Outbreak of adenovirus D8 in a neonatal intensive care unit involving multiple simultaneous transmission pathways.

BACKGROUND: Adenovirus (ADV) outbreaks in neonatal intensive care units (NICU) can lead to durable transmission and serious adverse outcomes. This study describes the investigation and control of an ADV-D8 outbreak in an NICU, associated with ophthalmologic equipment used during retinopathy of prematurity (ROP) screening. Cases were observed in neonates, parents and nurses. METHODS: The outbreak investigation was performed including sampling patients, parents and health care workers as well as the environment for molecular detection of ADV DNA. The investigation was also conducted in the guest house where some parents were temporary residents. A retrospective cohort study focused on neonates hospitalized during the epidemic period to assess the risk associated with ROP examination. RESULTS: Fifteen cases were identified in neonates; all but one presented with conjunctivitis. Two healthcare workers and 18 parents acquired conjunctivitis. ADV DNA was identified on the RetCam and on the freezer shared by parents. All ADV-positive samples were typed as ADV-D8. ADV infections occurred more frequently in neonates who had ROP examinations (37.8% (14/37) vs (0.9% (1/110); P<0.001) (relative risk 41.6; (5.7-305.8)). The RetCam was disinfected between two examinations using a disinfectant that was virucidal on ADV after a 30-min contact. CONCLUSION: This outbreak was significantly associated with ROP examination with a RetCam that had a disinfection protocol ill-adapted to rapid patient turnover. In addition, nosocomial transmission via the parents to neonates and parent-to-parent transmission is likely to have played a role in the dissemination of cases. No further cases were observed after the new disinfection procedure was enforced.

A venetoclax and azacitidine bridge-to-transplant strategy for NPM1-mutated acute myeloid leukaemia in molecular failure.

NPM1-mutated acute myeloid leukaemia (NPM1mut AML) represents a mostly favourable/intermediate risk disease that benefits from allogeneic haematopoietic stem cell transplantation (HSCT) in case of measurable residual disease (MRD) relapse or persistence after induction chemotherapy. Although the negative prognostic role of pre-HSCT MRD is established, no recommendations are available for the management of peri-transplant molecular failure (MF). Based on the efficacy data of venetoclax (VEN)-based treatment in NPM1mut AML older patients, we retrospectively analysed the off-label combination of VEN plus azacitidine (AZA) as bridge-to-transplant strategy in 11 NPM1mut MRD-positive fit AML patients. Patients were in MRD-positive complete remission (CRMRDpos ) at the time of treatment: nine in molecular relapse and two in molecular persistence. After a median number of two cycles (range 1-4) of VEN-AZA, 9/11 (81.8%) achieved CRMRD -negative (CRMRDneg ). All 11 patients proceeded to HSCT. With a median follow-up from treatment start of 26 months, and a median post-HSCT follow-up of 19 months, 10/11 patients are alive (1 died from non-relapse mortality), and 9/10 patients are in MRDneg status. This patient series highlights the efficacy and safety of VEN-AZA to prevent overt relapse, achieve deep responses and preserve patient fitness before HSCT, in patients with NPM1mut AML in MF.

Decreased muscle strength is associated with proinflammatory cytokines but not testosterone levels in men with diabetes.

The aim of this study was to compare muscle strength in male subjects with type 2 diabetes mellitus (DM2) with and without low plasma testosterone levels and assess the relationship between muscle strength, testosterone levels, and proinflammatory cytokines. Males (75) aged between 18 and 65 years were divided into 3 groups: control group that did not have diabetes and had a normal testosterone plasma level (>250 ng/dL), DnormalTT group that had DM2 with normal testosterone levels, and the DlowTT group that had DM2 and low plasma testosterone levels (<250 ng/dL). The age (means±SD) of the groups was 48.4±10, 52.6±7, and 54.6±7 years, respectively. Isokinetic concentric and isometric torque of knee flexors and extensors were analyzed by an isokinetic dynamometer. Plasma testosterone and proinflammatory cytokine levels were determined by chemiluminescence and ELISA, respectively. Glycemic control was analyzed by glycated hemoglobin (HbA1C). In general, concentric and isometric torques were lower and tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1ß plasma levels were higher in the groups with diabetes than in controls. There was no correlation between testosterone level and knee torques or proinflammatory cytokines. Concentric and isometric knee flexion and extension torque were negatively correlated with TNF-α, IL-6, and HbA1C. IL-6 and TNF-α were positively correlated with HbA1C. The results of this study demonstrated that muscle strength was not associated with testosterone levels in men with DM2. Low muscle strength was associated with inflammatory markers and poor glycemic control.

