Management Approaches in WHO Grade III Meningioma: A National Oncology Trainees' Collaborative for Healthcare Research (NOTCH) UK Multi-Centre Retrospective Study.

AIMS: WHO Grade 3 (G3) meningiomas are rare tumours with limited data to guide management. This retrospective study documents UK management approaches across 14 centres over 11 years. MATERIALS AND METHODS: Patients with WHO G3 meningioma between 01/01/2008 and 31/12/2018 were identified. Data were collected on demographics, management strategy, adjuvant radiotherapy, approach in recurrence setting and survival. RESULTS: 84 patients were identified. 21.4% transformed from lower-grade disease. 96.4% underwent primary surgical resection, with 20.8% having evidence of residual disease on their post-op MRI. 59.3% of patients underwent adjuvant radiotherapy (RT) following surgical resection. Overall median PFS and OS were 12.6 months and 28.2 months, respectively. Median OS in the group who underwent complete surgical resection was 34.9 months, compared to 27.5 months for those who had incomplete resection (HR 0.58, 95% CI 0.27-1.23, p = 0.15). Median OS was 33.1 months for those who underwent adjuvant RT and 14.0 months for those who did not (HR 0.48, 95% CI 0.27-0.84, p = 0.004). Median adjuvant RT dose delivered was 60Gy (range 12Gy-60Gy), 45.8% of adjuvant RT was delivered using IMRT. At disease relapse, 31% underwent salvage surgery and 29.3% underwent salvage RT. Of those treated with salvage RT, 64.7% were re-treats and all were treated with hypofractionated RT. CONCLUSION: Surgery continues to be the preferred primary management strategy. Post-operative MRI within 48 hours is indicated to assess presence of residual disease and guide further surgical options. Adjuvant radiotherapy plays an important part of the management paradigm in these patients with the data supporting an attached survival advantage. Further surgery and re-irradiation is an option in the disease recurrence setting with radiosurgery frequently utilised in this context.

Regression approaches in the test-negative study design for assessment of influenza vaccine effectiveness.

Influenza vaccination is the most practical means available for preventing influenza virus infection and is widely used in many countries. Because vaccine components and circulating strains frequently change, it is important to continually monitor vaccine effectiveness (VE). The test-negative design is frequently used to estimate VE. In this design, patients meeting the same clinical case definition are recruited and tested for influenza; those who test positive are the cases and those who test negative form the comparison group. When determining VE in these studies, the typical approach has been to use logistic regression, adjusting for potential confounders. Because vaccine coverage and influenza incidence change throughout the season, time is included among these confounders. While most studies use unconditional logistic regression, adjusting for time, an alternative approach is to use conditional logistic regression, matching on time. Here, we used simulation data to examine the potential for both regression approaches to permit accurate and robust estimates of VE. In situations where vaccine coverage changed during the influenza season, the conditional model and unconditional models adjusting for categorical week and using a spline function for week provided more accurate estimates. We illustrated the two approaches on data from a test-negative study of influenza VE against hospitalization in children in Hong Kong which resulted in the conditional logistic regression model providing the best fit to the data.

Light echoes reveal an unexpectedly cool η Carinae during its nineteenth-century Great Eruption.

η Carinae is one of the most massive binary stars in the Milky Way. It became the second-brightest star in our sky during its mid-nineteenth-century 'Great Eruption', but then faded from view (with only naked-eye estimates of brightness). Its eruption is unique in that it exceeded the Eddington luminosity limit for ten years. Because it is only 2.3 kiloparsecs away, spatially resolved studies of the nebula have constrained the ejected mass and velocity, indicating that during its nineteenth-century eruption, η Car ejected more than ten solar masses in an event that released ten per cent of the energy of a typical core-collapse supernova, without destroying the star. Here we report observations of light echoes of η Carinae from the 1838-1858 Great Eruption. Spectra of these light echoes show only absorption lines, which are blueshifted by -210 km s(-1), in good agreement with predicted expansion speeds. The light-echo spectra correlate best with those of G2-to-G5 supergiants, which have effective temperatures of around 5,000 kelvin. In contrast to the class of extragalactic outbursts assumed to be analogues of the Great Eruption of η Carinae, the effective temperature of its outburst is significantly lower than that allowed by standard opaque wind models. This indicates that other physical mechanisms such as an energetic blast wave may have triggered and influenced the eruption.

