SLC35B4, an Inhibitor of Gluconeogenesis, Responds to Glucose Stimulation and Downregulates Hsp60 among Other Proteins in HepG2 Liver Cell Lines.

SLC35B4, solute receptor for UDP-N-acetylglucosamine and UDP-xylose, is associated with diabetes and predisposing conditions. This study investigated the localization of SLC35B4 and compared the differential expression between a knockdown of SLC35B4 and controls in HepG2. Responsiveness to glucose, expression, and localization were assayed using Western blot and immunostaining. Localization was confirmed using a proximity ligation assay. Two-dimensional (2D) gel electrophoresis and MALDI-TOF were used to identify differentially expressed proteins and pathway analysis was performed. SLC35B4 was increased by 60% upon glucose stimulation and localized in Golgi apparatus and endoplasmic reticulum. Presence of SLC35B4 in the Golgi apparatus suggests its involvement in the biosynthesis of glycoconjugate proteins. Four proteins were markedly under-expressed (Hsp60, HspA8, TUBA1A, and ENO1) and linked to the pathogenesis of diabetes or post-translationally modified by O-GlcNAc. Glucose levels activate SLC35B4 expression. This triggers a downstream effect via Hsp60 and other proteins. We hypothesize that the downstream effect on the proteins is mediated via altering the glycosylation pattern inside liver cells. The downstream cascade ultimately alters the ability of cultured liver cells to inhibit endogenous glucose production, and this could play a role in the association of the above-listed genes with the pathogenesis of diabetes.

Effective targeting of chronic myeloid leukemia initiating activity with the combination of arsenic trioxide and interferon alpha.

Imatinib is the standard of care in chronic meloid leukemia (CML) therapy. However, imatinib is not curative since most patients who discontinue therapy relapse indicating that leukemia initiating cells (LIC) are resistant. Interferon alpha (IFN) induces hematologic and cytogenetic remissions and interestingly, improved outcome was reported with the combination of interferon and imatinib. Arsenic trioxide was suggested to decrease CML LIC. We investigated the effects of arsenic and IFN on human CML cell lines or primary cells and the bone marrow retroviral transduction/transplantation murine CML model. In vitro, the combination of arsenic and IFN inhibited proliferation and activated apoptosis. Importantly, arsenic and IFN synergistically reduced the clonogenic activity of primary bone marrow cells derived from CML patients. Finally, in vivo, combined interferon and arsenic treatment, but not single agents, prolonged the survival of primary CML mice. Importantly, the combination severely impaired engraftment into untreated secondary recipients, with some recipients never developing the disease, demonstrating a dramatic decrease in CML LIC activity. Arsenic/IFN effect on CML LIC activity was significantly superior to that of imatinib. These results support further exploration of this combination, alone or with imatinib aiming at achieving CML eradication rather than long-term disease control.

Diagnostic and therapeutic advances in blastic plasmacytoid dendritic cell neoplasm: a focus on hematopoietic cell transplantation.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is an exceedingly rare disorder categorized under acute myeloid leukemia by the World Health Organization. Phenotypically, malignant cells coexpress CD4(+) and CD56(+) without coexpressing common lymphoid or myeloid lineage markers. BPDCN frequently expresses CD123, TCL1, BDCA-2, and CD2AP. Restriction of CD2AP expression to plasmacytoid dendritic cells makes it a useful tool to help confirm diagnosis. Clonal complex chromosome aberrations are described in two-thirds of cases. Eighty percent of BPDCN cases present with nonspecific dermatological manifestations, prompting inclusion in the differential diagnosis of atypical skin rashes refractory to standard treatment. Prognosis is poor, with a median survival of less than 18 months. No prospective randomized data exist to define the most optimal frontline chemotherapy. Current practice considers acute myeloid leukemia-like or acute lymphoblastic leukemia-like regimens acceptable for induction treatment. Unfortunately, responses are short-lived, with second remissions difficult to achieve, underscoring the need to consider hematopoietic cell transplantation early in the disease course. Allografting, especially if offered in first remission, can result in long-term remissions. Preclinical data suggest a potential role for immunomodulatory agents in BPCDN. However, further research efforts are needed to better understand BPDCN biology and to establish evidence-based treatment algorithms that might ultimately improve overall prognosis of this disease.

