Prognostic significance of cyclooxygenase-2 expression in pediatric Hodgkin and non-Hodgkin lymphomas with or without Epstein–Barr virus latent infection

The aim of this study is to investigate the expression of cyclooxygenase-2 (COX-2) and the relationship between COX-2, prognosis and Epstein–Barr virus (EBV) in pediatric Hodgkin (pHL) and non-Hodgkin lymphomas (pNHL). Cyclooxygenase-2 is frequently overexpressed in various tumors. COX-2 inhibitors’ potential use as a therapeutic target in onco-hematology and its relation with EBV is a contemporary question. A potential role for COX-2 in EBV-associated human cancers was suggested. The role of COX-2 in EBV-related oncogenesis of pHL and pNHL is not studied. The expression of COX-2, latent membrane protein 1 and Epstein–Barr virus-encoded early RNA was detected by immunohistochemistry and in situ hybridization in 63 pHL and 70 pNHL. The results were scored as positive or negative. The statistical significance was assessed by correlation, survival analysis, and nonparametric tests. In pNHL cases, the mean age was 7.16 ± 3.72. EBV was positive in 18 cases, COX-2 in 16 cases (22.8%). Expression of COX-2 did not correlate with age, sex, stage, phenotype, histology, EBV positivity, and response rate to therapy. In pHL cases, the mean age was 8.46 ± 3.54. EBV was positive in 52(82.5%), COX-2 in 36(57.1%) cases. The expression of COX-2 protein did not correlate with age, sex, stage of disease, EBV positivity, and response to therapy. COX-2 expression was higher in nodular sclerosing and lymphocyte-rich histology. In log rank analysis, no statistical significance for prognosis was observed. In this series, COX-2 was found to be expressed in the minority of pNHL tissues and more than half in pHL cases especially in NS type. This might be related with fibrosis of NS cases. It did not correlate with prognosis. COX-2 expression did not seem to have a synergistic role with EBV in the pathogenesis of pediatric lymphomas. It will be useful studying the relationship of COX-2 expression with apoptotic and other factors in pediatric lymphomas.     

Expression of Smac/DIABLO in B-cell non-Hodgkin and Hodgkin lymphomas

Inhibitor of apoptosis proteins (IAPs) are upregulated in cancers and suppress cell death, in part, through their ability to directly inhibit caspases. Inhibitor of apoptosis proteins are differentially expressed in B-cell lymphomas. The functions of some IAPs are counteracted by the cell death inducer, second mitochondrial-derived activator of caspases/direct IAP binding protein with low pI (Smac/DIABLO). In this study, we investigated the expression levels of Smac/DIABLO in 14 lymphoma cell lines by Western blot analysis. We also assessed 247 B-cell non-Hodgkin's lymphoma (NHL) and 40 Hodgkin's lymphoma (HL) tumors using immunohistochemical methods. Smac/DIABLO was expressed in most NHL and all HL cell lines. In NHL, Smac/DIABLO was expressed in 117 (47%) tumors and was differentially expressed in various NHL types. In most NHLs, from 29% to 68% of tumors were positive; however, Smac/DIABLO was not detected in small lymphocytic lymphoma/chronic lymphocytic leukemia and Burkitt lymphoma, and was rare in extranodal marginal zone B-cell lymphoma. In HL, Smac/DIABLO was positive in 25 (63%) tumors. Unlike NHL, all types of HL were positive for Smac/DIABLO, although nodular sclerosis was least often positive. The differential expression of Smac/DIABLO in NHLs suggests that apoptotic mechanisms are differentially involved in their pathogenesis. These results may also have implications for using Smac/DIABLO or its agonists as therapeutic agents.     