Increased maternofetal calcium flux in parathyroid hormone-related protein-null mice.

The role of parathyroid hormone-related protein (PTHrP) in fetal calcium homeostasis and placental calcium transport was examined in mice homozygous for the deletion of the PTHrP gene (PTHrP-/- null; NL) compared to PTHrP+/+ (wild-type; WT) and PTHrP+/- (heterozygous; HZ) littermates. Fetal blood ionized calcium was significantly reduced in NL fetuses compared to WT and HZ groups at 18 days of pregnancy (dp) with abolition of the fetomaternal calcium gradient. In situ placental perfusion of the umbilical circulation at 18 dp was used to measure unidirectional clearance of (45)Ca across the placenta in maternofetal ((Ca)K(mf)) and fetoplacental ((Ca)K(fp)) directions; (Ca)K(fp) was < 5% of (Ca)K(mf) for all genotypes. At 18 dp, (Ca)K(mf) across perfused placenta and intact placenta ((Ca)K(mf(intact))) were similar and concordant with net calcium accretion rates in vivo. (Ca)K(mf) was significantly raised in NL fetuses compared to WT and HZ littermates. Calcium accretion was significantly elevated in NL fetuses by 19 dp. Placental calbindin-D(9K) expression in NL fetuses was marginally enhanced (P < 0.07) but expression of TRPV6/ECaC2 and plasma membrane Ca2+-ATPase (PMCA) isoforms 1 and 4 were unaltered. We conclude that PTHrP is an important regulator of fetal calcium homeostasis with its predominant effect being on unidirectional maternofetal transfer, probably mediated by modifying placental calbindin-D(9K) expression. In situ perfusion of mouse placenta is a robust methodology for allowing detailed dissection of placental transfer mechanisms in genetically modified mice.

Effect of acute restraint on hypothalamic pro-vasotocin mRNA expression in flounder, Platichthys flesus.

Arginine vasotocin (AVT) stimulates release of adenocorticotrophin hormone (ACTH) in trout. However, AVT's role in fish hypothalamic-pituitary-interrenal-axis (HPIA) is not fully understood. Here, we examined distribution of AVT and glucocorticoid receptor (GR) in the magnocellular preoptic nucleus (PM) and the AVT/cortisol response to acute restraint in flounder. The GR/AVT distribution in the PM was determined using double immunohistochemistry (IHC). Flounder were confined in nets, immersed in water for 30m, with plasma and tissue samples taken prior to, 3, 24 and 48h post-confinement. Plasma osmolality, Na(+), Cl(-) and cortisol were taken as indicators of HPIA activation. Plasma AVT was measured proVT mRNA expression in the PM was detected using in situ hybridisation (ISH) with a S35 labelled oligoprobe for homologous flounder proVT. Double IHC showed the presence of GR in AVT synthesising neurones of the PM. Plasma Na(+), Cl(-), osmolality and cortisol (1.0+/-0.9 to 183.6+/-3.1mM; p<0.001) increased significantly 3h post-restraint: recovering to control levels after 48h. Plasma AVT levels did not change. However, a concomitant increase in proVT mRNA expression in the magnocellular (PMm) and gigantocellular (PMg) neurones of the PM was observed (11.1+/-1.8 to 55.2+/-9.1% 24h post-restraint; p<0.001) and levels still remained significantly elevated at 48h (p<0.01). This suggests that PMm and PMg AVT neurones are associated with HPIA activation following acute restraint, including potential cortisol negative feedback. The extended elevation of hypothalamus proAVT mRNA expression following a single acute stressor affords a possible mechanism to moderate sensitivity of the HPIA to subsequent challenges.

Artificial perfusion of the fetal circulation of the in situ mouse placenta: methodology and validation.

