IL-25 Impact on Malignant B Cells Survival and T Cells Activation in Chronic Lymphocytic Leukemia.

T cell dysregulation and shift to T helper 2 responses, boosting tumor microenvironment support, contributes to the survival of leukemic B cells in Chronic Lymphocytic Leukemia. Interleukin (IL)-25 is involved in the initiation of T helper 2 cell responses. Signal transduction of IL-25 begins with the heterodimer receptor (IL-17RA/IL-17RB). The presence of IL-25 in the tumor microenvironment may affect the supportive effects of T cells in the surrounding tumor cell environment. The purpose of this study was to evaluate the role of IL-25 in the biology of CLL. IL-17RB expression in CD3+ and CD19+ cells was assessed in isolated peripheral blood mononuclear cells (PBMCs) of nine CLL patients and nine healthy subjects by real-time polymerase chain reaction and flow cytometry. B cells were positively enriched from PBMCs using magnetic-activated cell sorting (MACS). PBMCs and purified leukemic B cells were cultured with recombinant human IL-25 (20ng/ml) for 72 hours, then the viability and apoptosis of cultured cells were measured by MTT assay and AnnexinV/7AAD. Furthermore, the levels of CD69 expression on T lymphocytes and IL-17RB in T and B cells were determined by flow cytometry. The basal level of IL-17RB expression in CLL patients was significantly higher than that in control individuals. In addition, the percentage of IL-17RB+/CD3+, IL-17RB+/CD19+ cells and CD69+/CD3+ cells increased after 72 hours of culture with IL-25 in CLL patients compared to healthy subjects. IL-25 also reduces the apoptosis rate of tumor cells. We found that IL-25 could stimulate T cells in CLL patients and lower B cell death. This suggests that IL-25 might have a role in enhancing the survival of tumor cell by expressing receptors for inflammation, such as IL-17RB, and might be involved in the development of CLL.

Genetic Study of Hereditary Angioedema Type I and Type II (First Report from Iranian Patients: Describing Three New Mutations).

BACKGROUND: Hereditary Angioedema (HAE) is a rare autosomal dominant immunodeficiency disease with mutation in C1 inhibitor gene (SERPING1) which deficient and dysfunction of C1-INH protein result in HAE type I or type II, respectively. The present study aimed to define the genetic spectrum of HAE type I and type II among Iranian patients. METHODS: Thirty-four patients with clinical phenotype of recurrent edematous attacks in face, upper and lower limbs, hands, and upper airway entered the study. Mutations in SERPING1 were analyzed using PCR and Sanger Sequencing. In addition, Multiplex Ligation-dependent Probe Amplification (MLPA) was performed to discover large deletions or duplications in negative screening samples by Sanger. RESULTS: Twenty-three patients were diagnosed with HAE type I and 11 with HAE type II. Fourteen distinctive pathogenic variations including five frameshift (p.G217Vfs*, p.V454Gfs*18, p.S422Lfs*9, p.S36Ffs*21, p.L243Cfs*9), seven missense (p.A2V, p.G493R, p.V147E, p.G143R, p.L481P, p.P399H, p.R466C), one nonsense (p.R494*), and one splicing defect (C.51 + 2 T˃C), which three of these mutations were identified novel. However, no mutation was found in seven patients by Sanger sequencing and MLPA. CONCLUSION: Final diagnosis with mutation analysis of HAE after clinical evaluation and assessment of C1INH level and function can prevent potential risks and life-threatening manifestations of the disorder. In addition, genetic diagnosis can play a significant role in facilitating early diagnosis, pre-symptomatic diagnosis, early diagnosis of children, asymptomatic cases, and those patients who have the borderline biochemical results of C1-INH deficiency and/or C4.

Serum Level and Gene Expression of Interleukin-15 Do Not Correlate with Villous Atrophy in Celiac Disease Patients.

