Vascular endothelial growth factor [VEGF-C] signaling through KDR [VEGFR-2] and FLT-4 [VEGFR-3] mediates leukemic cell proliferation and survival

VEGF, a key angiogenic molecule, is a multifunctional cytokine that acts both as a potent inducer of vascular permeability and as a specific endothelial cell mitogen. Because of its effects on endothelial cell growth and microvascular permeability, VEGF is believed to be an important mediator of tumor angiogenesis. Leukemic cells not only release VEGF but also express its receptors, resulting in the establishment of an autocrine loop that supports their migration and survival. VEGF-C may play an important role in the pathophysiology of hematopoietic malignancies by not only regulation of lymphangiogenesis, in vivo, but also by promotion of angiogenesis invasion of neoplastic cells into lymphatic vessels and enhancing lymphatic metastasis during tumor progression. although it is well established that growth in solid tumors is dependent on the formation of neovasculature, the role of angiogenesis in hematopoietic neoplasms has not been determined. The present study was undertaken to identify whether VEGF-C and its receptors VEGFR-2 [KDR] and VEGFR-3 [FLT-4] were expressed in patients with denovo acute leukemia by RT-PCR and to evaluate the relationship between their expression and clinical, laboratory findings and prognosis. Using reverse transcription polymerase chain reaction analysis [RT-PCR], 30 de novo acute leukemia patients [20 ALL patients and 10 AML patients] as well as 10 controls were tested for the expressions of VEGF-C, VEGFR-3 [FLT 4] and VEGFR-2 [KDR] genes. In the current study, VEGF-C, FLT-4 and KDR were detected in 10% of control samples. In ALL patients VEGF C was expressed in 65% of cases, FLT-4 in 70% of cases and KDR in 30% of cases. The expressions of VEGF-C, FLT-4 and KDR in ALL patients were associated with increased risk of leukemia [with OR 16.7 and 95% CI 1.7-160.4, OR 21.0 and 95% CI 2.2-204.6 and with OR 3.9 and 95% CI 0.4-37.6 respectively]. In AML patients, VEGF-C was expressed in 60% of cases, FLT-4 in 70% of cases and KDR in 40% of cases. The expression of VEGF-C, FLT-4 and KDR in AML patients was associated with increased risk of leukemia [with OR 13.5 and 95% CI 1.2-152.2, OR 21.0 and 95% CI 1.8-248.1 and OR 6.0 and 95% CI 0.5-67.7 respectively]. In the 6 followed-up ALL patients, 3 [50%] were in remission, three of them were VEGF-C negative, 2 were FLT4 positive and 1 was KDR positive. 2 of the 6 ALL patients [33.3%] were resistant to treatment, both were VEGF-C positive, FLT-4 positive and 1 was KDR positive. One of the 6 ALL patients [16.6% died during induction, this patient was VEGF-C positive and FLT-4 and KDR negative. In the 4 followed-up AML patients, 3 of them [75%] were in remission, 1 of the 3 was VEGF-C positive and 2 were FLT-4 positive. One of the four AML patients [25%] was resistant to treatment, this patient was VEGF-C, FLT-4 and KDR positive. The number of VEGF-C positive patients with no treatment failure was lower than the number of VEGF-C positive patients with treatment failure. Also, the risk of failed induction was found to be greater in VEGF-C positive patients than in VEGF-C negative patients, thus, the expression of VEGF-C and its receptors [FLT-4 and KDR] in ALL and AML patients was associated with increased risk of leukemia and unfavorable treatment outcome. VEGF-C and its receptors KDR [VEGFR-2] and FLT-4 [VEGFR-3] may play an important role in the pathophysiology of hematopoietic malignancies and may actually contribute to the development of leukemia. Also, owing to the importance of angiogenesis in tumor progression and the effects of VEGF-C, KDR and FLT-4 in chemotherapy-treated leukemias, inhibition of VEGF-C signaling represents an attractive cancer treatment

Molecular screening for G6PD gene mutations in children with glucose-6-phosphate dehydrogenase deficiency

