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The screening for JAK2 V617F mutation in patients with polycythemia vera, essential thrombocythemia, and idiopathic myelofibrosis offers crucial information for the final diagnosis of these disorders. Recently, several JAK2 exons 12 and 14 mutations have been detected in V617F-negative patients with idiopathic erythrocytosis. The need for a rapid and accurate assay for the mutation screening in both exons 12 and 14 prompted us for the application of a method for the analysis of the entire coding region between exons 11 and 15. We applied the non-isotopic RNase cleavage assay and the accuracy of the method was verified in a series of V617F-positive, V617F-negative patients and healthy individuals, with no false results. This method can be applied in any laboratory without the requirement of specific sophisticated equipment.

It has been suggested that MEVF mutations offer advantage against infections, including tuberculosis. Bearing in mind the central role of TLR-2 and TLR-4 in the recognition of pathogens, we conducted this study to examine whether the TLR2-R753Q, TLR4-D299G, TLR4-T399I common polymorphisms are associated with susceptibility to familial Mediterranean fever (FMF) or affect the course of the disease. A cohort of 169 FMF patients and 245 healthy bone marrow donors were enrolled in the study. FMF patients appeared with a significantly lower frequency of the TLR4-D299G mutated allele (3.2% vs 6.9%, p = 0.032). No association was observed with the other analyzed polymorphisms. Moreover, we found no association between polymorphisms and the frequency of attacks or the development of amyloidosis. Our results may reinforce the hypothesis that FMF patients display a better defense against pathogens, providing an additional mechanism and suggesting a positive selection advantage in the area of the Mediterranean basin.

RASSF1A, a key cell cycle related gene, is expressed in all hematopoietic cells, it is implicated in ras signaling pathway and its promoter hypermethylation is observed in a wide variety of solid tumors. Till now, RASSF1A methylation status has not been investigated in patients with chronic myeloid leukemia (CML). In this study, we analyzed 41 patients carrying the BCR-ABL rearrangement, in different stages of the disease. No patient displayed RASSF1A promoter methylation, although the K562 erythroleukemia cell line, bearing the BCR-ABL rearrangement, was found methylated. Thus, our findings indicate that RASSF1A methylation does not appear to represent a critical step in the pathogenesis and/or the progression of CML.

Background: Primary eosinophlia associated with the FIP1L1-PDGFRA rearrangement represents a subset of chronic eosinophilic leukaemia (CEL) and affected patients are very sensitive to imatinib treatment. This study was undertaken in order to examine the prevalence and the associated clinicopathologic and genetic features of FIP1L1-PDGFRA rearrangement in a cohort of 15 adult patients presenting with profound eosinophilia (> 1.5 x 109/L)., Methods: Reverse transcriptase-polymerase chain reaction (RT-PCR) was used for the detection of FIP1L1-PDGFRA rearrangement and the results confirmed by direct sequencing. C-KIT-D816V mutation was analysed retrospectively by PCR and restriction-fragment-length-polymorphism (PCR-RFLP), in all cases with primary eosinophilia., Results: Two male patients with splenomegaly carried the FIP1L1-PDGFRA rearrangement, whilst 2 others were ultimately classified as suffering from idiopathic hypereosinophlic syndrome (HES) and one from systemic mastocytosis. These patients were negative for the C-KIT-D816V mutation and received imatinib (100-400 mg daily). Patients with CEL and HES responded to imatinib and remained in complete haematological, clinical and molecular (for carriers of FIP1L1-PDGFRA rearrangement) remission for a median of 28.2 months (range: 11-54), whilst the patient with systemic mastocytosis did not respond. Interestingly, in both patients with FIP1L1-PDGFRA rearrangement, the breakpoints into PDGFRA were located within exon 12 and fused with exons 8 and 8a of FIP1L1, respectively., Conclusion: An early diagnosis of FIPIL1-PDGFRA-positive CEL and imatinib treatment offer to the affected patients an excellent clinical therapeutic result, avoiding undesirable morbidity. Moreover, although the molecular mechanisms underlying disease pathogenesis remain to be determined, imatinib can be effective in patients with idiopathic HES.