Background and Aims: Interleukin-15 (IL-15) is a key player in the pathogenesis of celiac disease (CD). We investigated the functional role of IL-15 in the process of epithelial cell phenotypic modification at different stages of CD. Materials and Methods: In this study, we looked for correlations between the IL-15 mRNA levels in duodenal tissue and serum protein levels in a cohort of Iranian patients affected by CD based on the degree of histopathology. Ninety-five formalin-fixed, paraffin-embedded duodenal tissue specimens were collected: 23 with a Marsh I value; 30 with a Marsh II value; 32 with a Marsh III value; and 10 normal controls. The expression levels of the IL-15 gene in these biopsy specimens were determined by real-time quantitative polymerase chain reaction (qPCR), and IL-15 serum protein concentrations were determined by enzyme-linked immunosorbent assay and compared to tissue expression. Results: The IL-15 mRNA levels were higher in patients with Marsh II compared with the control group, and the Marsh I, and Marsh III groups. The differences between the Marsh II and Marsh I patients were statistically significant (p = 0.03). Similarly, the serum concentration of IL-15 was higher in Marsh II patients compared to those with Marsh I and Marsh III lesions, although the differences were not statistically significant (p = 0.221). Conclusions: Our results demonstrate that IL-15 gene expression might be elevated only in the early stages of CD onset (and histological damage) and that IL-15 serum levels do not significantly correlate with its tissue expression whatever the degree of histopathology.

Evaluating the mechanism underlying antitumor effect of interleukin 27 on B cells of chronic lymphocytic leukemia patients.

Chronic lymphocyte leukemia (CLL) is a B-cell malignancy resisted to apoptosis. Recently, some studies indicated that cytokines such as interleukin 27 (IL-27) can reduce B-cell proliferation. The aim of this study is to evaluate the mechanism underlying the proapoptotic effect of IL-27 on B cells of patients with CLL in comparison with B cells of normal subjects. The effect of IL-27 on the antitumor activity of natural killer (NK) and T cells was also evaluated. Peripheral blood mononuclear cells (PBMCs) were isolated from 35 patients with CLL and 15 normal subjects. B cells and PBMCs were cocultured with IL-27 and B cells apoptosis to evaluate proliferation. Both messenger RNA and protein expression of IL-27 and IL-27 receptor were determined using flow cytometry and real-time polymerase chain reaction analysis. To evaluate the apoptotic effect of IL-27 on B cells of patients with CLL, Annexin V-FITC and 7-AAD (BioLegend) fluorescent dyes were used. In addition, the IL-27 effect on activation of T cell and NK cell was determined by determining CD96 molecule expression. IL-27 and IL-27 receptor expression in patients with CLL was significantly lower than that of normal subjects (p < .05). IL-27 enhanced apoptosis of B cells in patients with CLL (p < .05) but this effect was not significantly observed in B cells of normal subjects (p > .05). Consequently, IL-27 reduced the proliferation of B cells and enhanced NK cell activity (p < .05). IL-27, through inducing apoptosis, can exert an inhibitory effect on cancer B cells of CLL patients with minimal effect on normal B cells.

Evaluation of Interleukin 25 and Interleukin 25 Receptor Expression in Peripheral Blood Mononuclear Cells of Breast Cancer Patients and Normal Subjects.

[Figure: see text] Interleukin 25 (IL-25) is a ligand for IL-25 receptor (IL-25R) with apoptotic effect on breast cancer epithelial cells that are produced by peripheral blood mononuclear cells (PBMCs). In this study, we aimed to evaluate IL-25/IL-25R mRNA expression in PBMCs, and also investigate correlation of IL-25/IL-25R with tumor stages/grades in patients with breast cancer. PBMCs and serum were isolated from 30 patients with breast cancer and 18 normal subjects. ELISA test was conducted for IL-25 cytokine. Total RNA was isolated from 2 × 106 PBMCs and reverse transcribed to cDNA. Quantitative PCRs were performed for IL-25, IL-25R, and GAPDH genes. IL-25 mRNA expression in PBMCs of breast cancer patients (malignant and benign) was significantly lower than that in normal subjects, Also IL-25 expression in breast cancer patients with malignant tumor was significantly lower than that in nonmalignant patients. IL-25R expression in malignant patients was significantly higher than that of benign and normal subjects (P < 0.05). IL-25 in serum of normal subjects was higher than that of benign and malignant patients. There was a direct association between IL-25R expression and tumor grade/stage of cancer. In conclusion, IL-25 seems as a potential prognostic factor in the serum of breast cancer patients and reduction of IL-25 is associated with a higher grade/stage of cancer.

In Vitro Evaluation of CMV Specific CD8+T Cells Function in CMV+ Colorectal Cancer Patients Compared to Healthy Controls.