Glucose-6-phosphate dehydrogenase [G6PD] deficiency is a heterogeneous enzyme abnormality with a high frequency among people of African, Mediterranean and Southeast Asian origins. In almost every group studied in the Middle East, only three to four different G6PD mutations were detected. Among these, the G6PD Mediterranean mutation [563C -> T] was by far the most common. Apart from this mutation, little is known about the genetic heterogeneity of G6PD deficiency in Egypt. To screen for G6PD gene mutations in a group of Egyptian children with G6PD-deficiency who were previously screened for the Mediterranean [563C -> T] mutation. This work was conducted on twenty-one unrelated Egyptian children [17 males and 4 females] presenting with G6PD deficiency previously screened for the G6PD Mediterranean mutation [563C -> T]. Carefully-preserved DNA of patients refrigerated at -20°C and DNA of 21 age-matched normal subjects extracted from blood leukocytes by saltingout technique were screened for mutations in the G6PD gene by PCR-single strand conformation polymorphism [SSCP] analysis followed by DNA sequencing. In addition to the G6PD Mediterranean mutation 563C -> T previously identified by PCR-RFLP analysis in 6/21 male patients [28.6%], a further of 2 different mutations; G6PD A- mutation and G6PD Chatham were observed in 2/21 [9.5%] and 1/21 [4.8%] patients respectively. Twelve patients [57.1%] remained uncharacterized at the genetic level, a normal African G6PD A genotype was detected in one patient of them. Patients with G6PD Mediterranean mutation were more susceptible to hemolysis than were patients with G6PD A- and G6PD Chatham mutations. The lower prevalence of G6PD Mediterranean mutation in our patients and the finding of three different mutations in a relatively small number of G6PD-deficient subjects reflect the considerable genetic heterogeneity of G6PD deficiency of the Egyptian population

Chlamydia pneumoniae [TWAR strain] in respiratory tract infections: a comparative study between different methods for its isolation

Chlamydia pneumoniae [C. peumoniae], is a common pathogen found in the respiratory tract, it has a seroprevalence rate of > 70% in the adult population, and it is considered responsible for about 10% of the cases of community-acquired pneumonia. Owing to the difficulties in the laboratory diagnosis of C. pneumoniae infection, a routine diagnostic service for this pathogen is not currently available. Moreover, diagnostic differences between the various techniques are not fully resolved. The present study was conducted to evaluate the utility of Direct Fluorescent Antibody [DFA], Tissue Culture [TC], PCR and microimmunofluorescence [MIF] assays for the diagnosis of C. pneumoniae infection in patients having respiratory symptoms. A total of 45 throat and retropharyngeal swabs as well as broncho-alveolar lavage specimens -collected from patients suffering from respiratory tract infections [RTIs]- were tested for the presence of C. pneumoniae by Direct Fluorescent Antibody staining [DFA, Bio Merieux], tissue culture on Hep-2 cell monolayer [Vacsera], PCR using species specific primer pair HR-1/MH-1 [Dia-sorin], and their sera were tested for the presence of IgG antibodies against C. pneumoniae by microimmunofluorescence assays [Vircell, S.L]. PCR gave the highest positive detection rate [44.4%], followed by DFA [28.8%], then TC [22.2%], while MIF gave the least [15.5%]. According to the clinical conditions; acute exacerbation of bronchial asthma, acute bronchitis, pneumonia and asthmatic bronchitis gave positive results with DFA, TC and PCR. Acute exacerbation of chronic obstructive pulmonary disease [COPD] gave positive results with DFA and PCR. PCR is the only assay that gave positive results with chronic bronchitis. In conclusion the difference between the various techniques applied for the diagnosis of C. pneumoniae infection is not fully resolved and it is difficult to define a perfect gold standard

Response of plasma adrenocortical steroids to low doses adrenocorticotrophic hormone compared to standard dose in normal Egyptian subjects

The short ACTH stimulation test, using 250 micro g synthetic adrenocorticotropic hormone [ACTH], has been advocated as a safe alternative procedure to the dangerous insulin stress test, for assessment of the function of the hypothalamic-pituitary-adrenal [HPA] axis. However, it has been suggested that a maximal cortisol response can be achieved with a much lower ACTH dose, hinting at the possibility that reducing the dose might further enhance the sensitivity of the procedure. Aim of work: The aim of this study was to assess the potential role of the low dose ACTFI challenge in the evaluation of the HPA axis function, compared to the standard 250.micro g ACTH test in normal adults. Subjects and The study was conducted on 21 apparently healthy Egyptian adult volunteers, who underwent stimulation with three different doses [1 micro g, 5 micro g and 250 micro g] of synthetic ACTH [1-24]. Blood samples were obtained at 0, 15, 30 and 60mm for measurement of serum cortisol concentration and 0. 60min serum samples were also used to measure adrenal precursors Delta-4-androstendione [delta-4A], Dehydroepiandrosterone [DHEA] and 17- hydroxyprogesterone [17 OHP] levels, to investigate the efficiency of the low doses of ACTH for detection of late-onset congenital adrenal hyperplasia [LOCAH]. The study revealed no significant difference between mean cortisol level at 15, 30 and 60min in response to the three different doses. Using the 1 or 5 micro g doses, all subjects [100%] reached adequate cortisol response at 30min, while using 250 micro g dose, 90% of subjects reached adequate cortisol response at 60min. The steroid precursors showed no significant difference at 60min in response to the three doses. During the study, 4 cases, two males and two females, were accidentally found to have elevated steroid precursors in response to ACTH. These cases were found to have 21- hydroxylase deficiency, as evidenced by the elevated levels of stimulated 17 OHP, thus, were diagnosed as LOCAH. The low doses [1micro g and 5 micro g] ACTH test responses were found almost comparable to the 250 micro g doses on normal subjects, and can be used to induce adequate adrenal stimulation. They were also useful in detecting latent cases of congenital adrenal hyperplasia