Decreased muscle strength is associated with proinflammatory cytokines but not testosterone levels in men with diabetes

The aim of this study was to compare muscle strength in male subjects with type 2 diabetes mellitus (DM2) with and without low plasma testosterone levels and assess the relationship between muscle strength, testosterone levels, and proinflammatory cytokines. Males (75) aged between 18 and 65 years were divided into 3 groups: control group that did not have diabetes and had a normal testosterone plasma level (>250 ng/dL), DnormalTT group that had DM2 with normal testosterone levels, and the DlowTT group that had DM2 and low plasma testosterone levels (<250 ng/dL). The age (means±SD) of the groups was 48.4±10, 52.6±7, and 54.6±7 years, respectively. Isokinetic concentric and isometric torque of knee flexors and extensors were analyzed by an isokinetic dynamometer. Plasma testosterone and proinflammatory cytokine levels were determined by chemiluminescence and ELISA, respectively. Glycemic control was analyzed by glycated hemoglobin (HbA1C). In general, concentric and isometric torques were lower and tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1β plasma levels were higher in the groups with diabetes than in controls. There was no correlation between testosterone level and knee torques or proinflammatory cytokines. Concentric and isometric knee flexion and extension torque were negatively correlated with TNF-α, IL-6, and HbA1C. IL-6 and TNF-α were positively correlated with HbA1C. The results of this study demonstrated that muscle strength was not associated with testosterone levels in men with DM2. Low muscle strength was associated with inflammatory markers and poor glycemic control.

Pelvic myeloid sarcoma: a multidisciplinary approach.

Myeloid sarcoma (MS) is a rare tumor mass derived from the extramedullary proliferation of blasts of one or more of myeloid lineages. It usually occurs at an anatomical site other than the bone marrow (BM). Among the anatomical site which may be involved, female genital tract is a rare localization. When MS follows a previous history of myeloid pathology it is usually associated to a poor prognosis. To date this disease was managed with exploratory laparotomy or with surgical debulking. The authors report a case of laparosc6pic diagnosis of a pelvic myeloid sarcoma in a patient previously affected by acute mycloid leukemia, evidencing the importance of minimally invasive diagnosis and subsequent multidisciplinary management.

The association of specific traumatic experiences with cannabis initiation and transition to problem use: Differences between African-American and European-American women.

INTRODUCTION: To examine the contribution of trauma exposure to cannabis initiation and transition to first cannabis use disorder (CUD) symptom in African-American (AA) and European-American (EA) emerging adults. METHODS: Data are from the Missouri Adolescent Female Twins Study [(N=3787); 14.6% AA; mean age=21.7 (SD 3.8)]. Trauma exposures (e.g. sexual abuse, physical abuse, witnessing another person being killed or injured, experiencing an accident, and experiencing a disaster) were modeled as time-varying predictors of cannabis initiation and transition to CUD symptom using Cox proportional hazards regression. Other substance involvement and psychiatric disorders were considered as time-varying covariates. RESULTS: Analyses revealed different trauma-related and psychiatric predictors for cannabis use supporting racially distinct etiologic models of cannabis involvement. For AA women, history of witnessing injury/death or experiencing a life-threatening accident was associated with cannabis initiation across the complete emerging adult risk period while sexual abuse predicted cannabis initiation only before 15 years old. For EA women, history of sexual or physical abuse and major depressive disorder (MDD) predicted cannabis initiation and physical abuse and MDD predicted transition from initiation to first CUD symptom. No association was discovered between trauma exposures and transition to first CUD symptom in AA women. CONCLUSIONS: Results reveal trauma exposures as important contributors to cannabis initiation and to a lesser extent transition to CUD symptom, with different trauma types conferring risk for cannabis involvement in AA and EA women. Findings suggest the importance of considering racial/ethnic differences when developing etiologic models of cannabis involvement.

Cannabis or alcohol first? Differences by ethnicity and in risk for rapid progression to cannabis-related problems in women.