The oncogenic role of human cytomegalovirus (HCMV) has been recently shown in different cancers like colorectal cancer (CRC). According to the recent immunotherapy approach to target the CMV-expressing tumor cells, we investigated the CMV peptide-stimulated CD8+T cells functions in CRC patients compared to healthy individuals. All sixteen patients and seven controls were CMV seropositive. Blood samples were obtained from patients without chemotherapy and radiotherapy before surgery. Cytotoxic CD8+ T cells were generated using 14-day culture of PBMCs in the presences of CMV peptide epitopes and rhIL-2. In addition to the supernatant evaluations for TNF-α and IFN-γ, the functionality of CD8+ T cells was examined by detecting CD107a and intracellular IFN-γ using flow cytometry. CMV DNA was detected in tissues by Real Time PCR. CMV DNA was found in 31% of tumor tissues, while it was not seen in the adjacent non-tumor tissues. There was a close association between CMV in tumor tissue and tumor grade. Surface expression of CD107a and intracellular IFN-γ in CMV-stimulated CD8+T cells and the level of IFN-γ production in patient and control groups increased significantly after culture. The number of functions increased in patients (p<0.05) and healthy individuals after culture. Followingstimulation, expressions of CD107a and intracellular IFN-γ were elevated in tumor CMV positive patients while the TNF-α secretion was decreased. In vitro stimulation of PBMC in the presence of CMV peptide epitopes and IL-2 can be an applicable method to generate cytotoxic CD8+ T cells in CRC patients for future T cell therapy.

Th1, Th2, Th17 cell subsets in two different immunosuppressive protocols in renal allograft recipients (Sirolimus vs mycophenolate mofetil): A cohort study.

Long-term use of calcineurin inhibitors (CNI) is associated with nephrotoxicity, which is an important cause of renal dysfunction. Therefore, CNI-minimization strategies which decrease the CNI nephrotoxicity under the protection of additional immunosuppressant drugs have been developed. The aim of current cohort study was to compare the effect of two immunosuppressive protocols [tacrolimus (TAC) in combination with mycophenolate mofetil (MMF) and prednisolone (PRED) versus TAC in combination with sirolimus (SRL) and prednisolone] on the frequency of T helper cell subsets (Th1, Th2 and Th17 cells) and their associated cytokine (IFN-γ, IL-4 and IL-17A) levels in renal allograft recipients. In this study, renal transplant recipients who received induction therapy (Antithymocyte globulin) and were also on triple immunosuppressive therapy were included and divided in to two groups: Group A was comprised 14 patients who received TAC, MMF and PERD whereas group B was composed of 10 patients who received TAC, SRL and PERD. The frequency of Th1, Th2 and Th17 cells in the peripheral blood mononuclear cells (PBMCs) of the patients was analyzed by flow cytometry before and 4 months after transplantation. In addition, IFN-γ, IL-4 and IL-17A concentrations in PBMC culture supernatants of patients before and 4 months after transplantation were quantified by ELISA. The results of our study showed that TAC, MMF and PRED protocol did not diminish the frequency of Th17 cells at 4 months post-transplantation (5% ±â€¯2.5) compared with pre-transplantation (2.3% ±â€¯1; P < 0.05). However, Th17 (3.6% ±â€¯1.5 pre-transplantation vs 2.2% ±â€¯0.9 at 4 months post-transplantation; P < 0.05), Th2 (1.4% ±â€¯0.3 pre-transplantation vs 0.8% ±â€¯0.4 at 4 months post-transplantation; P < 0.05) cell subsets and IL-4 concentration (71.5 pg/ml ±â€¯12 pre-transplantation vs 62.5 pg/ml ±4.4 at 4 months post-transplantation; P < 0.05) were significantly decreased after transplantation in patients who had received SRL, TAC and PRED. In conclusion, the data of the current study suggest that using reduced dose of TAC in SRL, TAC and PRED protocol is in favor of allograft survival; however a cohort study with larger sample size is needed for confirming our results.

Comparing Serum Levels of Th17 and Treg Cytokines in Women with Unexplained Recurrent Spontaneous Abortion and Fertile Women.