Bone mineral density in healthy Tunisian women

Interpretation of densitometric results requires a comparison with reference bone mineral density (BMD) values of normal age and sex-matched persons. Thus the aim of this study was to determine these values for healthy Tunisian women, to estimate the prevalence of osteoporosis and to compare our findings with other populations. A cross-sectional study of 1378 Tunisian women aged between 20 and 96 years was carried out using DXA (GE-Lunar Prodigy). Subjects with suspected conditions affecting bone metabolism were excluded. Measurements were taken at the lumbar spine and femoral neck. These values were expressed at T-scores, with reference to the mean BMD values of the group aged 20–40 years. The peak bone mass, estimated in this age group was 1.174 + 0.127 g/cm² at the lumbar spine and 1.016 ± 0.118 g/cm² at the femoral site. It was attained respectively within the age of 25 years and 36 years. For both sites, the expected decline in BMD was shown when the successive age groups [40–49 years] and [50–59 years] were compared. Bone loss was rapid during the first 5 years after menopause. Thereafter BMD declined slowly but continually. The prevalence of osteoporosis in the women over 50 years of age, taking account of peak bone mass observed in our cohort, was 23.3% at the spine and 17.3% at the femoral neck with a combined prevalence of 23.4%. These rates attained respectively 30.4%, 11.8% and 32.9% when we considered the Italian values, which demonstrate the variability of osteodensitometric depending to the reference population adopted.     

Clonality testing of malignant lymphomas with the BIOMED-2 primers in a large cohort of 1969 primary and consultant biopsies

The introduction of the BIOMED-2 primers allowed for reliable comparisons of clonality testing data of malignant lymphomas from different laboratories. This study undertook a retrospective analysis of a large cohort of cases; 1862 cases involved the immunoglobulin heavy chain locus (IGH V_H–J_H), and 1527 cases involved the T cell receptor gamma locus (TCRG). We confirmed previously published clonality rates in various B cell, T cell, and Hodgkin lymphoma cases. In reactive lesions, clonality for the IGH locus was frequently accompanied by additional polyclonal background. Clonality for TCRG was found in a subgroup of diffuse large B cell lymphomas. On closer morphologic inspection, seven cases appeared to have arisen from an underlying peripheral T-cell lymphoma. Five cases with monoclonal TCRG rearrangements, originally diagnosed as Hodgkin lymphomas, were reclassified as T-cell lymphomas. TCRG clonality was very rarely only observed in Hodgkin lymphoma. In case of clear TCRG clonality a T-cell neoplasia must be ruled out on morphological grounds. By careful examination of the rearrangement patterns, including an assessment of a co-amplified polyclonal background, clonality testing provides a powerful tool which in concert with morphologic and immunohistochemical parameters can lead to a firm diagnosis.     

南京2504例0～3岁婴幼儿的骨密度

目的 探讨年龄、性别、孕期母亲补钙、佝偻病早期症状与骨密度Z值的关系,为更好地预防儿童佝偻病和低骨密度工作提供依据和建议。 方法 对南京市秦淮区妇幼保健所儿保门诊的2504例0～3岁婴幼儿,用定量超声骨密度仪对其骨密度进行检测,分析骨密度不足检出率与年龄、性别、孕期补钙情况、佝偻病早期症状之间的关系。 结果 0～3岁女童骨密度Z值总体低于男童（P＜0.001）。在0～3月,3～6月,6月～1岁3个年龄段中,女童骨密度不足的发生率均明显高于男童（P＜0.001）。1～3岁年龄段中,男女童骨密度不足检出率差异无统计学意义（P＞0.05）。3～6月年龄段的婴儿骨密度不足检出率最高,达83.5%,其中女童骨密度不足检出率占该年龄总案例的51.0%。佝偻病早期症状与骨密度不足检出率差异有统计学意义（P＜0.001）。母亲孕期补钙与骨密度不足检出率差异无统计学意义（P＞0.05）。 结论 要定期监测婴幼儿骨密度,关注1岁以内小儿的骨密度,尤其关注女童。     

B-cell non-Hodgkin lymphomas in Uganda: an immunohistochemical appraisal on tissue microarray