Tobacco smoking has been considered the most important risk factor for chronic obstructive pulmonary disease (COPD) development. However, not all smokers develop COPD and other environmental and genetic susceptibility factors underlie disease pathogenesis. Recent studies have indicated that the impairment of TLR signaling might play a crucial role in the development of emphysema. For this purpose we investigated the prevalence and any possible associations of common TLR polymorphisms (TLR2-R753Q, TLR4-D299G, and TLR4-T399I) in a group of 240 heavy smokers (>20 pack years), without overt atherosclerosis disease, of whom 136 had developed COPD and 104 had not. The presence of TLR4-T399I polymorphism was associated with a 2.4-fold increased risk for COPD development (P = .044), but not with disease stage or frequency of exacerbations. Considering that infections contribute to COPD and emphysema pathogenesis, our findings possibly indicate that dysfunctional polymorphisms of innate immune genes can affect the development of COPD in smokers. Although this finding warrants further investigation, it highlights the importance of impaired innate immunity towards COPD development.

Mutations in the SLC40A1 gene result in a dominant genetic disorder [ferroportin disease; hereditary hemochromatosis type (HH) IV], characterized by iron overload with two different clinical manifestations, normal transferrin saturation with macrophage iron accumulation (the most prevalent type) or high transferrin saturation with hepatocyte iron accumulation (classical hemochromatosis phenotype). In previous studies, the mutational analysis of SLC40A1 gene has been performed at the genomic DNA level by PCR amplification and direct sequencing of all coding regions and flanking intron-exon boundaries (usually in 9 PCR reactions). In this study, we analyzed the SLC40A1 gene at the mRNA level, in two RT-PCR reactions, followed by direct sequencing and/or NIRCA (non-isotopic RNase cleavage assay). This protocol turned out to be rapid, sensitive and reliable, facilitating the detection of the SLC40A1 gene mutations in two patients with hyperferritinemia, normal transferrin saturation and iron accumulation predominantly in macrophages and Kupffer cells. The first one displayed the well-described alteration V162 Delta and the second a novel mutation (R178G) that was further detected in two relatives in a pedigree analysis. The proposed procedure would facilitate the wide-range molecular analysis of the SLC40A1 gene, contributing to better understanding the pathogenesis of the ferroportin disease.

In this report we describe a patient suffering from chronic myeloid leukemia (CML), who was treated for 4.5 years with imatinib and developed pneumonia caused by two Candida species, i.e., C. krusei and C. glabrata. The patient was in complete hematologic remission and molecular analyses did not display the presence of TLR2-R752Q, TLR4-D299G and TLR4-T399I polymorphisms that may predispose individuals to fungal infections. This case report indicates that in some patients, as previously observed, the long-term administration of targeted therapy might affect immunity and predispose patients to opportunistic and life-threatening fungal infections.

Objective: Transcription factor forkhead box protein 3 (Foxp3) specifically characterizes the thymically derived naturally occurring regulatory T cells (Tregs). Limited evidence indicates that it is also expressed, albeit to a lesser extent, in tissues other than thymus and spleen, while, very recently, it was shown that Foxp3 is expressed by pancreatic carcinoma. This study was scheduled to investigate whether expression of Foxp3 transcripts and mature protein occurs constitutively in various tumor types., Materials and Methods: Twenty five tumor cell lines of different tissue origins (lung cancer, colon cancer, breast cancer, melanoma, erythroid leukemia, acute T-cell leukemia) were studied. Detection of Foxp3 mRNA was performed using both conventional RT-PCR and quantitative real-time PCR while protein expression was assessed by immunocytochemistry and flow cytometry, using different antibody clones., Results: Foxp3 mRNA as well as Foxp3 protein was detected in all tumor cell lines, albeit in variable levels, not related to the tissue of origin. This expression correlated with the expression levels of IL-10 and TGFb1., Conclusion: We offer evidence that Foxp3 expression, characterizes tumor cells of various tissue origins. The biological significance of these findings warrants further investigation in the context of tumor immune escape, and especially under the light of current anti-cancer efforts interfering with Foxp3 expression.