BACKGROUND: Increased evidences have shown that unexplained recurrent spontaneous abortion (URSA) is associated with inflammatory responses and breakage of immunological autotolerance. Therefore, the balance between Th17 and Treg cells may elucidate the pathophysiology of URSA. OBJECTIVE: To investigate the serum concentration of regulatory and inflammatory cytokines associated with Treg and Th17 in both normal and URSA females. METHODS: Forty-six women with URSA and 28 non-pregnant control women with at least one successful pregnancy were included. Serum was obtained from both groups and stored at -7º C. The serum concentrations of IL-17, IL-21, IL-22, IL-10, and TGF-ß were quantitatively determined by ELISA. RESULTS: The levels of IL-17, IL-21, and IL-22 in sera were significantly higher (P<0.001, P=0.01 and P<0.001, respectively) and TGF-ß serum concentration was significantly lower (P=0.02) in URSA women compared with normal controls. CONCLUSION: Our results suggest that enhancement in Th17-associated cytokine levels and reduction in TGF-ß may be one of the factors involved in URSA.

Autologous T cells expressing the oncogenic transcription factor KLF6-SV1 prevent apoptosis of chronic lymphocytic leukemia cells.

Crosstalk between leukemic cells and the tumor microenvironment is of importance in chronic lymphocytic leukemia (CLL). T cells seem to sustain the survival of CLL cells by various mechanisms. The Krüppel-like family of transcription factors (KLFs) are identified as regulators of proliferation and cell death. In the present study, we analyzed the expression of the wild type (WT) gene KLF6 and the oncogenic splice variant 1 (KLF6-SV1) at the mRNA level in subsets of T cells from CLL patients (n = 29), multiple myeloma patients (n = 6) and normal donors (n = 10). RNA Silencing was used for wtKLF6 and KLF6-SV1. Tumor cell apoptosis was measured. A significant overexpression of wtKLF6 and KLF6-SV1 in T cells of CLL patients compared to normal donors and myeloma patients was noted (p<0.002). Western blot showed that both wtKLF6 and KLF6-SV1 were expressed in purified T cells from CLL patients. KLF6-SV1 siRNA transfection induced a significant down-regulation of KLF6-SV1 in CLL T cells, which lost the capability to sustain the growth of leukemic cells. However, no such a significant effect was seen after wtKLF6 transfection of the autologous T cells. The results suggest that KLF6-SV1 may play a role in the regulation of survival CLL cells.

Suppression of protein tyrosine phosphatase PTPN22 gene induces apoptosis in T-cell leukemia cell line (Jurkat) through the AKT and ERK pathways.

The aim of this study was to investigate the effect of specific PTPN22 small interfering RNAs (siRNAs) on the viability and induction of apoptosis in Jurkat cells and to evaluate apoptosis signaling pathways. In this study, Jurkat cells were transfected with specific PTPN22 siRNA. Relative PTPN22 mRNA expression was measured by Quantitative Real-time PCR. Western blotting was performed to determine the protein levels of PTPN22, AKT, P-AKT, ERK, and P-ERK. The cytotoxic effects of PTPN22 siRNA were determined using the MTT assay. Apoptosis was quantified using TUNEL assay and flow cytometry. Results showed that in Jurkat cells after transfection with PTPN22 siRNA, the expression of PTPN22 in both mRNA and protein levels was effectively reduced. Moreover, siRNA transfection induced apoptosis on the viability of T-cell acute leukemia cells. More importantly, PTPN22 positively regulated the anti-apoptotic AKT kinase, which provides a powerful survival signal to T-ALL cells as well as the suppression of PTPN22 down regulated ERK activity. Our results suggest that the PTPN22 specific siRNA effectively decreases the viability of T-cell acute leukemia cells, induces apoptosis in this cell line, and therefore could be considered as a potent adjuvant in T-ALL therapy.

Evaluation of interleukin 12 and CD56+ lymphocyte cells in pediatric hematopoietic stem cell transplantation for early diagnosis of acute graft versus host disease.