The most common non-Hodgkin lymphomas in Uganda are neoplasms of B-cell derivation. The field of B-cell lymphoma immunophenotype has rapidly progressed because of the increasing availability of markers applicable to routine sections. Although the latter have allowed the identification of distinctive lymphoma entities in the developed countries, such approach has not yet been used in Uganda. One hundred twenty-nine formalin-fixed, paraffin-embedded tissue samples from the Department of Pathology of Makerere University were used for tissue micro-array (TMA) construction. Four-micrometer-thick sections were cut from TMAs and stained with hematoxylin and eosin and Giemsa. They were also used for immunohistochemistry and in situ hybridization. According to morphology and immunohistochemistry, lymphoid neoplasms were classified as Burkitt's lymphoma (BL) (95 cases), diffuse large B-cell lymphoma (19 cases), mantle cell lymphoma (4 cases), and B-cell lymphoblastic lymphoma (1 case). In BL, a homogeneous phenotype (CD10(+), Bcl-6(+), Bcl-2(-), MUM1/IRF4-, and Ki-67 approximately 100%) and a stable Epstein-Barr virus integration were found. A distinctive and unusual feature was the frequent plasma cellular differentiation, along with the positivity for CD30 and CD138 (recorded in 35 and 43 cases, respectively). According to our findings, most non-Hodgkin B-cell tumors in Uganda are endemic BLs followed by diffuse large B-cell lymphomas. The rest consist of rare but clinically important entities such as mantle cell lymphoma and B-cell lymphoblastic lymphoma. The availability of TMAs and immunohistochemistry has enabled us to precisely categorize tumors that have so far been diagnosed in Uganda as "high-grade/aggressive" lymphomas on the basis of cell morphology alone.     

丙戊酸对癫痫儿童骨密度的影响及VitD防治作用

目的观察丙戊酸对癫痫儿童骨密度的影响及VitD的预防作用。方法 6-14岁癫痫儿童63例,分两组,一组以单药丙戊酸治疗,另一组以丙戊酸+VitD治疗,应用CT分别测量两组治疗前及治疗后6个月第四腰椎及股骨颈骨密质和骨松质的骨密度值。结果丙戊酸治疗组和丙戊酸+VitD治疗组治疗前骨松质、骨密质的骨密度无明显差异。在第4腰椎和股骨颈,丙戊酸治疗组疗后6个月骨松质的骨密度明显低于疗前,差异有明显意义;丙戊酸+VitD治疗组疗后6个月骨松质的骨密度与治疗前无明显差异。丙戊酸+VitD治疗组治疗6个月后第四腰椎骨松质的骨密度变化率显著低于丙戊酸治疗组,添加VitD能提高骨松质的骨密度。结论丙戊酸可致癫痫儿童的骨松质密度明显降低。VitD对此副作用有预防作用。     

Serum levels of angiogenic cytokines decrease after radiotherapy in non-Hodgkin lymphomas

PURPOSE: Serum levels of angiogenic cytokines decrease after radiotherapy in patients with cancer, and this may be relevant for treatment response and progression-free survival. Herein, we set out to determine whether circulating fibroblast growth factor-2 (FGF-2), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF) and platelet-derived growth factor-beta (PDGF-beta) decrease after radiotherapy in patients with non-Hodgkin lymphomas (NHLs) and if so, whether their decrease correlates with age, tumour histotype and stage, and radiation dose. MATERIAL AND METHODS: The serum levels of FGF-2, VEGF, HGF and PDGF-beta were evaluated before and after radiotherapy by an enzyme-linked immunosorbent assay (ELISA). These levels were correlated both reciprocally and with age, histotype, stage and radiation dose. RESULTS: After radiotherapy, FGF-2, VEGF and PDGF-beta, but not HGF, significantly decreased in relation to the radiation dose and response. No correlation was established between cytokine levels, except for VEGF and PDGF-beta, which decreased in parallel. Haemoglobin levels did not decrease after radiotherapy, while FGF-2, VEGF, HGF and PDGF-beta levels did not correlate with age, NHL stage and histotype. CONCLUSIONS: Soluble FGF-2, VEGF and PDGF-beta levels decline after radiotherapy in NHLs, and may have predictive significance for response to treatment and recurrence.     

Flow cytometric DNA analysis of 199 histologically favourable or unfavourable non-Hodgkin lymphomas

We have studied the nuclear DNA content of histologically favourable (n = 82) or unfavourable (n = 117) non-Hodgkin lymphomas (NHLs) diagnosed between 1957 and 1978 in the Tampere University Central Hospital. The DNA analysis was done by applying a trypsin digestion method to archival tumour samples. DNA aneuploidy was seen in 40 per cent of the unfavourable cases and in 10 per cent of the favourable cases, but varied considerably between different histological subtypes. The unfavourable cases showed high proliferative activity (S-phase fraction, SPF), while considerable variation in the SPF among the favourable NHLs was noted. Among the unfavourable NHLs, cases with DNA-aneuploid tumours had significantly (P less than 0.01) worse prognosis than stage and treatment matched cases with DNA-diploid tumours. In general, survival of the patients who had high SPF tumours was significantly lower compared with patients with low SPF tumours (P less than 0.01). However, SPF was not related to the prognosis in the unfavourable NHLs. We conclude that the flow cytometric DNA analysis revealed characteristic features in the favourable and unfavourable NHLs and may be useful in predicting the clinical outcome of patients.     