Implementation of an intensive risk-stratified treatment protocol for children and adolescents with acute lymphoblastic leukemia in Lebanon.

With modern risk-adapted therapy, over 80% of children with acute lymphoblastic leukemia (ALL) in high-income countries (HICs) are cured. In countries with limited resources, however, therapy results for pediatric ALL are still not encouraging. We describe our experience in treating children with ALL using a risk-adapted protocol at a tertiary referral center in Lebanon. From May 2002 to August 2009, 111 consecutive patients 1-21 years of age with newly diagnosed ALL received the CCCL ALL protocol which was based on the St. Jude Children's Research Hospital Total XV Study. The median age at diagnosis was 5 years 5 months. The male to female ratio was 1.5. Forty-six patients received the intermediate-/high-risk arm and 65 received the low-risk arm. Only one patient (0.9%) died during induction therapy. Relapse occurred in 8 (7.2%) patients. Eight (7.2%) patients died, 4 of whom were in remission. The median follow-up of the patients was 38 months. The 5-year overall survival and event-free survival were and 88.5% (95% CI: 77.1-94.4) and 78.7% (95% CI: 69.8-88.4), respectively. Our results are comparable to those in HICs in spite of the limited resources and the relatively low socioeconomic status of the Lebanese population. Children treated on this protocol experienced significant toxicity necessitating expert supportive care, but benefited from improved cure rates and prolonged survival.

Should we screen for hereditary hemochromatosis in healthy Lebanese: a pilot study.

Hereditary hemochromatosis (HHC) is a genetic disorder of iron metabolism characterized by abnormal accumulation of iron that may lead to organ damage and death. Diagnosis is usually based on various genetic and phenotypic criteria. The study goals were to perform mutation analysis for 18 different mutations associated with HHC in healthy Lebanese, determine their allele frequency, and compare iron-overload status in identified carriers versus those found to be wild-type for mutations analyzed. 116 healthy adults (59 males and 57 females) underwent DNA testing for 18 different HHC mutations, and biochemical testing for percent transferrin saturation (%TS) and ferritin. C282Y mutation was not detected. Only H63D mutation (rs1799945) was found with an overall carrier frequency of 25.8% (24.1% heterozygous and 1.7% homozygous). %TS and ferritin differed significantly between genders. %TS and ferritin were significantly higher in males with H63D mutation when compared to males with wild-type (P=0.001, 0.019; respectively); but not in females. The proportion of subjects with increased %TS and serum ferritin was not statistically different between those with H63D mutation and the wild-type in either gender. In addition, none of the subjects had concurrent increase in %TS and ferritin. In conclusion, the H63D carrier frequency in healthy Lebanese is comparable to other populations in the region, and it does not result in significant biochemical iron overload. Moreover, in the absence of the C282Y mutation, genetic screening for HHC is not recommended according to this preliminary study in healthy Lebanese.

Comparison of neutrophil volume distribution width to C-reactive protein and procalcitonin as a proposed new marker of acute infection.

BACKGROUND: The aim of this study was to assess the use of neutrophil distribution width (NDW) and to compare it to C-reactive protein (CRP) and procalcitonin (PCT), in the detection of early sepsis in the intensive care unit. METHODS: Subjects (N = 166) were divided into 4 groups: healthy, acute inflammatory non-infectious (AINI), localized infection, and systemic infection, according to clinical history and cultures. NDW, CRP, and PCT were compared among the different groups using multivariate analysis of variance (MANOVA). Diagnostic efficacy was assessed using receiver operating characteristic curves and areas under the curves (AUC). RESULTS: The lowest mean(NDW) was found in the healthy group (n = 41), followed by the AINI (n = 20), localized infection (n = 55), and systemic infection (n = 50) groups. AUC(NDW) was 0.877 for infected (localized + systemic) vs non-infected (healthy + AINI) groups, and 0.965 for systemic infection vs non-infected groups. A cut-off of 21.9 resulted in 90% sensitivity, 92% specificity, 90% positive predictive value, and 92% negative predictive value (AUC(NDW) = 0.965, 95% confidence interval 0.935-0.995). According to MANOVA, only NDW was able to differentiate an acute inflammatory process from early infection in postoperative patients, but not healthy from AINI subjects. CONCLUSIONS: NDW had the highest diagnostic accuracy and is available with the complete blood count with differential (CBC). It may be a promising parameter to aid in the diagnosis of acute infection in adults, provided the possibility of haematological disorders is first ruled out.

