mature cells normally producing growth-inhibitory substances (chalones). Alternatively, the culture medium may contain an excess of growth-stimulatory substances. The results also suggest that the proliferative response spreads in a centrifugal direction during in vitro healing of a central corneal abrasion.

Cell kinetics of the rat corneal epithelium.

In the rat corneal epithelium the mitotic rate (MR) is almost equal throughout the epithelium in the morning (Haaskjold et al. 1988). The labelling index (LI) shows a marked reduction in the central cornea, which could suggest a lack of uptake of tritiated thymidine via the salvage pathway (Haaskjold et al. 1989). In the present study we have used [3H]deoxycytidine, and [3H]thymidine after prior treatment with a methotrexate regimen to elucidate this discrepancy. Deoxycytidine is incorporated into DNA independent of thymidine kinase, while methotrexate, which depletes the cells of reduced folates, makes the cells completely dependent on the salvage pathway. With both techniques the same pattern of labelling was observed, confirming that in the morning the ratio between the MR and the LI differs throughout the cornea. Based on previous observations, an analysis of the MR/LI ratio during 24 h was performed, showing that these parameters were strongly correlated. This suggests that there may be different circadian variations in the cell proliferation parameters throughout the corneal epithelium. The methotrexate regimen may be a useful tool to investigate the salvage pathway.

The early cell kinetic response during healing of corneal epithelial wounds.

The labeling index and the mitotic rate were measured at 4-hour intervals during the first 24 hours after a central abrasion had been made in the corneal epithelium of six groups of four rats each. The proliferative response was noted in the conjunctival, the limbal, and the corneal epithelium. After 24 hours, the density of epithelial cells was equal throughout the corneal epithelium, but there was only half the normal number of cells. Physiological mechanisms seem strongly to regulate the total number of cells per square unit throughout the healing corneal epithelium, but the nature of these mechanisms is unknown.

Colony-forming unit-granulocyte-macrophage and DNA synthesis of human bone marrow are circadian stage-dependent and show covariation.

Bone marrow samples from sternum and iliac crests were harvested every 4 hours during 19 24-hour periods from 16 healthy male volunteers, and myeloid progenitor cells were cultured by the colony-forming unit-granulocyte-macrophage (CFU-GM) assay. A large interindividual variation was observed in the mean number of colonies during each 24-hour period, with the highest 24-hour mean colony number being about 600% greater than the lowest (range: 16 +/- 2.3 to 100.3 +/- 4.5). For each individual the difference between the lowest and highest colony number throughout the day ranged from 47.4% to 256.3% of the mean colony number of each series. A circadian stage-dependent variation in the number of colony-forming units of myeloid progenitor cells (CFU-GM) of human bone marrow was demonstrated, with values 150% higher, on the average, during the day as compared with the night. The overall data (891 CFU-GM replicates) exhibited a significant 24-hour rhythm (P less than .001) with an acrophase at midday (12.09 hours with 95% confidence limits from 10.32 to 13.49 hours) and a trough at midnight. This 24-hour variation was found to covary with DNA synthesis in the total proliferating bone marrow cell population. A seasonal effect on CFU-GM numbers was detected by ANOVA (P = .014) and by the least squares fit of a 1-year cosine (P = .015), with the highest number found in summer. The potential relevance of these findings should be examined in relation to cytotoxic cancer therapy, use of hematopoietic growth factors, and bone marrow transplantation.

DNA synthesis in human bone marrow is circadian stage dependent.

Fraction of human bone marrow (BM) cells in DNA synthesis has been studied by sampling BM from the sternum or the iliac crests every 4 hours during one 24-hour period in 16 healthy male volunteers. Three of the subjects underwent the sampling procedure twice, resulting in 19 24-hour profiles. The percentage of cells in DNA synthesis measured by flow cytometry demonstrated a large variation along the circadian time scale for each 24-hour profile, with a range of variation from 29% to 339% from lowest to highest value. Seventeen profiles (89.5%) had the highest DNA synthesis during waking hours between 08:00 hours and 20:00 hours, and the lowest percentage of cells in DNA synthesis between 00:00 hours and 04:00 hours. The mean value of the lowest DNA synthesis for each 19 24-hour period was 8.7% +/- 0.6%, while the mean value of the highest DNA synthesis was 17.6% +/- 0.6%, ie, a twofold difference. There was no difference in DNA synthesis between winter and summer. A significantly higher DNA synthesis was demonstrated for samples obtained from sternum as compared with the iliac crests, but the same circadian pattern was demonstrated for both localizations. By taking circadian stage-dependent variations in DNA synthesis into account it may be possible to reduce BM sensitivity to cytotoxic chemotherapy, to increase the effect of hematopoietic growth factors as well as increase the fraction of proliferating cells with careful selection of time of day for harvesting BM cells for auto- or allografting.

The IgG FcRII and the PI-linked IgG FcRIII trigger cytoplasmic calcium fluxes independently in human granulocytes.

The aim of this study was to investigate signal transduction through the two Fc receptors for IgG on human granulocytes. Using flow cytometry and the calcium indicator Fluo-3, we measured changes in leucocyte cytoplasmic calcium concentrations following cross-linking of cellular Fc receptors with specific antibodies. Two different approaches were used in order to study the two Fc receptors independently of each other. One was to avoid the presence of IgG Fc fragments, capable of binding to both types of receptors. The other was to use leucocytes from a patient with paroxysmal nocturnal haemoglobinuria (PNH) deficient in granulocyte FcRIII. In contrast to earlier reports, both approaches showed that the two types of IgG Fc receptors on granulocytes are capable of increasing cytoplasmic free calcium concentrations independently of each other. The results suggest that free cytoplasmic calcium ions are involved in the signal transduction pathway of both types of IgG Fc receptors on human granulocytes.

Circadian variation in cell proliferation and maturation. A hypothesis for the growth regulation of the rat corneal epithelium.

The rat corneal epithelium has been chosen as a model for studying growth regulation. In this epithelium a large single cohort of cells enters the S phase during a fairly short time period once a day. The factor responsible for this wave of cell proliferation is unknown, but it may be a chemical signal from the central nervous system (the suprachiasmatic nucleus or the corpus pineale). The mature cell compartment of the corneal epithelium is assumed to produce a negative feedback factor (chalone), counteracting the effect of the circadian proliferative factor on the local cell proliferation. When no circadian factor is being produced, during most of the 24 h, the chalone seems to enhance the maturation process. During diminished chalone production (e.g. after cell injury and subsequent regeneration), we will get a more or less unrestricted cell proliferation in the tissue with a delayed maturation process prolonging the chalone depletion. This interaction between the circadian proliferative factor and the negative feedback factor for regulation of proliferation with its accompanying stimulatory effect on maturation, may represent a general mechanism in the regulation of cell proliferation in any tissue. Since in at least some organs virtually all cells entering the S phase do this as a single wave once a day, this mechanism may be enough to explain the regulation of cell proliferation during both normal and regenerative conditions.