BACKGROUND: Initiation of cannabis use typically follows alcohol use, but the reverse order does occur and is more common for African-Americans (AAs) than European-Americans (EAs). The aim of this study was to test for differences in the order of initiation of cannabis and alcohol use between AA and EA women and to determine whether order and ethnicity contribute independently to risk for rapid progression to cannabis-related problems. Method Data were drawn from structured psychiatric interviews of 4102 women (mean age = 21.6 years), 3787 from an all-female twin study and 315 from a high-risk family study; 18.1% self-identified as AA, 81.9% as EA. Ethnicity and order of initiation of cannabis and alcohol use were modeled as predictors of transition time from first use to onset of cannabis use disorder symptom(s) using Cox proportional hazards regression analyses. RESULTS: AA women were nearly three times as likely as EA women to initiate cannabis use before alcohol use. Using cannabis before alcohol [hazard ratio (HR) 1.44, 95% confidence interval (CI) 1.08-1.93] and AA ethnicity (HR 1.59, 95% CI 1.13-2.24) were both associated with rapid progression from first use to cannabis symptom onset even after accounting for age at initiation and psychiatric risk factors. CONCLUSIONS: The findings indicate that AA women are at greater risk for rapid development of cannabis-related problems than EA women and that this risk is even higher when cannabis use is initiated before alcohol use. Prevention programs should be tailored to the various patterns of cannabis use and relative contributions of risk factors to the development of cannabis-related problems in different ethnic groups.

Effect of image resolution manipulation in rearfoot angle measurements obtained with photogrammetry

The aim of this study was to investigate the influence of image resolution manipulation on the photogrammetric measurement of the rearfoot static angle. The study design was that of a reliability study. We evaluated 19 healthy young adults (11 females and 8 males). The photographs were taken at 1536 pixels in the greatest dimension, resized into four different resolutions (1200, 768, 600, 384 pixels) and analyzed by three equally trained examiners on a 96-pixels per inch (ppi) screen. An experienced physiotherapist marked the anatomic landmarks of rearfoot static angles on two occasions within a 1-week interval. Three different examiners had marked angles on digital pictures. The systematic error and the smallest detectable difference were calculated from the angle values between the image resolutions and times of evaluation. Different resolutions were compared by analysis of variance. Inter- and intra-examiner reliability was calculated by intra-class correlation coefficients (ICC). The rearfoot static angles obtained by the examiners in each resolution were not different (P > 0.05); however, the higher the image resolution the better the inter-examiner reliability. The intra-examiner reliability (within a 1-week interval) was considered to be unacceptable for all image resolutions (ICC range: 0.08-0.52). The whole body image of an adult with a minimum size of 768 pixels analyzed on a 96-ppi screen can provide very good inter-examiner reliability for photogrammetric measurements of rearfoot static angles (ICC range: 0.85-0.92), although the intra-examiner reliability within each resolution was not acceptable. Therefore, this method is not a proper tool for follow-up evaluations of patients within a therapeutic protocol.

Avaliação das perdas sensório-motoras do pé e tornozelo decorrentes da neuropatia diabética / Assessment of motor sensory losses in the foot and ankle due to diabetic neuropathy

OBJETIVOS: Identificar déficits sensório-motores de pés de pacientes diabéticos neuropatas e comparar os déficits do grupo neuropata com um grupo de sujeitos saudáveis. MÉTODO: 49 diabéticos neuropatas (GD) e 22 controles foram submetidos a um protocolo de três estágios: (1) entrevista por meio de questionário, que caracterizou a neuropatia e sintomas, (2) avaliação da função muscular, amplitude de movimentos e testes funcionais dos pés e tornozelos, (3) avaliação da sensibilidade tátil e térmica. Os grupos foram comparados por meio dos testes Qui-quadrado, Mann-Withney e Teste T (p<0,05). RESULTADOS: O GD mostrou perda significativa das sensibilidades tátil e térmica em comparação ao grupo controle, principalmente nos calcanhares (49,0 por cento no GD e 97,3 por cento no GC). A função muscular está diminuída no GD, com predomínio da perda do grau 5. Os músculos mais afetados são os interósseos (23,4 por cento), extensor do hálux (42,5 por cento) e tríceps sural (43,2 por cento), enquanto que o GC teve todos os músculos preservados. Todas as ADMs do GD estão diminuídas em relação ao GC. O GD apresentou os testes funcionais de tornozelo diminuídos em 50 por cento. CONCLUSÃO: Houve diferenças significativas entre os grupos quanto às perdas sensitivas, de função muscular, amplitude de movimento e funcionais. Essas diferenças podem ser atribuídas à neuropatia diabética.