The present study tried to explain CD56+ lymphocyte cells activities and possible prognostic role of these cells in Graft-Versus-Host-Disease (GVHD). The role of IL-12 activation and function is of interest in this study. Peripheral blood samples of 51 Hematopoietic Stem Cell Transplantation (HSCT) recipients collected at before (day -8) and after (days 7 and 14). PBMC were collected by Ficoll separation and analyzed by Flow Cytometry using triple antibody (CD45-PerCP, CD56-FITC, and CD69-PE staining and control antibody. Levels of the cytokine IL-12 in the patient's serum were evaluated by ELISA. Percentage of CD56+ lymphocytes (CD56+bright) cells was significantly increased at day 14 in patients with acute GVHD and percentage of lymphocytes expressing CD69 was significantly increased at days 7 and 14 posts HSCT in patients with acute GVHD in comparison to those in non-GVHD patients. Baseline serum IL-12 levels (pre-HSCT, day -8) were significantly higher in those HSCT recipients who did not develop GVHD. This study showed that post-transplant CD56+ lymphocytes and pre-transplant serum levels of IL-12 play significant roles in the induction of and protection against GVHD, respectively. The increase in the percentage of CD69+ cells indicates the activation of lymphocyte in acute GVHD group.

Full Intelligent Cancer Classification of Thermal Breast Images to Assist Physician in Clinical Diagnostic Applications.

Breast cancer is the most common type of cancer among women. The important key to treat the breast cancer is early detection of it because according to many pathological studies more than 75% - 80% of all abnormalities are still benign at primary stages; so in recent years, many studies and extensive research done to early detection of breast cancer with higher precision and accuracy. Infra-red breast thermography is an imaging technique based on recording temperature distribution patterns of breast tissue. Compared with breast mammography technique, thermography is more suitable technique because it is noninvasive, non-contact, passive and free ionizing radiation. In this paper, a full automatic high accuracy technique for classification of suspicious areas in thermogram images with the aim of assisting physicians in early detection of breast cancer has been presented. Proposed algorithm consists of four main steps: pre-processing & segmentation, feature extraction, feature selection and classification. At the first step, using full automatic operation, region of interest (ROI) determined and the quality of image improved. Using thresholding and edge detection techniques, both right and left breasts separated from each other. Then relative suspected areas become segmented and image matrix normalized due to the uniqueness of each person's body temperature. At feature extraction stage, 23 features, including statistical, morphological, frequency domain, histogram and Gray Level Co-occurrence Matrix (GLCM) based features are extracted from segmented right and left breast obtained from step 1. To achieve the best features, feature selection methods such as minimum Redundancy and Maximum Relevance (mRMR), Sequential Forward Selection (SFS), Sequential Backward Selection (SBS), Sequential Floating Forward Selection (SFFS), Sequential Floating Backward Selection (SFBS) and Genetic Algorithm (GA) have been used at step 3. Finally to classify and TH labeling procedures, different classifiers such as AdaBoost, Support Vector Machine (SVM), k-Nearest Neighbors (kNN), Naïve Bayes (NB) and probability Neural Network (PNN) are assessed to find the best suitable one. These steps are applied on different thermogram images degrees. The results obtained on native database showed the best and significant performance of the proposed algorithm in comprise to the similar studies. According to experimental results, GA combined with AdaBoost with the mean accuracy of 85.33% and 87.42% on the left and right breast images with 0 degree, GA combined with AdaBoost with mean accuracy of 85.17% on the left breast images with 45 degree and mRMR combined with AdaBoost with mean accuracy of 85.15% on the right breast images with 45 degree, and also GA combined with AdaBoost with a mean accuracy of 84.67% and 86.21%, on the left and right breast images with 90 degree, are the best combinations of feature selection and classifier for evaluation of breast images.

Breast cancer detection and classification in digital mammography based on Non-Subsampled Contourlet Transform (NSCT) and Super Resolution.

Breast cancer is one of the most perilous diseases among women. Breast screening is a method of detecting breast cancer at a very early stage which can reduce the mortality rate. Mammography is a standard method for the early diagnosis of breast cancer. In this paper, a new algorithm is proposed for breast cancer detection and classification in digital mammography based on Non-Subsampled Contourlet Transform (NSCT) and Super Resolution (SR). The presented algorithm includes three main parts including pre-processing, feature extraction and classification. In the pre-processing stage, after determining the region of interest (ROI) by an automatic technique, the quality of image is improved using NSCT and SR algorithm. In the feature extraction part, several features of the image components are extracted and skewness of each feature is calculated. Finally, AdaBoost algorithm is used to classify and determine the probability of benign and malign disease. The obtained results on Mammographic Image Analysis Society (MIAS) database indicate the significant performance and superiority of the proposed method in comparison with the state of the art approaches. According to the obtained results, the proposed technique achieves 91.43% and 6.42% as a mean accuracy and FPR, respectively.