Bone mineral density and vitamin D status in systemic lupus erythematosus (SLE): A systematic review

Despite the improvement in the quality of life of patients with SLE due to scientific and technological advances, SLE remains a disease that over the years may produce irreversible damage to patients. Osteoporosis and secondary bone fractures are two of the major causes of irreparable injury in patients with SLE. Vitamin D insufficiency may play a vital role both in reduced bone mineral density (BMD) and in the appearance of fractures, although its mechanisms of action are still unclear. We performed a systematic review of the literature in order to determine the prevalence and predictors of reduced vitamin D plasma levels, bone loss and the presence of fractures in SLE patients. Our review encompassed all English-language publications using Medline and EMBase electronic databases from their inception (1966 and 1980, respectively) to December 2016. We included all intervention studies and observational studies in which vitamin D plasma levels, BMD and bone loss were measured and applied to patients with SLE. Previous studies suggested an increase in bone loss and fracture in patients with SLE compared with general population and although there is a high prevalence of vitamin D insufficiency in the general population, previous studies had demonstrated lower vitamin D levels in patients with SLE compared to age-matched controls. The etiology of reduced bone mass and reduced vitamin D plasma levels in SLE is multifactorial and includes a variety of intrinsic factors related to the disease itself and treatment side effects. SLE patients are at risk for developing these two comorbidities (reduced vitamin D plasma levels and low BMD) and it is therefore essential to study, monitor, prevent and treat bone metabolism disorders in SLE patients.     

Expression of Grb2 distinguishes classical Hodgkin lymphomas from primary mediastinal B-cell lymphomas and other diffuse large B-cell lymphomas

Growth factor receptor-bound protein 2 is an adaptor molecule that mediates B-cell receptor (BCR) signaling pathways, but the expression of growth factor receptor-bound protein 2 in lymphoma tissues has not been reported. We sought to characterize growth factor receptor-bound protein 2 protein expression in reactive tonsillar tissues and lymphoma tissues obtained from diagnostic biopsies of classical Hodgkin lymphoma, primary mediastinal large B-cell lymphoma, diffuse large B-cell lymphoma, nodular lymphocyte predominant Hodgkin lymphoma, and 20 low-grade B-cell lymphomas. Growth factor receptor-bound protein 2 expression was assessed in tissues by immunohistochemistry and in lymphoma cell lines by immunoblotting. In reactive lymphoid tissues, growth factor receptor-bound protein 2 was expressed in the cytoplasm of B-cells and histiocytes but not T-cells. Strong, cytoplasmic growth factor receptor-bound protein 2 expression was seen in the neoplastic cells of follicular lymphoma, chronic lymphocytic leukemia/small lymphocytic lymphoma, splenic marginal zone lymphoma, primary mediastinal large B-cell lymphoma, diffuse large B-cell lymphoma, and nodular lymphocyte predominant Hodgkin lymphoma. In contrast, only 10% of the classical Hodgkin lymphomas showed growth factor receptor-bound protein 2 expression in the neoplastic cells. Growth factor receptor-bound protein 2 protein expression was detected by Western blotting in all lymphoma cell lines tested with higher levels in primary mediastinal large B-cell lymphoma compared with classical Hodgkin lymphoma cell lines. These findings support a role for growth factor receptor-bound protein 2 in the diagnostically challenging workup of classical Hodgkin lymphoma versus primary mediastinal large B-cell lymphoma and warrant further studies to evaluate the biologic significance of growth factor receptor-bound protein 2 in the pathogenesis of classical Hodgkin lymphoma.     