Spectrophotometric determination of some drugs containing phenolic group using 3-methyl-2-benzothiazolinon hydrazone hydrochloride [MBTH]

A simple, rapid, sensitive and suitable method for analysis of etilefrineHCl [I], hexoprenaline.HCl [II], levodopa [III] and carbidopa [IV] either in pure form or in pharmaceutical formulations is described. The proposed method is based on an oxidative coupling reaction with 3-methyl-2-benzothiazolinon hydrazone hydrochloride [MBTH] in the presence of an oxidant [Ce+4 or F e+3] to give coloured species. The resulting coloures are well developed within 5 minutes at 20 +/- 5°C and stable for at least 2 hours. The gamma max were 517, 477 and 474 nm for I, II, both III and IV, respectively. A linear correlation was found between the absorbance and the concentration. Beer's law was obeyed for concentration ranges from 1-7 mg/ml, l-ll mg/ml, 1-8 mg/ml and 2-24 mg/ml for I, II, III and IV, respectively. The molar absorbtivities and Sandell sensitivities were calculated. The results of analysis of pure drugs by the proposed method are in good agreament with the official BP and USP methods. The results obtained showed recoveries of 99.80 +/- 0.27, 100.40 +/- 1.26, 99.89 +/- 0.21 and 99.96 +/- 0.36 for I, II, III and IV, in order. The proposed method was successfully applied to determine the studied drugs in their pharmaceutical dosage forms

Thyroid autoimmunity and dysfunction among chronic hepatitis C patients

Forty patients with untreated chronic hepatitis C virus [HCV] infection [22 males and 18 males, their mean age was 40.6 +/- 7.9 with a range of 26-42 years] were studied. Anti-thyroperoxidase [TPO- Ab] and antithyroglobulin [TG-Ab] antibodies were assessed together with the measurement of free T3, T4 and TSH. Fine needle aspirate from thyroid gland was performed for patients with positive thyroid autoantibodies. The study concluded that a considerable number of patients with HCV infection harbored thyroid TPO-Ab and TG-Ab antibodies and had an abnormal thyroid function. The histological evidence of subacute thyroiditis could partly explain the mechanism of thyroid dysfunction in such patients. Thyroid functions should be assessed in patients with HCV infection, even in the absence of thyroid autoantibodies, especially before the initiation of treatment with interferon

Prevalence of activated protein C resistance, factor V Leiden and prothrombin 20210A mutations among patients with venous thromboembolism

Venous thromboembolism [VTE] is a multifactorial disease. Multiple interactions between genetic and environmental factor contribute to the development of the disease. Factor V [F V] Leiden and factor II[F II] G 20210A mutations are two frequent genetic risk factors involved in VTE. The goal of this case control study which was carried on 30 young patients with VTE and 20 age matched controls was to determine the prevalence of activated protein C resistance [APC-R], FV Leiden mutation as well as F II 20210A mutation as risk factors for VTE in the studied groups. Both patients and control groups were subjected to proper history taking including personal and family history, routine laboratory and coagulation tests including liver function [SGOT, SGPT], random blood sugar, total cholesterol, PT, and APPT. Our results revealed the history of immobilization was the only acquired risk factor which showed a statistically significant difference [p=0.01] between the two studied group. Actifed protein C sensitivity ratio and the normalized sensitivity ratio [APC-SR and APC-SR] were all significantly lower in the patients' group as compared to the control group with p values of 0.002 and 0.002 respectively. Factor V Leiden was detected in 30% of patients and 5% of controls with F II 202140A mutation was only detected in 6.7% of significantly lower APC-SR and n-APC-SR when compared to patients without the emulation with p values < 0.001. Simultaneous detection of both studied mutation was not recorded in any of our cases

Gene frequency and linkage disequilibrium within the HLA-DRB region in Egyptians using PCR-based reversed hybridization oligonucleotide genotyping

HLA class II alleles were studied in 278 Egyptians. Gene frequency as well as linkage [haplotypic association] between HLA-DRB1, HLA- DRB3, HLA-DRB4 and HLA-DRB5 types were defined. A new unexpected haplotype linked with DRB5 was also detected. It was concluded that PCR based reversed hybridization genotyping is a reliable technique for DNA- based HLA-DR typing. In addition, this specific population study has important implications for unrelated bone marrow and other organ donor registries in Egypt