Apoptosis/Necrosis Induction by Ultraviolet, in ER Positive and ER Negative Breast Cancer Cell Lines.

BACKGROUND: Ultraviolet (UV) light exposure has been one of the major inducers of apoptosis. UV exposure has caused pyrimidine dimers and DNA fragmentation which might lead to cell cycle arrest and apoptosis signals activation. UV induced apoptosis has investigated in MDA-MB 468 as an ER negative breast adenocarcinoma and MCF-7 as an ER positive breast cancer cell line. Apoptosis induction rate by UV might be different in these two types of cells due to different biological characteristics of the cell. OBJECTIVES: In this paper we have evaluated serial dose of UV-B exposure on ER positive and ER negative breast cancer cell lines and its effect on apoptosis or necrosis induction in these cells. MATERIALS AND METHODS: MDA-MB468 and MCF-7 cell lines have cultured for 24 hours and UV exposure has carried out at 290 nm at dose of 154 J/m(2) to 18 KJ/m(2) using UV lamp. UV exposed cells have incubated in cell culture condition for 24 or 48 hours following UV exposure and the cells have stained and analyzed by flow cytometry for apoptosis evaluation by Annexin V/PI method. RESULTS: Apoptosis rate (PI and Annexin V double positive cells) after 24 hours incubation was higher in 24 hours in comparison with 48 hours incubation in both cell lines. The frequency of PI positive MDA-MB 468 cells was higher than PI and Annexin V double positive cells after 48 hours. PI positive MDA-MB 468 cells were significantly higher than MCF-7 cells in 24 hours incubation time. CONCLUSIONS: The results have shown that MDA-MB 468 cells were more sensitive to UV exposure and DNA fragmentation and necrosis pathway was dominant in these cells.

T cells from indolent CLL patients prevent apoptosis of leukemic B cells in vitro and have altered gene expression profile.

T cells may have a role in sustaining the leukemic clone in chronic lymphocytic leukemia (CLL). In this study, we have examined the ability of T cells from CLL patients to support the survival of the leukemic B cells in vitro. Additionally, we compared global gene expression of T cells from indolent CLL patients with healthy individuals and multiple myeloma (MM) patients. Apoptosis of purified leukemic B cells was inhibited in vitro when co-cultured with increasing numbers of autologous T cells (p < 0.01) but not autologous B and T cells of normal donors. The anti-apoptotic effect exceeded that of the anti-apoptotic cytokine IL-4 (p = 0.002) and was greater with CD8+ cells (p = 0.02) than with CD4+ cells (p = 0.05). The effect was depended mainly on cell-cell contact although a significant effect was also observed in transwell experiments (p = 0.05). About 356 genes involved in different cellular pathways were deregulated in T cells of CLL patients compared to healthy individuals and MM patients. The results of gene expression profiling were verified for 6 genes (CCL4, CCL5 (RANTES), XCL1, XCL2, KLF6, and TRAF1) using qRT-PCR and immunoblotting. Our results demonstrate that CLL-derived T cells can prevent apoptosis of leukemic B cells and have altered expression of genes that may facilitate the survival of the leukemic clone.

Tanzanian malignant lymphomas: WHO classification, presentation, ploidy, proliferation and HIV/EBV association.