Successful pregnancy involving a man with chronic myeloid leukemia on dasatinib.

Dasatinib is a highly potent Bcr-Abl inhibitor that is approved for the treatment of imatinib-resistant or -intolerant chronic myeloid leukemia (CML). The potential effects of dasatinib on sperm counts, sperm function, and fertility have not been studied yet. There is only one report in the medical literature of successful pregnancies while patients were taking dasatinib, thus making our case the second report. Here, we present the case of a 38-year-old man who conceived a healthy baby while on dasatinib therapy.

Chronic lymphocytic leukemia associated with myelodysplastic syndrome with ring sideroblasts.

Diagnosis of myelodysplastic syndrome/acute myeloid leukemia (MDS/AML) and chronic lymphocytic leukemia (CLL) in the same patient is extremely rare. We describe a 69-year-old CLL patient who developed MDS with ring sideroblasts 1 year after diagnosis of CLL and without any previous treatment. Diagnosis was based on flow cytometry, bone marrow aspirate morphology, and iron stain. Our findings indicate that the 2 disorders belong to 2 different hematopoietic clones in this patient. In cases of worsening anemia in CLL patients, it is recommended that an iron stain be performed on bone marrow aspirates to exclude a coexistent malignancy such as refractory anemia with ring sideroblasts.

Absence of JAK2 V617F mutation in thalassemia intermedia patients.

JAK2 is a cytoplasmic tyrosine kinase that has a vital role in signal transduction from several hemopoietic growth factor receptors. The JAK2 V617F mutation has been implicated in a variety of diseases mainly related to myeloproliferative disorders including polycythemia Vera, essential thrombocythemia, and idiopathic Myelofibrosis but has not been previously described in Thalassemia patients. We studied 36 Lebanese patients diagnosed with thalassemia intermedia and assessed the presence or absence of the JAK2 V617F mutation using JAK2 activating mutation assay (In VivoScribe Technologies) and Polymerase Chain Reaction (PCR). None of the thalassemia intermedia patients were positive for this mutation. To our knowledge, this study is the first to determine the status of JAK2 V617F mutation in thalassemia intermedia patients and expands the international published literature on JAK2. The latter's V617F mutation does not seem to play a role in this hematologically important clinical entity.

Prevalence of the prothrombin G20210A polymorphism in the Lebanese population: use of a reverse hybridization strip assay approach.

The factor II (prothrombin) G20210A gene polymorphism is the second most common SNP reported in VTE where it is associated with elevated plasma prothrombin levels and with a 3-fold increased risk. We studied the distribution of the G/G, G/A, and A/A genotypes of the Prothrombin G20210A gene mutation in the general Lebanese population using a novel technique in order to assess their prevalence, compare the results to previously reported data and to describe an available method that will permit easy and fast identification of the mutation. Prothrombin different genotypes were determined using the Cardiovascular Disease (CVD) StripAssay which is based on a Polymerase Chain Reaction-Reverse hybridization technique and DNA from 205 unrelated healthy donors from our HLA-bank was used. The prevalence of G/G, G/A, and A/A genotypes was found to be 98.54, 1.46, and 0%, respectively, with G and A allelic frequency of 99 and 1%, respectively. The sampled Lebanese population showed prothrombin genotypes distribution similar to Caucasians, and our results are comparable to other reports on the Lebanese healthy individuals. However, this is the first report on the prevalence of prothrombin G20210A mutation using this technique. Our results suggest that this approach is reliable and can be used as an assessment for thrombophilia profile. In addition, future investigations should be conducted to assess the contribution of the prothrombin G20210A mutation, on its own and in collaboration with other factors, in various clinical entities notably VTE.

Factor V HR2 haplotype: a risk factor for venous thromboembolism in individuals with absence of Factor V Leiden.