Introduction: Leptin is an adipocyte-derived cytokine primarily involved in the regulation of body weight and energy balance. In vivo studies suggest that leptin promotes platelet aggregation and thrombosis. Neutrophils are involved in the crosstalk between inflammation and thrombosis in clinical disorders. Leptin is also involved in the regulation of inflammation., Aim: We examined the in vitro effects of leptin on the expression of tissue factor (TF), the primary initiator of coagulation, in healthy neutrophils., Materials and Methods/results: The effects on TF expression were assayed functionally using a modified prothrombin time (mPT), as well as at mRNA and protein levels. The same experiments were performed in parallel with PBMC. Leptin induced functional TF and increased TF mRNA and protein expression in both cell types, as determined by mPT, real-time RT-PCR, western blot, flow cytometry, immunocytochemistry. Inhibition studies revealed that the effect of leptin on TF expression is mediated, at least in part, by JAK2 and PI3K. Our findings, after neutralising TNFalpha in supernatants of leptin-treated cells, also suggest the involvement of TNFalpha in the leptin-induced TF expression in leukocytes., Conclusions: This study indicates a novel link between inflammation, obesity and thrombosis by showing that leptin is able to trigger the extrinsic coagulation cascade. This work suggests a possible mechanism of the thrombotic effects of hyperleptinemic-associated clinical disorders.

Recently, the acquired mutation JAK2-V617F has been described in the majority of patients with myeloproliferative disorders (MPDs). In this study we evaluated its clinical and laboratory correlates in 166 patients with MPDs. The mutation was detected by allele-specific PCR in 119 patients: 81.4% (35/43) of those with polycythemia vera, 69.1% (77/111) of those with essential thrombocythemia and 58.1% (7/12) of those with idiopathic myelofibrosis. The patients carrying the mutation were older (p=0.02) and displayed higher levels of Ht (p<0.01) and Hb (<0.01) and lower erythropoietin levels (p<0.01). Moreover, mutation-positive patients displayed a higher probability of having leucocytosis, splenomegaly and thrombotic events (three-fold, two-fold and two-fold, respectively) than mutation-negative patients. These correlations imply that the JAK2-V617F mutation may be useful for the classification and the management of patients with MPDs.

Objectives: To investigate if pepsin is detected, with an activity assay, in the saliva of patients with a clinical diagnosis of laryngopharyngeal reflux (LPR) and can therefore be used as a diagnostic marker of laryngopharyngeal reflux., Study Design: Pilot, prospective study., Methods: Adult participants with a clinical diagnosis of LPR collected whole saliva samples on regular intervals for a day, and upon experiencing symptoms attributed to LPR. Patients were selected on the basis of presence of severe symptoms and laryngoscopic findings of laryngopharyngeal reflux and symptoms of gastroesopharyngeal reflux. They reported voice disorders, dysphagia, throat clearing, excessive secretions, breathing difficulties, cough, globus sensation and throat pain. Control participants reported the absence of pharyngeal and laryngeal symptoms and of symptoms of gastroesophageal reflux. Saliva samples were assayed with fibrinogen on an agarose gel plate. The detection of pepsin was based on the presence of peptic activity which was qualitatively evaluated., Results: The control participants had negative assays. No saliva samples from the LPR patients, collected at regular sampling, tested positive for pepsin. All the samples collected at the presence of symptoms and following regurgitation episodes tested negative for pepsin. Saliva samples pH ranged from 7 to 8., Conclusions: Pepsin was not detected, with an activity assay, in the saliva of patients with a clinical diagnosis of LPR. A concentration method might be more sensitive although saliva and swallowing physiology renders the detection of pepsin in the saliva difficult.