Here we present methodology and validation (including measurement of unidirectional maternofetal clearance (Kmf) of (45)Ca and (14)C-mannitol) for in situ perfusion of the mouse placenta. On day 18 of gestation (term=19 days) mice were anaesthetised and the uterus delivered into a saline bath (40 degrees C). A fetus was selected, the umbilical artery and vein catheterised and perfused with Krebs Ringer (pH 7.4) at 60 microl/min. (45)Ca/(14)C-mannitol (2 microCi/5 microCi in 50 microl saline) was injected via maternal tail vein. Perfusate samples were collected every 5 min for 45 min. Maternal carotid artery pressure was monitored throughout perfusion. A terminal maternal cardiac blood sample was taken and analysed. Placentas were immersion fixed and processed for electron microscopy. Kmf for (45)Ca and (14)C-mannitol was calculated as perfusate [(45)Ca or (14)C-mannitol] x perfusion rate/maternal plasma [(45)Ca or (14)C-mannitol]xplacental weight. Maternal cardiac blood chemistry at termination (n=8-15, mean+/-SEM) was as follows: pH 7.153+/-0.016, PCO(2) 45.48+/-2.06 mmHg, PO(2) 66.47+/-7.10 mmHg, Na(+) 151.4+/-1.2 mmol/l, K(+) 5.54+/-0.17 mmol/l, Ca(2+) 1.15+/-0.03 mmol/l, glucose 7.2+/-0.5 mmol/l, and lactate 1.76+/-0.77 mmol/l. A successful 45 min perfusion in which perfusate recovery was >95% occurred in >50% of animals. Perfusion did not alter placental morphology or carotid pressure. Kmf (microl/min/g placenta) for (45)Ca (66.0+/-8.4 (n=7)) was significantly higher than Kmf for (14)C-mannitol (20.0+/-2.4 (n=5)) (p<0.01). These data demonstrate physiological perfusion of the mouse placenta in situ and its usefulness for measurement of solute transfer.

Altered plasma and pituitary arginine vasotocin and hypothalamic provasotocin expression in flounder (Platichthys flesus) following hypertonic challenge and distribution of vasotocin receptors within the kidney.

Plasma AVT concentration, pituitary AVT content, hypothalamic provasotocin mRNA expression and other osmoregulatory parameters were measured in euryhaline flounder 4, 8, and 24 h after the hypertonic challenge of transfer from fresh water (FW) to seawater (SW). Osmolality and the concentration of major plasma ions, sodium and chloride, were significantly higher in fish transferred to SW by comparison with time matched controls, an effect evident within 4 h. By comparison with time matched controls, pituitary store of AVT was lower while plasma AVT concentration was higher 8 and 24 h after transfer to SW. Higher provasotocin mRNA expression in the hypothalamus was also seen at 4 and 8 h in flounder transferred from FW to SW compared with time matched controls. The lower pituitary store and higher circulating levels imply substantial AVT secretion occurs in the early phase response to this hypertonic challenge. Changes in the regulation of AVT synthesis and secretion appeared quickly following movement to SW, consistent with the rapid osmoregulatory response, including reduced urine production that fish require to accommodate the dehydrative water losses and salt loading on exposure to the new hyperosmotic environment. qPCR measures of whole kidney vasotocin receptor mRNA expression indicated similar levels in SW and FW. Immunohistochemistry for the vasotocin receptor in flounder kidney showed localisation on the afferent and efferent arterioles of the glomerulus and on the capillary bed that extends from the efferent arteriole to the smooth muscle surrounding the collecting duct. Localisation of the vasotocin receptor was comparable in SW and FW fish.

Diabetes in rat pregnancy alters renal calcium and magnesium reabsorption and bone formation in adult offspring.

AIMS/HYPOTHESIS: We tested the hypothesis that diabetes in pregnancy can result in the in-utero reprogramming of renal calcium and magnesium handling and of bone formation in the offspring, which persists into adulthood. METHODS: Male offspring of streptozotocin-treated diabetic rats (OD rats) and of control non-diabetic animals (OC rats) were investigated as neonates and at 8, 12 and 16 weeks of age. RESULTS: Compared with OC rats, urinary calcium and magnesium output was significantly reduced in OD rats at every age studied; Na+ and K+ outputs were unaffected. The renal expression of proteins involved in the tubular reabsorption of calcium (calcium ATPase, calbindin-D28k and epithelial calcium channel) was increased in OD animals compared with that in OC animals. Additionally, we observed that adult OD rats had lower trabecular and higher cortical femoral bone volumes, explained by deposition of bone on the endosteal surface. CONCLUSIONS/INTERPRETATION: These data show that diabetes in pregnancy has profound effects on male offspring in terms of renal tubular calcium and magnesium reabsorption and the normal pattern of bone formation. These effects persist into adulthood. Such long-lasting effects of diabetes on kidney and the skeleton were not suspected and could have important implications for the health of children born to diabetic women.

Plasma concentrations of arginine vasotocin and urotensin II are reduced following transfer of the euryhaline flounder (Platichthys flesus) from seawater to fresh water.