OBJECTIVE: To identify motor sensory deficits in the feet of neuropathic diabetic patients and compare their deficits with a group of healthy subjects. METHOD: 49 neuropathic diabetics (group NG) and 22 controls (group CG) underwent a three-stage protocol: (1) an interview using a questionnaire to characterize the neuropathy and symptoms; (2) assessment of muscle function and range of motion, and functional tests on the feet and ankles; (3) assessment of tactile and thermal sensitivity. The groups were compared using the chi-squared, Mann-Whitney and Student t tests (p<0.05). RESULTS: NG presented significant losses of tactile and thermal sensitivity in comparison with CG, especially in the heels (49.0 percent of NG and 97.3 percent of CG). Muscle function was decreased in NG, with predominance of loss of grade 5. The muscles most affected were the interossei (23.4 percent), extensor hallucis (42.5 percent) and triceps surae (43.2 percent), while all muscle function was preserved in CG. All ranges of motion in NG were reduced in comparison with CG. The functional tests on the ankles in NG presented a decrease of around 50 percent. CONCLUSION: There were significant differences between the groups with regard to sensitivity, muscle function, range of motion and functional losses. These differences can be attributed to the diabetic neuropathy.

Postmastectomy radiotherapy in women with breast cancer metastatic to one to three axillary lymph nodes.

The influence of postmastectomy radiotherapy on survival has long been debated. Early randomized trials established a clear role for adjuvant postmastectomy chest wall radiotherapy (PMCWRT) in reducing locoregional recurrence (LRR), and PMCWRT became standard therapy for patients at high risk of LRR: those with T3 or T4 tumors and four or more involved lymph nodes. However, without effective systemic therapy, distant metastases limited any effect of improved local control on overall outcome, and radiotherapy showed no benefit in survival. In fact, early meta-analyses showed a negative impact of radiotherapy on survival. As data and techniques matured, a favorable influence of PMCWRT on breast cancer-specific mortality emerged but was offset by a radiotherapy-related increase in vascular mortality. Improvements in radiotherapy delivery to increase efficacy and reduce toxicity, restriction of PMCWRT to patients at intermediate or high risk of LRR after mastectomy, and improved distant control of disease with systemic therapy are expected to bring the greatest likelihood of a survival advantage from locoregional control. Three randomized trials with sufficient follow-up meet these criteria. All demonstrate significant improvement in overall survival with PMCWRT. However, the trials were not designed to specifically address the benefit of PMCWRT in patients at intermediate risk of LRR (those with T1 or T2 tumors and one to three involved lymph nodes). These findings have been discussed in a host of publications and conferences in light of historical negative results. This review focuses on the recent data on PMCWRT in patients with one to three involved nodes.

Her4 mediates ligand-dependent antiproliferative and differentiation responses in human breast cancer cells.

The function of the epidermal growth factor receptor (EGFR) family member HER4 remains unclear because its activating ligand, heregulin, results in either proliferation or differentiation. This variable response may stem from the range of signals generated by HER4 homodimers versus heterodimeric complexes with other EGFR family members. The ratio of homo- and heterodimeric complexes may be influenced both by a cell's EGFR family member expression profile and by the ligand or even ligand isoform used. To define the role of HER4 in mediating antiproliferative and differentiation responses, human breast cancer cell lines were screened for responses to heregulin. Only cells that expressed HER4 exhibited heregulin-dependent antiproliferative responses. In-depth studies of one line, SUM44, demonstrated that the antiproliferative and differentiation responses correlated with HER4 activation and were abolished by stable expression of a kinase-inactive HER4. HB-EGF, a HER4-specific ligand in this EGFR-negative cell line, also induced an antiproliferative response. Moreover, introduction and stable expression of HER4 in HER4-negative SUM102 cells resulted in the acquisition of a heregulin-dependent antiproliferative response, associated with increases in markers of differentiation. The role of HER2 in these heregulin-dependent responses was examined through elimination of cell surface HER2 signaling by stable expression of a single-chain anti-HER2 antibody that sequestered HER2 in the endoplasmic reticulum. In the cell lines with either endogenously (SUM44) or exogenously (SUM102) expressed HER4, elimination of HER2 did not alter HER4-dependent decreases in cell growth. These results suggest that HER4 is both necessary and sufficient to trigger an antiproliferative response in human breast cancer cells.