BACKGROUND: In Tanzania, the International Working Formulation [WF] rather than the WHO Classification is still being used in diagnosing malignant lymphomas (ML) and the biological characterization including the HIV/EBV association is sketchy, thus restraining comparison, prognostication and application of established therapeutic protocols. METHODS: Archival, diagnostic ML biopsies (N = 336), available sera (N = 35) screened by ELISA for HIV antibodies and corresponding clinical/histological reports at Muhimbili National Hospital (MNH) in Tanzania between 1996 and 2006 were retrieved and evaluated. A fraction (N = 174) were analyzed by histopathology and immunohistochemistry (IHC). Selected biopsies were characterized by flow-cytometry (FC) for DNA ploidy (N = 60) and some by in-situ hybridization (ISH) for EBV-encoded RNA (EBER, N = 37). RESULTS: A third (38.8%, 109/281) of the ML patients with available clinical information had extranodal disease presentation. A total of 158 out of 174 biopsies selected for immunophenotyping were confirmed to be ML which were mostly (84. 8%, 134/158) non-Hodgkin lymphoma (NHL). Most (83.6%, 112/134) of NHL were B-cell lymphomas (BCL) (CD20+), of which 50.9%, (57/112) were diffuse large B-cell (DLBCL). Out of the 158 confirmed MLs, 22 (13.9%) were T-cell [CD3+] lymphomas (TCL) and 24 (15.2%) were Hodgkin lymphomas (HL) [CD30+]. Furthermore, out of the 60 FC analyzed ML cases, 27 (M:F ratio 2:1) were DLBCL, a slight majority (55.6%, 15/27) with activated B-cell like (ABC) and 45% (12/27) with germinal center B-cell like (GCB) immunophenotype. Overall, 40% (24/60) ML were aneuploid mostly (63.0%, 17/27) the DLBCL and TCL (54.5%, 6/11). DNA index (DI) of FC-analyzed ML ranged from 1.103-2.407 (median = 1.51) and most (75.0%) aneuploid cases showed high (>40%) cell proliferation by Ki-67 reactivity. The majority (51.4%, 19/37) of EBER ISH analyzed lymphoma biopsies were positive. Of the serologically tested MLs, 40.0% (14/35) were HIV positive, mostly with high (> or =40.0%) Ki-67 reactivity. CONCLUSIONS: According to the 2001 WHO Classification, most subtypes are represented in Tanzanian ML. Extranodal presentation was common among MNH lymphoma patients who also showed high aneuploidy, tumor proliferation (KI-67) and EBER positivity. DLBCL was frequent and phenotype heterogeneity appeared similar to observations in Western countries suggesting applicability of established intervention approaches. HIV was apparently associated with high ML cell proliferation but extended studies are needed to clarify this.

Human herpesvirus-8 (HHV-8) sero-detection and HIV association in Kaposi's sarcoma (KS), non-KS tumors and non-neoplastic conditions.

BACKGROUND: The association of the human herpesvirus-8/Kaposi's sarcoma (KS)-associated herpesvirus (HHV-8/KSHV) serology with various malignancies in Tanzania is not currently well established while previous studies were based on either PCR or immunofluorescence assays [IFA] but not with a sensitive enzyme-linked immunosorbent assay (ELISA). Selected archival diagnostic biopsies (n = 184) and sera from indigenous patients with KS (n = 120), non-KS tumors (n = 24) and non-neoplastic lesions (n = 40) at Muhimbili National Hospital (MNH), Tanzania, were evaluated by diagnostic histopathology, immunohistology [anti-HHV-8 latency-associated nuclear antigen (LANA)] and serology for HIV (ELISA) and HHV-8 (IFA and ELISA). RESULTS: About 66.3% (n = 122) cases including AIDS-associated Kaposi's sarcoma (AKS) (n = 93), reactive conditions (n = 28) and only one non-KS tumour were HIV positive. Endemic KS (EKS) patients were mostly males (96.3%, 26/27) who were less (69.9%, 65/93) predominant in AIDS-associated (AKS). A high (89%) percentage of patients with anti-HHV-8 antibodies was found in the cohort including the HIV positive (92%) cases, males (81.2%), KS patients (93%), non-KS tumors (92%), and reactive conditions (75%). All HHV-8 seronegative KS cases were nodular stage whereas both sera and corresponding biopsies from early stage KS were HHV-8+. Assay sensitivity, positive predictive value (PPV) and specificity were 98.6%, 93.5% and 16.7% for IFA and 93.5%, 98.6% and 50.0% for ELISA respectively. CONCLUSION: HHV-8 seroprevalence at MNH appears high as expected among AKS cases and males but also in non-KS patients. ELISA showed a combination of high HHV-8 sensitivity as well as higher PPV and specificity than IFA which however, showed higher sensitivity. The apparent stage-dependent, inverted serum HHV-8 immunoreactivity supports a notion of viral immune-segregation during KS development. Routine HHV-8 screening should be considered particularly in patients at risk of KS and for selection of blood/organ donations.

Kaposi's sarcoma herpesvirus load in biopsies of cutaneous and oral Kaposi's sarcoma lesions.