Plasma concentrations and stored levels of the neuroendocrine peptides arginine vasotocin (AVT) and urotensin II (UII) were measured in the euryhaline flounder (Platichthys flesus) following the acute hypo-osmotic challenge of direct seawater (SW) to fresh water (FW) transfer. Hormone measures, plasma osmolality, and ion concentrations and tissue water content were determined 1, 4, 8, 24, 72, and 144 h after transfer. Plasma AVT concentration fell initially following FW transfer but then returned toward pretransfer levels by day 6. Plasma UII concentration decreased while urophysial UII content was increased following hypo-osmotic challenge relative to SW time-matched controls, suggesting down regulation of the UII system during the initial stages after FW transfer. These changes in neuroendocrine activity were associated with a significant fall in plasma osmolality and major plasma ions. Positive correlations were observed between plasma AVT and osmolality and Cl- and Mg2+ concentrations, suggesting functional association of these plasma parameters with AVT action and/or control of AVT secretion. The initial response to hypotonic challenge involves reduced plasma AVT and UII levels consistent with the proposed role for these hormones, supporting flounder osmoregulation in hypertonic media.

The caudal neurosecretory system: control and function of a novel neuroendocrine system in fish.

The caudal neurosecretory system (CNSS) of fish was first defined over 70 years ago yet despite much investigation, a clear physiological role has yet to be elucidated. Although the CNSS structure is as yet thought to be confined to piscine species, the secreted peptides, urotensins I and II (UI and UII), have been detected in a number of vertebrate species, most recently illustrated by the isolation of UII in humans. The apparent importance of these peptides, suggested by their relative phylogenetic conservation, is further supported by the complex control mechanisms associated with their secretion. The CNSS in teleosts is known to receive extensive and diverse innervation from the higher central nervous system, with evidence for the presence of cholinergic, noradrenergic, serotonergic, and peptidergic descending inputs. Recent observations also suggest the presence of glucocorticoid receptors in the flounder CNSS, supporting previous evidence for a possible role as a pituitary-independent mechanism controlling cortisol secretion. The most convincing evidence as to a physiological role for the CNSS in fish has stemmed from the direct and indirect influence of the urotensins on osmoregulatory function. Recent advances allowing the measurement of circulating levels of UII in the flounder have supported this. In addition, there is evidence to suggest some seasonal variation in peptide levels supporting the notion that the CNSS may have an integrative role in the control of coordinated changes in the reproductive, osmoregulatory and nutritional systems of migratory euryhaline species.

Regulation of cell survival in CD95-induced T cell apoptosis: role of NF-kappa B/Rel transcription factors.

The activity of NF-kappa B/Rel transcription factors can inhibit the apoptosis induced by TNF, UV or cancer therapy drugs in a number of cell types, including human T lymphocytes. Furthermore, the NF-kappa B/Rel inducer, phorbol-12-myristate-13-acetate (PMA), has been reported to suppress the CD95-induced apoptosis of human T lymphocytes. To verify whether the survival-enhancing effect of PMA required NF-kappa B/Rel activity, we generated two Jurkat cell sublines (AL.7 and AL.8) transfected with a pCMV4-I kappa B alpha construct, and two (AL.3 and AL.5) with the void pCMV4 vector. Compared to wild type, AL.3 and AL.5 cells, the AL.7 and AL.8 sublines displayed markedly lower amounts of NF-kappa B/Rel nuclear complexes and a reduced expression of a kappa B-controlled CAT reporter gene after 1 and 4 h of incubation with PMA, respectively. All the five cell types displayed negligible levels of apoptosis when cultured with medium or PMA alone; when stimulated with the mAb CH-11, the AL.7 and AL.8 sublines displayed apoptotic responses only slightly (<0.5 fold) higher than control cells. On the other hand, the salvage activity of PMA was partially impaired in the AL.7 and AL.8 sublines. PMA inhibited apoptosis by >85% in wild type, AL.3 and AL.5 cells and by <60% in the AL.7 and AL.8 sublines; the apoptosis percentages in the mAb CH-11 + PMA cultures of the I kappa B alpha-transfected cells were >4-fold higher than in control cells. We conclude that the inhibition of the CD95-induced apoptosis by PMA relies on both NF-kappa B/Rel-dependent and -independent mechanisms. The partial contribution of these nuclear factors to the suppression of apoptosis indicates that the NF-kappa B/Rel activity can influence the extent of the CD95-induced T cell death.