Venous thromboembolism (VTE), including deep venous thrombosis (DVT) and pulmonary embolism (PE), occurs secondary to a number of hereditary and acquired disorders of hemostasis. A recently recognized polymorphism in Factor V (FV) gene H1299R (also named HR2) has been reported to be a possible risk factor for the development of VTE. The aim of this study is to evaluate the role of HR2 polymorphism in VTE in a group of Lebanese patients. Seventy-three VTE patients and 125 healthy subjects were examined for HR2. The average ages for the patients and controls were 45.0+/-19.1 years and 35.4+/-18.6 years, respectively. Sixty patients (82.2%) had DVT, eight patients (11%) had PE, and five patients (6.8%) had both. There was significant association between FV Leiden and VTE (p<0.001). HR2 haplotype had a prevalence of 16.4% in patients. VTE patients with normal FV were 2.7 times more likely to have the HR2 haplotype as compared to controls with normal FV (p=0.036, 95% CI=1.04-7.06). We conclude that the FV HR2 haplotype significantly affects the risk of VTE in subjects with normal FV. This finding entails that screening for the HR2 haplotype should be done in VTE patients with normal FV Leiden results.

High prevalence of MTHFR gene A1298C polymorphism in Lebanon.

BACKGROUND: Mutations in the 5,10-methylenetetrahydrofolate reductase (MTHFR) gene could reduce the enzyme activity and lead to hyperhomocysteinemia, a condition that has been associated with several vascular conditions, in particular, coronary artery disease and deep vein thrombosis. AIM: The aim of this study was to assess the prevalence of the two most common polymorphisms, C677T and A1298C, which have not been well studied in the Lebanese population. METHODS: We randomly selected 205 healthy individuals originating from different Lebanese provinces and religious communities. The CVD StripAssay was used to test for MTHFR gene polymorphisms. RESULTS: We found that for C677T, the prevalence of C/C, C/T, and T/T genotypes was 65.3%, 30.8%, and 3.9%, respectively, with an overall carrier rate of 34.6% and allelic frequency of 0.19. However, the A1298C genotypic prevalence of A/C, A/A, and C/C was 50.2%, 25.9%, and 23.9%, respectively, with an overall carrier rate of 74.14% and an allelic frequency of 0.49. CONCLUSIONS: Compared to all other populations reported so far, the Lebanese population harbors the highest prevalence of the MTHFR A1298C polymorphism. This is an important finding to be followed in terms of clinical significance and sheds light on an additional unique genetic feature in this community.

An overview of the pathogenesis and natural history of post-transplant T-cell lymphoma (corrected and republished article originally printed in Leukemia & Lymphoma, June 2007; 48(6): 1237 - 1241).

Post-transplantation lymphoproliferative disorders (PTLDs) are well recognized complications of solid organ transplantation. The vast majority of early PTLDs are B-cell non-Hodgkin lymphomas. PTLDs of T-cell origin occur much less frequently and account for only a minority of cases. T-cell PTLDs have been reported to occur primarily at extranodal sites, commonly affecting bone marrow or splenic tissues. The small bowel is an uncommon site of origin of these tumors with only seven cases of primary intestinal post-transplant T-cell lymphomas reported in the literature. We hereby report a new case of primary intestinal post-transplant T-cell lymphoma, arising 18 years following renal transplantation, along with a literature review of all published cases.

Recombinant activated factor VII for intractable bleeding post splenectomy in a patient with myeloproliferative disorder.

Recombinant activated factor VII has been Food and Drug Administration approved to treat hemorrhages in hemophiliac patients with inhibitors and in acquired hemophilia patients. Recombinant activated factor VII use has also been considered for the management of uncontrolled bleeding in a number of congenital and acquired hemostatic abnormalities. The myeloproliferative disorders are a group of clonal hematologic diseases where, frequently, abnormal platelet function is considered a hallmark. This is the first case report addressing the clinical benefit of off-label use of recombinant activated factor VII in an attempt to control intractable bleeding in a patient with a myeloproliferative disorder after splenectomy.

Prevalence of factor V Leiden, prothrombin and methylene tetrahydrofolate reductase mutations in women with adverse pregnancy outcomes in Lebanon.