Biological modifiers as potential radiosensitizers: targeting the epidermal growth factor receptor family.

The epidermal growth factor receptor family plays an important role in the pathogenesis of human epithelial tumors. Overexpression is associated with poor prognosis and resistance to therapy. Epidermal growth factor receptor family members activate signal transduction pathways that have been implicated in radioresistance, and inhibition of signal transduction pathways involved in epidermal growth factor receptor family member signaling causes radiosensitization. Recent encouraging results indicate that epidermal growth factor receptor family member inhibitors may be specific, effective radiosensitizers in tumors that overexpress one or more of these receptors.

Rate of PSA rise predicts metastatic versus local recurrence after definitive radiotherapy.

OBJECTIVE: A rising prostate specific antigen (PSA) following treatment for adenocarcinoma of the prostate indicates eventual clinical failure, but the rate of rise can be quite different from patient to patient, as can the pattern of clinical failure. We sought to determine whether the rate of PSA rise could differentiate future local versus metastatic failure. METHODS AND MATERIALS: Two thousand six hundred sixty-seven PSA values from 400 patients treated with radiotherapy for localized adenocarcinoma of the prostate were analyzed with respect to PSA patterns and clinical outcome. Patients had received no hormonal therapy or prostate surgery and had > 4 PSA values post-treatment. PSA rate of rise, determined by the slope of the natural log, was classified as gradual [< 0.69 log(ng/ml)/year, or doubling time (DT) > 1 year], moderate [0.69-1.4 log(ng/ml)/year, or DT 6 months-1 year], or rapid [> 1.4 log(ng/ml)/year, or DT < 6 months]. RESULTS: Sixty-one percent of patients had non-rising PSA following treatment; 25% of patients with rising PSA developed clinical failure, and 93% of patients with clinical failure had rising PSA. The rate of rise discerned different clinical failure patterns. Local failure occurred in 23% of patients with moderate rate of rise versus 7% with gradual rise (p = 0.0001). Metastatic disease developed in 46% of those with rapid rise versus 8% with moderate rise (p < 0.0001). By multivariate analysis, in addition to rate of rise, PSA nadir and rate of decline predicted local failure; those with post-treatment nadir of 1-4 ng/ml were five times more likely to experience local failure than nadir < 1 ng/ml (p = 0.0002). Rapid rate of rise was the most significant independent predictor of metastatic failure. CONCLUSIONS: The rate of PSA rise following definitive radiotherapy can predict clinical failure patterns, with a rapidly rising PSA indicating metastatic recurrence and moderately rising PSA local recurrence. This information could potentially direct therapy; if the rise predicts metastatic failure hormonal therapy could be considered, while aggressive salvage therapy may benefit subclinical local recurrence identified by a moderate rate of PSA rise.

Role of epidermal growth factor receptor and STAT-3 activation in autonomous proliferation of SUM-102PT human breast cancer cells.

This report describes the isolation and characterization of a new human breast cancer cell line, SUM-102PT, obtained from a minimally invasive human breast carcinoma. SUM-102PT cells have a near diploid karyotype, and early-passage cells had minor chromosomal abnormalities including a 5, 12 and a 6, 16 reciprocal translocation. The cells were isolated and have been continually cultured in three defined media, one of which contains exogenous epidermal growth factor (EGF). SUM-102PT cells have also been carried in an EGF-free medium supplemented with progesterone. All SUM-102PT cells require EGF receptor (EGFR) activation for continuous growth, because incubation of the cells with EGFR-neutralizing antibodies or with EGFR kinase inhibitors blocks growth of these cells. Southern analysis indicates that the EGFR gene is not amplified in these cells; however, these cells express high levels of EGFR mRNA. Thus, SUM-102PT is representative of a class of human breast cancers characterized by high level EGFR expression in the absence of gene amplification. SUM-102PT cells cultured in EGF-free, progesterone-containing medium express high levels of constitutively active EGFR. Conditioned medium from SUM-102PT cells contains an EGF-like mitogen that binds to a heparin-agarose affinity matrix with high affinity. Northern analysis for various EGF family members indicates that SUM-102PT cells synthesize heparin binding (HB)-EGF mRNA. HB-EGF protein is detectable on the surface of these cells by immunohistochemistry, and SUM-102PT cells are killed by diphtheria toxin, which acts by binding to HB-EGF. Furthermore, HB-EGF antibodies partially neutralize the mitogenic activity of the conditioned medium. Thus, EGFR activation in SUM-102PT cells is mediated, at least in part, by autocrine/juxtacrine stimulation by HB-EGF. SUM-102PT cells also express constitutively active STAT-3 homodimers. Constitutively tyrosine-phosphorylated STAT-3 homodimers were also detected in another breast cancer cell line, MDA468, which has an EGFR amplification and also has constitutive EGFR activity. Thus, SUM-102PT is a new human breast cancer cell line that expresses activated EGFR as a result of an autocrine/juxtacrine interaction with HB-EGF which, in turn, results in activation of STAT-3.