Safety and effectiveness of nurse telephone consultation in out of hours primary care: randomised controlled trial. The South Wiltshire Out of Hours Project (SWOOP) Group.

OBJECTIVE: To determine the safety and effectiveness of nurse telephone consultation in out of hours primary care by investigating adverse events and the management of calls. DESIGN: Block randomised controlled trial over a year of 156 matched pairs of days and weekends in 26 blocks. One of each matched pair was randomised to receive the intervention. SETTING: One 55 member general practice cooperative serving 97 000 registered patients in Wiltshire. SUBJECTS: All patients contacting the out of hours service or about whom contact was made during specified times over the trial year. INTERVENTION: A nurse telephone consultation service integrated within a general practice cooperative. The out of hours period was 615 pm to 1115 pm from Monday to Friday, 1100 am to 1115 pm on Saturday, and 800 am to 1115 pm on Sunday. Experienced and specially trained nurses received, assessed, and managed calls from patients or their carers. Management options included telephone advice; referral to the general practitioner on duty (for telephone advice, an appointment at a primary care centre, or a home visit); referral to the emergency service or advice to attend accident and emergency. Calls were managed with the help of decision support software. MAIN OUTCOME MEASURES: Deaths within seven days of a contact with the out of hours service; emergency hospital admissions within 24 hours and within three days of contact; attendance at accident and emergency within three days of a contact; number and management of calls in each arm of the trial. RESULTS: 14 492 calls were received during the specified times in the trial year (7308 in the control arm and 7184 in the intervention arm) concerning 10 134 patients (10.4% of the registered population). There were no substantial differences in the age and sex of patients in the intervention and control groups, though male patients were underrepresented overall. Reasons for calling the service were consistent with previous studies. Nurses managed 49.8% of calls during intervention periods without referral to a general practitioner. A 69% reduction in telephone advice from a general practitioner, together with a 38% reduction in patient attendance at primary care centres and a 23% reduction in home visits was observed during intervention periods. Statistical equivalence was observed in the number of deaths within seven days, in the number of emergency hospital admissions, and in the number of attendances at accident and emergency departments. Conclusions Nurse telephone consultation produced substantial changes in call management, reducing overall workload of general practitioners by 50% while allowing callers faster access to health information and advice. It was not associated with an increase in the number of adverse events. This model of out of hours primary care is safe and effective.

CD36 is rapidly and transiently upregulated on phytohemagglutinin (PHA)-stimulated peripheral blood lymphocytes. Analysis by a new monoclonal antibody (UN7).

The monoclonal antibody (mAb) UN7, clustered as an anti-CD36 mAb, has been used to test the cell surface expression of CD36 on peripheral blood lymphocytes (PBL) following mitogenic stimulation. CD36, scarcely expressed on resting cell membranes, was rapidly upregulated on PBL after phytohemagglutinin (PHA) stimulation. The antigen was detected on the cell surface after 15 min of stimulation, increased rapidly by 60 min and peaked between 3 and 12 h, declining thereafter. The inhibition of protein synthesis by cycloheximide did not modify the PHA-induced expression of CD36. Neither the anti-CD3 OKT3 mAb nor the anti-CD2 BIL 2.29 and 9.1 mAbs induced any significant upregulation of the molecule. The addition of anti-CD28 15E8 mAb or IL-2 or IFN-gamma to PHA or anti-CD3 or anti-CD2 mAbs did not influence the pattern of CD36 expression. The phorbol-2-myristate-13-acetate (PMA), alone or in combination with ionomycin, was unable to activate the expression of CD36, while it inhibited the PHA-induced upregulation. The PHA-induced upregulation of CD36 was partially inhibited by the addition of LY294002 or wortmannin, while not affected by that of calphostin C. Thus, CD36 was found to be early and transiently upregulated by PHA stimulation on PBL. The rapid modulation of the molecule was not related to new protein synthesis, but was probably due to the insertion into the plasma membrane of a presynthetized protein pool.

Purification and characterization of a human sialoglycoprotein antigen expressed in immature thymocytes and fetal tissues.