OBJECTIVE: The purpose of this study was to determine the prevalence of factor V Leiden, prothrombin, and methylene tetrahydrofolate reductase gene mutations in women with adverse pregnancy outcome compared with women who had uneventful pregnancies. STUDY DESIGN: Between 2003 and 2005, pregnant women with > or = 1 unexplained second trimester abortion, > or = 1 intrauterine fetal death, severe preeclampsia, or severe intrauterine growth restriction (study subjects) were compared with control subjects (uneventful pregnancy) for the frequency of the mutations. RESULTS: The cases of 91 patients in each arm were analyzed. Obstetric complications were second trimester abortions (16.5%), intrauterine fetal death (53.8%), preeclampsia (8.8%), and severe intrauterine growth restriction (20.9%). Study subjects were more likely to be older and multiparous compared with control subjects. The 2 groups showed no difference in the incidence of smoking or family history of thrombosis, but study subjects were more likely to have a positive family history of obstetric complications. The prevalence of factor V Leiden (12.1% vs 18.7%; P = .304), prothrombin (7.7% vs 5.5%; P = .765), methylene tetrahydrofolate reductase gene mutations (53.8% vs 65.9%; P = .130), and > 1 mutation (11.0% vs 17.6%; P = .290) was not significantly different between study subjects and control subjects. CONCLUSION: Factor V Leiden, prothrombin, and methylene tetrahydrofolate reductase gene mutations did not seem to play a significant role in adverse pregnancy outcome in our population.

Prevalence of factor V R2 (H1299R) polymorphism in the Lebanese population.

AIMS: A recently identified polymorphism in factor V gene (His1299Arg; also named HR2) has been reported to be a possible risk factor for the development of venous thromboembolism (VTE), with a high prevalence of 9.5-15.2% in patients of different ethnic groups in different parts of the world. The aim of this study is to assess the prevalence of HR2 haplotype in Lebanon. METHODS: We randomly selected 125 samples from unrelated donors logged into our HLA registry; these represent healthy Lebanese individuals originating from different provinces and religious communities of the country. Their DNA was extracted using the Pel-Freez extraction kit and stored at -80 degrees C for later use. The CVD StripAssay was used for PCR and reverse hybridisation. It screens for several gene mutations including factor V H1299R. RESULTS: A total of 125 controls were studied: 72 males and 53 females with a median age 42 years. Thirteen (10.4%) had the HR2 haplotype; 11 (8.8%) were heterozygous (R1/R2), and two (1.6%) were homozygous (R2/R2), with an allelic frequency of 0.06. CONCLUSIONS: Our study is the first report from Lebanon that describes the prevalence of HR2 haplotype and the frequency of its alleles. We are reporting a high prevalence of the HR2 in our population (10.4%). The hypothesis that A4070G polymorphism might contribute to the expression of a thrombotic phenotype deserves to be tested in our population through larger studies.

First report on HLA-DPA1 gene allelic distribution in the general Lebanese population.

AIMS: HLA-DPA1 is an important marker in bone marrow and organ transplantation and a highly emerging screening parameter in histocompatibility laboratories. Being highly polymorphic, it has another significant value in detecting population origins and migrations. This is the first study to assess DPA1 allele frequencies in an Arab population. METHODS: The HLA DPA1 alleles were identified using the One-Lambda assays on a Luminex reverse SSO DNA typing system. Our study included 101 individuals coming from different Lebanese geographical areas representing the different communities and religious sects of the country. RESULTS: We compared the results of this study to 16 different populations and found very interesting similarities and differences between Lebanese people and individuals of European ancestry. CONCLUSION: This study is the first to describe the different allelic frequencies of HLA-DPA1 in the Lebanese population and will serve as a template that can be later used for disease association studies both at the level of the country and internationally.

Potential role of killer immunoglobulin receptor genes among individuals vaccinated against hepatitis B virus in Lebanon.