Fibrosis in Hodgkin and non-Hodgkin lymphomas

There is little quantitative information about the amount of fibrosis in lymphomas. The aim of the present study was to investigate the amount of fibrosis in lymphomas and to highlight the relationship between fibrosis and mast cells, the key players of fibrosis. Tissue sections of 60 patients with diagnosis of lymphoma were reevaluated for classification. The mean fibrotic-stained area percentage (F-SAP) was determined in van Gieson-stained digital images using image analysis (Mediscope, Dokuz Eylul University, Clinical Engineering, Turkey). Mast cells were visualized using streptavidin peroxidase immunohistochemistry with anti-tryptase staining. Twenty-seven (44%) cases were Hodgkin's lymphoma (HL). F-SAP was 11.09+/-8.96 and 1.72+/-1.76 for HL and non-HL cases (Mann-Whitney U, p<0.000), and the mean mast cell count (MMCC) was 24.63+/-13.58 and 8.03+/-8.07, respectively (Mann-Whitney U test, p<0.000). There was a significant difference between F-SAP and MMCC concerning different types of lymphomas (Kruskal-Wallis test, p>0.000). F-SAP was highest in nodular sclerosis HL, and MMCC was highest in mixed cellular HL. There was a strong positive correlation between MMCC and F-SAP (Pearson Correlation test, p<0.000, r=0.51). These results suggest that the amount of fibrosis demonstrates differences in subtypes of lymphomas, and mast cells are increased in fibrosing lymphomas. However, it seems likely that more than one cell type is involved.     

Evaluation of methods for prediction of bone mineral density by clinical and biochemical variables in perimenopausal women

Objectives: to predict spinal and femoral bone mineral density (BMD) in perimenopausal women from simple clinical and biochemical variables. Methods: 2016 women 3–24 months past last menstrual bleeding. Mean age 50.1±2.8 years. Age, height, weight, number of full term pregnancies, weekly hours of physical activity, sunbathing habits, use of sun bed, daily intake of calcium and vitamin D, smoking habits, consumption of alcohol, coffee, and tea, history of forearm or femoral neck fractures among the parents, serum osteocalcin (S-OC), serum bone specific isoenzyme of alkaline phosphatase (BSAP), and urine hydroxyproline/creatinine ratio (U-OHP) were used as predictors in three different mathematical models. Lumbar spine (L2–L4) and femoral neck BMD were measured by DEXA. Three mathematical models (multiple regression, logistic regression, and discriminant analysis) were applied. Results: the multiple regression explained 19–21% of the total variation, and the logistic regression and discriminant function had a sensitivity between 53 and 67% with specificity ranging from 67 to 80%. Age, S-OC, serum bone specific alkaline phosphatase, and a maternal history of forearm or femoral neck fractures seemed to be reproducible risk factors for low bone mineral density irrespective of the mathematical model applied. When applied to a separate population, the models performed poorly. Conclusions: Simple clinical and biochemical variables are not useful to predict spinal and femoral BMD in the individual perimenopausal woman.     

Classical Hodgkin lymphoma concurrently evolving in a patient with marginal zone B-cell lymphoma of the spleen

Combination of the splenic marginal zone B-cell lymphoma (SMZL) and classical Hodgkin lymphoma (cHL) is extremely rare. We report a unique case with concurrent SMZL and cHL. The patient was a 63-year-old man who presented with fatigue and anemia, showing a splenomegaly and retroperitoneal lymphadenopathy. A splenectomy revealed monotonous marginal zone lymphocytic infiltrates and numerous large Reed-Sternberg–like cells. Flow cytometry revealed a κ light-chain–restricted CD5 (−), CD23 (−) B-cell population. DNA polymerase chain reaction analysis confirmed the presence of clonal rearrangement of the immunoglobulin heavy-chain gene. Immunohistochemical studies revealed that the large atypical cells were CD30 (+), CD15 (weakly +), CD20 (−), CD45 (−), Pax5 (weakly +), BOB.1 (−), and Oct2 (−), indicating the coexistence of SMZL with cHL. After the chemotherapy, the patient achieved a clinical/radiologic remission, whereas cHL was detected in liver and bone marrow subsequently. The case indicates that both components of lymphoma can present concurrently as a composite form of lymphoma and both need to be treated adequately.     