Prevalence surveys of nosocomial infections using a random sampling method in Marseille hospitals.

In 1992, we conducted two prevalence surveys on hospital-acquired infections (HAIs) in a group of eight university affiliated hospitals with a total of 4462 acute care beds. Most of the intensive care units (ICUs) already had a prospective surveillance scheme. The need for HAI rates that could be estimated easily and economically led us to develop a prevalence survey by using a stratified sampling method. The units were distributed into four groups: ICU; clean surgery; other surgery; and medical. In each group a sample of units was randomly selected using different sampling fractions. The first survey was conducted in May and involved 1220 patients. The second survey in November included 1389 patients. The HAI rates in May and November were 8.6% (95% CI 7.4-9.8%) and 7.1% (95% CI 6-8.2%), respectively. This study allowed us to direct resources towards high risk units and clean surgical procedures. Based on the results, prospective surveillance was implemented in units of the clean surgery group.

Granulocyte colony-stimulating factor (G-CSF) production and G-CSF receptor structure in patients with congenital neutropenia.

Congenital neutropenia (Kostmann's syndrome [KS]) is an autosomal recessive syndrome that is characterized by profound neutropenia, resulting in major clinical infections and death. Since the neutropenia and symptoms in KS improve in response to exogenous administration of granulocyte colony-stimulating factor (G-CSF), we studied bone marrow cytokine (G-CSF, granulocyte-macrophage CSF [GM-CSF], and interleukin-6) production under both basal and stimulated conditions. No differences in G-CSF, GM-CSF, or IL-6 gene expression were found in bone marrow stromal cells between normal controls and KS patients, and all three cytokines were detected by enzyme-linked immunosorbent assay (ELISA) in medium conditioned by bone marrow stromal cells from normal donors and patients with KS. Each KS patient tested had detectable, functional G-CSF in their own serum before exogenous G-CSF administration. Since G-CSF production appeared normal in KS patients, we then asked whether we could detect structural defects in the signaling portion of G-CSF receptor genes. Polymerase chain reaction (PCR) amplification of the G-CSF receptor transmembrane region alone, and of the transmembrane plus cytosolic portions of the receptor, yielded the size products predicted from the sequences of the normal G-CSF receptor. Single-strand conformational polymorphism (SSCP) analysis of G-CSF receptor PCR products demonstrated no variance in structural conformation between KS patients and normal subjects. These results demonstrate that bone marrow stromal cells in patients with KS secrete normal concentrations of functional G-CSF and suggest that the neutropenia in KS patients is caused by an inability of neutrophilic progenitor and precursor cells to respond to normal, physiologic levels of G-CSF. Such a defect, clinically responsive to pharmacologic doses of G-CSF, might be caused by defects in the post-G-CSF receptor signal transduction pathway.

Bone marrow stromal fibroblasts secrete interleukin-6 and granulocyte-macrophage colony-stimulating factor in the absence of inflammatory stimulation: demonstration by serum-free bioassay, enzyme-linked immunosorbent assay, and reverse transcriptase polymerase chain reaction.