The aim of the study was to evaluate the role of hypochromic erythrocytes (HYPO%) compared to "traditional" and novel markers of iron status and erythropoiesis in recognizing iron-restricted erythropoiesis (IRE) and predicting response to erythropoietin (rHuEPO) in anemic patients with myeloma and lymphoma. Forty-one newly diagnosed patients who received epoetin-beta at a subcutaneous weekly dose of 30,000 IU for 6 weeks were studied. Response to rHuEPO was observed in 27 patients (65.8%). Twelve non-responders received, additionally, 200 mg of IV iron sucrose, weekly, for 4 weeks. Evaluation of markers was performed at baseline and on weeks 1, 2 and 6 for all patients and also on weeks 7-10 for non-responders to rHuEPO. Baseline HYPO%, at a cut-off value of <5%, and an increment in reticulocyte absolute number (RETICS-AB) >or= 50,000/microl and reticulocyte hematocrit (RETICS-Hct) >or= 50%, between baseline and week 2, were independent predictive factors for response to rHuEPO. We found that these markers had superior predictive value for response to rHuEPO than four widely used predictive models. Furthermore, a baseline HYPO% count of above 5% proved superior over serum ferritin < 100 ng/ml and transferrin saturation < 20% in recognizing IRE. In conclusion, baseline HYPO% either alone or in combination with RETICS-AB or RETICS-Hct after 2 weeks of rHuEPO treatment could be reliably used in predicting response to rHuEPO. Additionally, HYPO% has proved a reliable marker for recognizing IRE before rHuEPO treatment and, thus, could be used for identifying patients who will benefit from IV iron supplementation.

Familial Mediterranean fever (FMF) is a disease characterized by recurrent, self-limiting bouts of fever and serositis and caused by altered pyrin due to mutated MEFV gene. FMF is common in the Mediterranean Basin populations, although with varying genetic patterns. The spectrum and clinical significance of MEFV alterations in Greece has yet not been elucidated. The aim of this study was to analyze the spectrum of MEFV alterations in FMF patients and healthy individuals in Greece. A cohort of 152 Greek FMF patients along with 140 Greek healthy controls was enrolled. Non-isotopic RNase cleavage assay (NIRCA) and sequencing allowed mutational and haplotypic analysis of the entire coding sequence of MEFV. The ARLEQUIN 2.0, DNASP 4.0 and PHYLIP software were used for population genetics analysis. Among patients, 127 (83.6%) carried at least one known mutation. The most common mutations identified were M694V (38.1%), M680I (19.7%), V726A (12.2%), E148Q (10.9%) and E230K (6.1%). The total carrier rate among healthy individuals was 0.7%. The presence of R202Q homozygosity in 12 of the remaining 25 MEFV negative FMF patients might be considered as disease related in Greeks. Population genetics analysis revealed that Greeks rely closer to the eastern rather than western populations of the Mediterranean Basin.

The purpose of this study was to evaluate the sTfR-F index and hypochromic erythrocytes (HYPO%) as potential predictors of response to recombinant human erythropoietin (r-HuEPO) of anemic patients with multiple myeloma (MM) before treatment, as well as early in the course of treatment. Twenty-six newly diagnosed anemic MM patients received r-HuEPO 30,000 IU/wk sc, for six weeks. The sTfR-F index and HYPO% were determined at baseline and at weeks 2 and 6. Patients were classified in 1 of 4 categories of a diagnostic plot, according to erythropoietic state (ES I-IV), defined by the combination of sTfR-F index and HYPO%. Sixteen of 20 patients in ES I and II before treatment responded to r-HuEPO, whereas none of the 6 patients in ES III and IV responded (P < .001). At week 2, 44% of patients who responded and 60% of the nonresponders were in functional iron deficiency (FID) and the proportion increased to 69% and 80%, respectively, by week 6. Seven of the patients who did not respond received in addition 200 mg iron sucrose IV weekly, for the next 4 weeks, and 6 of them responded. These results suggest that combination of sTfR-F index and HYPO% in a diagnostic plot can be used as a predictive model to recognize patients who will benefit from r-HuEPO and identify FID requiring iron supplementation, before treatment and early in the course of treatment, contributing thus to optimization of r-HuEPO therapy.