Effect of natural early menopause on bone mineral density

OBJECTIVES: Early menopause (EM) is included among the risk factors for osteoporosis. Several studies have shown that women with early menopause have lower bone mineral density (BMD) than those with normal expected age of menopause. The aim of our cross-sectional study was to investigate the effects of time of menopause on vertebral bone mass in healthy postmenopausal women and to evaluate if early menopause is a risk factor for lower vertebral BMD. METHOD: We studied 782 who had never received drugs acting on bone mass. The study population was divided into three groups: women with early, normal (NM), and late (LM) menopause. Our study population was further categorized in 5-year age segments between 45 and >75. RESULTS: The three groups examined did not differ for age, age at menarche, body mass index (BMI), and vertebral BMD, while there were significant differences in age at menopause and years since menopause. Our study showed that women with EM presented significantly lower vertebral BMD than NM and LM in 50-54 age segments. Beyond 55 years, EM, NM, and LM women had no differences in lumbar BMD values. CONCLUSIONS: In conclusion, controversial data demonstrated that the absolute amount of bone loss is greater after early menopause than after normal or late menopause, even if a slight effect of early menopause on bone mass cannot be excluded.     

The high frequency of EBV infection in pediatric Hodgkin lymphoma is related to the classical type in Bahia, Brazil

Pediatric Hodgkin lymphoma (HL) occurring in developing regions is different from HL in industrialized countries due to the higher frequency of association with Epstein–Barr virus (EBV) infection. This infection is related to classical HL (cHL) but is virtually absent in nodular lymphocyte predominant HL (nLPHL). We studied the phenotype and the expression of EBV gene products in 90 pediatric cases by immunohistochemistry and in situ hybridization. EBV-positive tumor cells were found exclusively in cHL. The infection occurred with high frequency in all cHL subtypes, but it predominated in the mixed cellularity and lymphocyte depletion subtypes. These results reinforce the hypothesis that EBV plays a major role in the etiology of pediatric cHL in developing areas. Curiously, the frequency of EBV infection in HL was identical to the previously described for Burkitt’s lymphoma in the same pediatric population. As both lymphomas have a postulated precursor cell in the germinal center (GC), the pattern of latently EBV-infected GC cells previously described in Bahia may be related to the development of these lymphomas.     

Malignant lymphomas in HIV-seropositive patients. Frequency,features, and prognosis. Report on 31 cases

Summary In a random HIV-seropositive population, malignant lymphomas were diagnosed in 31 patients, of whom 24 (77%) had non-Hodgkin lymphoma (NHL) and 7 (23%) Hodgkin lymphoma (HL). The prevalence of NHL among AIDS patients was 8% (23/279 cases), with a prevalence of 17% among autopsied patients (16/96 cases). No patient with HL had AIDS at the time of diagnosis. In 7 of 23 AIDS patients with NHL (30%) the diagnosis was made only post mortem; among these were all 5 patients with primary CNS NHL. Median survival from the time of diagnosis was 1 month for patients with NHL and 3 months for those with HL. In individual patients, survival for several years may be possible with chemotherapy. Certain patients with NHL appear to benefit from intensive chemotherapy with a combination of methotrexate, doxorubicin, cyclophosphamide, vincristine, prednisone, and bleomycin (MACOPB protocol). Appropriate, therapeutic strategies taking into account the patients' individual conditions, including the overall prognosis, urgently requires development. Metastatic CNS involvement, which was the primary cause of death in 5 of 11 patients with NHL (45%) receiving chemotherapy, represents a serious limitation to successful treatment.Abbreviations AIDS acquired immunodeficiency syndrome - CB centroblastic - CDC Centers for Disease Control - CHOP cyclophosphamide, doxorubicin, vincristine, prednisone - CMV cytomegalovirus - CNS central nervous system - COPBLAM cyclophosphamide, vincristine, prednisone, bleomycin, doxorubicin, procarbazine - COPP/ABVD cyclophosphamide, vincristine, prednisone, procarbazine/doxorubicin, bleomycin, vinblastine, darcarbazine - CR complete remission - CT computerized tomography - ELISA enzyme-linked immunosorbent assay - HIV human immunodeficiency virus - HL Hodgkin lymphoma - IT intrathecal - IMVP16 ifosfamide, methotrexate, etoposide - KC Kiel classification - MACOP-B methotrexate, doxorubicin, cyclophosphamide, vincristine, prednisone, bleomycin - ML malignant lymphoma - NHL non-Hodgkin lymphoma - OI opportunistic infection - PCP Pneumocystis carinii pneumonia - PD progressive disease - PR partial remission - RT radiation therapy - WBC white blood cells - WF Working Formulation