Bone marrow (BM) stromal fibroblasts produce hematopoietic growth factors (HGFs) in response to inflammatory mediators such as tumor necrosis factor-alpha or interleukin-1 alpha (IL-1 alpha). In the absence of such inflammatory stimuli, production of HGFs by BM stromal cells has been problematic and controversial. In vivo, however, basal hematopoiesis maintains blood counts within a normal homeostatic range even in the absence of inflammation, and HGFs are required for progenitor cell differentiation in vitro. To better ascertain the contribution of BM stromal fibroblasts to basal hematopoiesis, we therefore studied HGF production in quiescent BM stromal fibroblasts by three sensitive assays: serum-free bioassay, enzyme-linked immunosorbent assay, and reverse transcriptase polymerase chain reaction. Stromal fibroblasts were cultured in the presence or absence of normal human serum to determine if serum factor(s) present in the noninflammatory (basal) state induce secretion of HGFs. Human serum was found to induce or enhance transcription and secretion of granulocyte-macrophage colony-stimulating factor (GM-CSF) and enhance secretion of constitutively expressed IL-6. In contrast, no secretion of either granulocyte-CSF (G-CSF) or IL-3 was found. These data indicate that factors in normal human serum are active in enhancing GM-CSF and IL-6 production by stromal fibroblasts and suggest that these growth factors contribute to the maintainance of normal, basal hematopoiesis in vivo.

Interleukin-6 in psoriasis: expression and mitogenicity studies.

Interleukin-6 (IL-6) is a multifunctional cytokine which has been suggested to function as an autocrine mitogen in psoriatic epidermis. We report here the results of several experiments designed to further examine this hypothesis. Blot hybridization was unable to detect 1.3 kb IL-6 transcripts in RNA extracted from normal or psoriatic epidermal (keratome) biopsies, suggesting that IL-6 expression is very low in normal and psoriatic epidermis. Therefore, qualitative and semiquantitative PCR/Southern blot analyses were performed on keratome-derived RNA, and revealed variable but significantly increased IL-6 mRNA levels in lesional psoriatic relative to normal tissue. To further examine the ability of normal human keratinocytes (NHK) to express IL-6, RNA was extracted from rapidly proliferating secondary NHK cultures. IL-6 transcripts were nearly undetectable by blotting in keratinocytes grown in low-calcium serum-free medium, but low levels could be induced by treatment with 1.8 mM CaCl2. IL-6 transcripts were strongly superinduced after cycloheximide treatment, suggesting that a labile protein regulates IL-6 mRNA levels in these cells. Finally, the mitogenic activity of IL-6 was examined in NHK under varying conditions of cell density and added growth factors. IL-6 did not stimulate high density keratinocyte growth in the presence or absence of other growth factors, but did stimulate clonal growth in epidermal growth factor (EGF)-deficient media at high concentrations (> or = 10 ng/ml). The proliferative effects of IL-6, but not of basic fibroblast growth factor, were abrogated by monoclonal antibodies directed against the EGF receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Nuclear proteins that bind the human gamma-globin gene promoter: alterations in binding produced by point mutations associated with hereditary persistence of fetal hemoglobin.

The molecular mechanisms responsible for the human fetal-to-adult hemoglobin switch have not yet been elucidated. Point mutations identified in the promoter regions of gamma-globin genes from individuals with nondeletion hereditary persistence of fetal hemoglobin (HPFH) may mark cis-acting sequences important for this switch, and the trans-acting factors which interact with these sequences may be integral parts in the puzzle of gamma-globin gene regulation. We have used gel retardation and footprinting strategies to define nuclear proteins which bind to the normal gamma-globin promoter and to determine the effect of HPFH mutations on the binding of a subset of these proteins. We have identified five proteins in human erythroleukemia cells (K562 and HEL) which bind to the proximal promoter region of the normal gamma-globin gene. One factor, gamma CAAT, binds the duplicated CCAAT box sequences; the -117 HPFH mutation increases the affinity of interaction between gamma CAAT and its cognate site. Two proteins, gamma CAC1 and gamma CAC2, bind the CACCC sequence. These proteins require divalent cations for binding. The -175 HPFH mutation interferes with the binding of a fourth protein, gamma OBP, which binds an octamer sequence (ATGCAAAT) in the normal gamma-globin promoter. The HPFH phenotype of the -175 mutation indicates that the octamer-binding protein may play a negative regulatory role in this setting. A fifth protein, EF gamma a, binds to sequences which overlap the octamer-binding site. The erythroid-specific distribution of EF gamma a and its close approximation to an apparent repressor-binding site suggest that it may be important in gamma-globin regulation.