AIM: To explore the role of killer immunoglobulin receptor (KIR) genes in responsiveness or non-responsiveness to vaccination against hepatitis B virus. METHODS: We recruited 101 voluntary participants between March 2010 and December 2011. Sera samples from vaccinated and non-vaccinated participants were tested for the presence of anti-HBs antibodies as a measure of protection against hepatitis B, hepatitis B surface antigen and hepatitis B core antibody as indicators of infection by enzyme-linked immunosorbent assay. KIR gene frequencies were determined by polymerase chain reaction. RESULTS: Sera samples from 99 participants were tested for the levels of anti-HBs as an indicator of protection (≥ 10 mIU/mL) following vaccination as defined by the World Health Organization international reference standard. Among the vaccinated participants, 47% (35/74) had anti-HBs titers above 100 mIU/mL, 22% (16/74) had anti-HBs ranging between 10-100 mIU/mL, and 20% (15/74) had values of less than 10 mIU/mL. We report the lack of significant association between the number of vaccine dosages and the titer of antibodies among our vaccinated participants. The inhibitory KIR2DL1, KIR2DL4, KIR3DL1, KIR3DL2, and KIR3DL were detected in more than 95%, whereas KIR2DL2, KIR2DL3, KIR2DL5 (KR2DL5A and KIR2DL5B) were expressed in 56%, 84% and 42% (25% and 29%) of participants, respectively. The observed frequency of the activating KIR genes ranged between 35% and 55% except for KIR2DS4, detected in 95% of the study participants (40.6% 2DS4*001/002; 82.2% 2DS4*003/007). KIR2DP1 pseudogene was detected in 99% of our participants, whereas KIR3DP*001/02/04 and KIR3DP1*003 had frequencies of 17% and 100%, respectively. No association between the frequency of KIR genes and anti-HBs antibodies was detected. When we compared the frequency of KIR genes between vaccinated individuals with protective antibodies titers and those who lost their protective antibody levels, we did not detect a significant difference. KIR2DL5B was significantly different among different groups of vaccinated participants (group I > 100 mIU/mL, group II 10-100 mIU/mL, group III < 10 mIU/mL and group IV with undetectable levels of protective antibodies). CONCLUSION: To our knowledge, this is the first study screening for the possible role of KIR genes among individuals vaccinated against hepatitis B virus (HBV). Our results can be used to design larger studies to better understand the role of KIR genes in protection against or susceptibility to HBV post vaccination.

Thymoma with Concomitant Pure Red Cell Aplasia, Good's Syndrome and Myasthenia Gravis Responding to Rituximab.

Thymomas are often associated with a variety of autoimmune diseases, mostly myasthenia gravis. The association of thymomas with both pure red cell aplasia (PRCA) and Good's syndrome is exceedingly rare. To the best of our knowledge, the combination of a thymoma with manifestations of myasthenia gravis, PRCA, and Good's syndrome, as in our case herein, has not been described before in the medical literature. We present a 90-year-old man initially diagnosed with an asymptomatic thymoma. Later, he developed generalized muscle weakness and was found to have severe anemia. He was diagnosed with PRCA, myasthenia gravis and Good's syndrome. He responded to rituximab with restoration of bone marrow erythroid maturation and stabilization of red blood cell counts.

Vitamin D receptor biochemical and genetic profiling and HLA-class II genotyping among Lebanese with multiple sclerosis - A pilot study.

BACKGROUND: Multiple sclerosis (MS) is an autoimmune demyelinating disease affecting mostly young adult females with multifactorial etiology. Recent studies suggested that adequate vitamin D levels may lower the risk of developing MS. OBJECTIVES: Our aim was to explore the relationship between vitamin D receptor (VDR) polymorphism, HLA-DR locus genotype, and serum vitamins D and A levels in the Lebanese population. METHODS: Fifty MS patients were recruited for this study. The control group consisted of 48 healthy and 51 patients with other neurological disorders (non-MS). Biochemical analysis included serum 25 hydroxyvitamin D (25OHD) and vitamin A. Molecular analysis targeted VDR genotypes (ApaI, TaqI and BsmI) and low resolution HLA typing for DRB1 locus. RESULTS: Healthy and non-MS groups had comparable parameters and were combined into one control group. No significant differences were found between MS and control groups for VDR genotypes. The frequency of HLA-DRB1*15 was significantly higher in MS patients (22%) compared to controls (8%) (p=0.018). Odds ratio for MS in the presence of DRB1*15 allele was 3.21 (p=0.018). Cosegregation with A (ApaI) and b (BsmI) alleles did not influence the risk for MS. 25OHD levels were significantly higher in MS patients compared to controls (p=0.002), due to more frequent oral supplementation (p=0.005). Vitamin A levels were comparable between the two groups. When all parameters were included in a logistic regression model adjusted for supplementation, only HLA-DRB1*15 (OR=3.42; p=0.027) contributed significantly to MS risk. CONCLUSION: There was no association between serum vitamin D or A or VDR genotypes and MS. HLA-DRB1*15 was the major factor imposing more than 3 folds greater risk for developing MS among Lebanese.