The monoclonal antibody UN1 was previously produced in our laboratory on the basis of selective reactivity with human thymocytes and has been classified as unclustered by the 5th and 6th International Workshop and Conference on Human Leukocyte Differentiation Antigens. The antigen recognized by mAb UN1 was found to be expressed on the cell surface of immature human thymocytes, a subpopulation of peripheral T lymphocytes and on several fetal tissues including thymus. The UN1 antigen is purified from children's thymus by ion-exchange and affinity chromatography. Two-dimensional electrophoresis shows that the purified antigen displays microheterogeneity appearing as multiple spots over a pI range 4.4-5.0 at 100-120 kDa. Treatment with neuraminidase results in a retarded migration in SDS-PAGE, an increase in isoelectric point and a reduction in carbohydrate content, indicating a substantial content of sialic acid. Glycosidase digestion and lectin-binding analysis indicate that the carbohydrate residues are essentially O-linked. A preliminary analysis has detected the UN1 antigen in human breast carcinoma tissues but not in normal breast. The biochemical features and the pattern of expression of the UN1 antigen indicate that this molecule may have the characteristics typical of the family of cell-membrane-associated mucin-like glycoproteins; a number of these molecules are thought to have a role in cell-cell interaction, tumor progression and metastasis.

A 5' regulatory sequence containing two Ets motifs controls the expression of the Wiskott-Aldrich syndrome protein (WASP) gene in human hematopoietic cells.

The recently-identified Wiskott-Aldrich syndrome protein gene (WASP) is responsible for the Wiskott-Aldrich X-linked immunodeficiency as well as for isolated X-linked thrombocytopenia (XLT). To characterize the regulatory sequences of the WASP gene, we have isolated, sequenced and functionally analyzed a 1.6-Kb DNA fragment upstream of the WASP coding sequence. Transfection experiments showed that this fragment is capable of directing efficient expression of the reporter chloramphenicol acetyltransferase (CAT) gene in all human hematopoietic cell lines tested. Progressive 5' deletions showed that the minimal sequence required for hematopoietic-specific expression consists of 137 bp upstream of the transcription start site. This contains potential binding sites for several hematopoietic transcription factors and, in particular, two Ets-1 consensus that proved able to specifically bind to proteins present in nuclear extracts of Jurkat cells. Overexpression of Ets-1 in HeLa resulted in transactivation of the CAT reporter gene under the control of WASP regulatory sequences. Disruption of the Ets-binding sequences by side-directed mutagenesis abolished CAT expression in Jurkat cells, indicating that transcription factors of the Ets family play a key role in the control of WASP transcription.

Identification by differential display of transcripts regulated during hematopoietic differentiation.

The polymerase chain reaction-based differential display method (DDRT-PCR) was used to identify mRNAs differentially expressed during the maturation of human CD34+ progenitor cells stimulated to differentiate in vitro towards granulomonocytic or erythroid lineages with a mixture of hemopoietins (kit ligand + interleukin 3 + GM-CSF in the absence or presence of erythropoietin, respectively). Three cDNA transcripts (B32, B41, and B56) display differential expression during cytokine-induced maturation of CD34+ cells. These clones have no homology with already-described sequences. Primer extension cofirmed the presence of the corresponding mRNA. The levels of mRNA corresponding to B32 are enhanced in the later phases of the granulomonocytic as well as in the erythroid differentiation of CD34+ cells. The mRNA identified by B41 was induced by a late stage in only granulomonocytic differentiation of CD34+ cells. The mRNA corresponding to B56 was instead present in nonstimulated CD34+ cells, declined in the early stages of differentiation, and reappeared at later stages in cells treated with both combinations of cytokines. Expression of these genes was detected in a number of acute myelogenous leukemias, as well as in some leukemic cell lines. B32 and B41 were downregulated in KG-1 cells induced to differentiate towards the monocytic lineage, whereas the levels of B56 were unchanged. In K562 cells, clones B41 and B56 were downregulated only in the late phases of PMA-induced megakaryocytic differentiation and during erythroid differentiation. B32 was rapidly downregulated when K562 cells were induced to differentiate towards either megakaryocytic or erythroid phenotypes. These transcripts represent novel hematopoietic cDNAs that should prove of value for the study of human blood cells and their disorders.

Expression of growth hormone receptor by peripheral blood lymphocytes in children: evaluation in clinical conditions of impaired growth.