Setting: In many cases of extra-pulmonary tuberculosis (EPTB), with the exception of paucibacillary analysed specimens, the suspected site of mycobacterial infection is relatively inaccessible or unknown, making laboratory confirmation of TB laborious and problematic., Objective: Two different polymerase chain reaction (PCR) based methods were compared to investigate the validity of bone marrow aspiration material as an easily accessible alternative sample for molecular analysis in EPTB., Design: We amplified the same sequence of IS6110 of Mycobacterium tuberculosis complex in 19 confirmed cases of EPTB using two different nested PCR techniques: one in-house 'classic' PCR and another based on LightCycler technology., Results: Both methods demonstrated the same reliability when performed in samples of infected tissue. However, the LightCycler protocol was superior to the in-house system when applied in bone marrow aspiration material, revealing positivity in 18/19 compared to 13/19 samples of 'classic' PCR., Conclusion: The application of an optimised LightCycler nested amplification protocol in bone marrow aspirates may promote diagnostic accuracy in difficult and/or urgent cases of EPTB.

Members of the transforming growth factor (TGF)-beta family play pivotal roles in the control of differentiation, proliferation and tolerance in peripheral T cells. Recently, they have been implicated in thymic selection, but their role is so far not well characterized. In the present study, we demonstrate that specific thymocyte populations are under the influence of either the TGF-beta and/or Activin pathway, and transduce signals into the nucleus via phosphorylated Smad2 (pSmad2). Thymocytes in the medulla and in the subcapsular zone expressed nuclear translocated pSmad2, a hallmark of active TGF-beta/Activin receptor signaling. When analyzed at the cellular level, the pSmad2(+) cells were confined to the double-negative (DN) and single-positive (SP) subpopulations. Moreover, the most immature DN thymocytes (CD44(+)CD25(-) and CD44(+)CD25(+)) expressed higher levels of pSmad2 compared to the more mature DN. In vitro stimulation demonstrated that pure CD44(+)CD25(-), CD44(+)CD25(+) and CD44(+)CD25(+) thymocytes respond to ActivinA, while the mature CD4(+) and CD8(+) SP thymocytes respond to TGF-beta stimulation measured as enhanced phosphorylation of Smad2. Double staining of pSmad2(+) cells with either the Activin type I receptor, ALK4, or the TGF-beta type I receptor, ALK5, demonstrated that pSmad2(+) DN cells exhibited high levels of immunoreactivity to ALK4 and moderate levels of immunoreactivity to the TGF-beta-responsive ALK5 receptor. In sharp contrast, the SP pSmad2(+) cells were predominately ALK5(+). Collectively, our results demonstrate that early and late thymocytes express pSmad2 in the nuclei in vivo. The functional experiments in vitro suggest that members of the TGF-beta family (TGF-beta or Activin) may play important non-redundant roles during different stages of thymopoiesis.

The frequencies of the hereditary hemochromatosis gene (HFE) mutations C282Y and H63D vary between different populations. There are a limited number of reports regarding the frequency of these mutations in populations of southeastern Europe. Two hundred and sixty-four adult individuals of Greek origin were examined for the C282Y and H63D mutations to determine the allele and genotype frequencies. The HFE gene region of DNA samples extracted from peripheral leukocytes was amplified by the polymerase chain reaction. Restriction enzyme analysis was performed using RSAI for C282Y and MBOI for H63D. None of the 264 individuals carried the mutation C282Y. Forty-three individuals (16.2%) were heterozygous carriers of the H63D allele and 2 were homozygous for this mutation (0.75%). The overall H63D allele prevalence is thus estimated at 8.9%. HFE mutation frequencies were low in the population studied and this may explain, in part, the relative rarity of clinical cases of hereditary hemochromatosis in Greece., (Copyright 2003 S. Karger AG, Basel)