OBJECTIVES: To evaluate human herpesvirus 8/Kaposi's sarcoma associated herpesvirus (HHV-8/KSHV) viral load in diagnostic, (formalin fixed, paraffinised) biopsies and patient serum during tumour progression of oral and cutaneous AIDS-related Kaposi's sarcoma (AKS), and endemic Kaposi's sarcoma (EKS) by a sensitive and specific quantitative real time polymerase chain reaction (qRT-PCR) assay. STUDY DESIGN: Eighty six biopsies of both AKS (oral and cutaneous AKS, 68) and EKS (cutaneous EKS, 18) were evaluated by qRT-PCR and immunohistochemistry (IHC). The viral load in human tumour tissue and serum of some individual patients were compared. RESULTS: Higher viral load as well as frequency of latency-associated nuclear antigen (LANA)+ tumour spindle cells (SC) and number of LANA granules per SC was found in oral AKS compared to cutaneous AKS. Although few cases were available, serum viral load appeared to decrease compared to tumour tissue during KS progression. CONCLUSIONS: The higher viral load in oral rather than cutaneous AKS is consistent with the well recognised reservoir function of the oral mucosa. Decrease of serum HHV-8 load during KS progression may indicate decreased virus release and/or increased virus clearance.

Oral Kaposi's sarcoma in Tanzania: presentation, immunopathology and human herpesvirus-8 association.

Oral Kaposi's sarcoma (OKS) from Tanzanian patients (78) at Muhimbili National Hospital/Muhimbili University College of Health Sciences corresponding to approximately 10% of KS registered during 1990-2005, were diagnosed (ELISA) as HIV-infected (OAKS) (74/78) and endemic KS (4/78). Females were 69.2% (54/78) with median age 31 and males 30.8% (24/78) with median age 38. More males (50%) had systemic KS than females (37%) and 4-times more multicentric OKS. All tested (34) oral KS patients sera had HHV-8 antibodies. Available (31/78) blood showed very low CD4+ T-lymphocyte counts. Most OKS (61.5%) had nodular histology. Immunostaining showed adult male nodular OAKS to have a significantly higher frequency of viral LANA+, endothelial CD34+ tumour spindle-cells (SC) and more Ki-67+ (median =24.1%) proliferating cells compared to females (17.2%). Juvenile nodular OAKS had more LANA+ and Ki-67+ cells than corresponding adult cases. Significantly more LANA+ and Ki-67+ cells were found in nodular OAKS compared to cutaneous HIV/AIDS Kaposi's sarcoma (CAKS). A positive correlation (60%) was found between the proliferation index (Ki-67+ cell frequency) and LANA+/CD34+ SC. OKS in Tanzania is since 1990, mostly seen in females, associated with HIV/AIDS and advanced (nodular) histopathology. Males have more systemic tumour burden while more females develop primary OAKS. HHV-8+ cells were more frequent in nodular male than female and in juvenile than adult nodular OAKS than cAKS. Higher tumoral HHV-8 content appeared to be correlated to proliferation index.

KSHV/HHV-8 and HIV infection in Kaposi's sarcoma development.

Kaposi's sarcoma (KS) is a highly and abnormally vascularized tumor-like lesion affecting the skin, lymphnodes and viscera, which develops from early inflammatory stages of patch/plaque to late, nodular tumors composed predominant of spindle cells (SC). These SC are infected with the Kaposi's sarcoma-associated herpesvirus or human herpesvirus-8 (KSHV/HHV-8). KS is promoted during HIV infection by various angiogenic and pro-inflammatory factors including HIV-Tat. The latency associated nuclear antigen type 1 (LANA-1) protein is well expressed in SC, highly immunogenic and considered important in the generation and maintenance of HHV-8 associated malignancies. Various studies favour an endothelial origin of the KS SC, expressing "mixed" lymphatic and vascular endothelial cell markers, possibly representing hybrid phenotypes of endothelial cells (EC). A significant number of SC during KS development are apparently not HHV8 infected, which heterogeneity in viral permissiveness may indicate that non-infected SC may continuously be recruited in to the lesion from progenitor cells and locally triggered to develop permissiveness to HHV8 infection. In the present study various aspects of KS pathogenesis are discussed, focusing on the histopathological as well as cytogenetic and molecular genetic changes in KS.