OBJECTIVE: It is widely accepted that the haematopoietic system is a target of growth hormone action and that GH may act as a lymphokine. The expression of GH receptors (GHR) on human peripheral blood lymphocytes (PBL) has been reported previously in adult donors by dual fluorochrome flow cytometry. The aim of this study was to apply the cytofluorimetric method to the analysis of GHR expression on PBL in various human conditions characterized by different patterns of growth due to age or physiopathological conditions. SUBJECTS AND DESIGN: PBL from 38 normal (control) subjects (7 newborns, 18 prepubertal children, 13 adults) were studied in order to provide age-related physiological data. Twenty-two short children (18 with idiopathic short stature, 4 with Ullrich-Turner syndrome) were studied to determine the expression of GHR in conditions of impaired longitudinal growth which may or may not require GH treatment. METHODS: Analysis was performed using a fluorescein isothiocyanate (FITC)-conjugated antibody specific for the GHR (mAb263) and phycoerythrin (PE)-anti CD2 (T and natural killer cells) or PE-anti CD2 (B cells) in dual fluorochrome flow cytometric assays. Results were expressed as mean fluorescent intensity (MFI). RESULTS: Adult CD2+ coils exhibited a significantly higher GHR expression (MFI 347 +/- 40) than that expressed in children and newborns (MFI 285 +/- 36 and 299 +/- 41, respectively, P < 0.001). A significantly increased expression of GHR on CD2+ cells was also found in short children (MFI 330 +/- 42 vs 285 42- 36, respectively; P < 0.002), whereas Ullrich-Turner syndrome patients did not show any difference from their age and gender matched controls (254 +/- 52 and 288 +/- 40, respectively). A negative relationship was found between GHR expression on CD2+ cells and height-SDS (r - 0.54, P < 0.0001) or BMI (r - 0.4, P < 0.015) in controls and short children, independent of their GH secretory status. Expression of GHR and CD20+ cells was higher than that expressed on CD2+ cells in all subjects. No appreciable differences were found in the MFI levels of GHR expression on CD20+ cells either among the different age group controls or between short children or Ullrich-Turner syndrome patients. A significant downregulation of expression was shown in CD20+ (P < 0.008) but not CD2+ cells after 6 months of GH treatment in 6 short children who had a poor response to GH provocative tests. CONCLUSIONS: GH receptor expression on immune cells in non-syndromic short children appears to be inversely related to the linear growth expression and BMI of the subjects, contrary to findings with hepatic derived serum GHBP. This finding may reflect alternate exon usage in lymphoid cells, and indicates that GH has a distinctive role in the immune system.

CD69 expression on primitive progenitor cells and hematopoietic malignancies.

CD69 is an early activation antigen of peripheral blood lymphocytes and is constitutively expressed on a wide variety of bone marrow-derived cells. To further characterize the distribution and understand the potential biological role of the molecule in normal and malignant hematopoiesis, we used a novel high affinity anti-CD69 mAb (UN6) and analyzed hematopoietic progenitor cells together with a panel of myeloid and lymphoid malignancies. We report that mobilized peripheral blood CD34+ cells display detectable levels of CD69 and that the density of membrane expression correlates with the immature phenotype CD34bright Thy-1bright cells. Furthermore, during cytokine-induced differentiation, the expression of CD69 is moderately down-regulated. Analysis of hematopoietic malignancies revealed that CD69 expression correlates with the immature myeloid phenotype. Taken together these data suggest a role of CD69 during the early phase of hematopoiesis and in the leukemic transformation.

Differential regulation of the expression of interleukin-2 receptor gamma-chain during the in vitro differentiation of human myeloid cells.

The common gamma-chain (gamma c) is a shared component of cell-surface receptors for the interleukins- 2, -4 and -7, and possibly others. We studied its expression in cells and cell lines of myeloid origin and found ubiquitous presence of gamma c mRNA in all cells examined. Differential regulation of gamma c expression was observed in myeloid cell lines induced to differentiate in vitro. In K-562 erythromyeloid cells, a sharp rise in the levels of gamma c mRNA and protein accompanied megakaryocytic, but not erythroid, differentiation. Surface binding of interleukin-2, as well as the transcripts for cognate receptor chains, were scarcely detectable in K-562 cells, whereas a significant increase in the binding of granulocyte-macrophage colony-stimulating factor specifically occurred during their megakaryocytic maturation. Our data indicate that expression of gamma c is a common feature of human myeloid cells, and suggest that its expression may be a requirement for human myelopoiesis.