Histiocytic necrotizing lymphadenitis (HNL), or Kikuchi-Fujimoto disease, is a benign, self-limited disease that predominantly occurs in women. The etiology remains undetermined, although a viral or autoimmune hypothesis has been suggested. The disease usually emerges with cervical lymphadenopathy with or without fever. The diagnosis can be confirmed only by histological findings of lymph node biopsy, characterized by necrosis and histiocytic infiltration without neutrophils. We report a case of a 28-year-old woman with a medical history of two episodes of unexplained pulmonary embolisms (3 and 2 years previously) who was admitted to our hospital because of unilateral cervical lymphadenopathy and mild fever that presented 1 week before admission. A diagnosis of HNL was performed by lymph node biopsy. In parallel, whereas the laboratory tests for inherited thrombophilia were negative, a progressive elevated titer of anti-beta(2) glycoprotein I (GPI) antibodies was established. Because of persistent fever, the patient received a short course of corticosteroid therapy and she recovered completely from the HNL after 2 months. It is noteworthy that to date the patient has displayed an elevated titer of anti-beta(2) GPI antibodies (18 months after the recovery from the HNL). Thus, considering the previous history of venous thrombosis and the presence of antiphospholipid antibodies, the diagnosis of primary antiphospholipid syndrome associated with HNL was made. To our knowledge, this is the first report in the literature describing antiphospholipid syndrome associated with HNL. Moreover, a brief literature review is provided with emphasis on the etiology, clinical course, and pathogenesis of this rare disease entity.

Chronic myeloid leukaemia (CML) is a malignant clonal disorder of the haematopoietic stem cell. Treatment of CML patients with interferon alpha (IFN-alpha) has induced haematological and cytogenetic remission. Interferons transcriptionally activate target genes through the JAK-STAT and interferon regulated factors (IRFs) family pathways. Interferon regulated factor-1 (IRF-1) is a transcriptional activator of genes critical for cell growth, differentiation and apoptosis. The skipping of exons 2 or 2 and 3 of IRF-1 in patients with myelodysplastic syndromes and acute myelogenous leukaemia suggests that this factor may have a critical role in leukaemogenesis. The role of IRF-1 in CML is currently unknown. Therefore, mutational analysis of IRF-1 was performed and its expression pattern was also studied in CML patients. We studied IRF-1 in peripheral blood mononuclear cells of 21 patients in chronic phase CML. No point mutations were identified at the cDNA level. Surprisingly, fourfold reduction of full-length IRF-1 mRNA expression was established in 17/21 patients compared with normal individuals. Low expression of full-length IRF-1 was observed in conjunction with high levels of aberrantly spliced mRNAs, reported for the first time. In three patients who were also analysed during blastic transformation, further reduction of full-length IRF-1 mRNA was observed. These findings demonstrate that, in CML patients, IRF-1 can produce high levels of aberrant spliced mRNAs with subsequent reduction in the levels of full-length IRF-1 mRNA. This observation is consistent with the notion that exon skipping may constitute another mechanism of tumour suppressor gene inactivation in this disease.

Bone morphogenetic proteins (BMPs) are pleiotropic secreted proteins, structurally related to transforming growth factor (TGF)-beta and activins. BMPs play pivotal roles in the regulation of embryonic lung development and branching of airways and have recently been considered to influence inflammatory processes in adults due to their chemotactic activity on fibroblasts, myocytes, and inflammatory cells. In this study, we have investigated the possible involvement of BMPs in a model of experimental allergic-airway inflammation in situ using antibodies that detect activated Smad proteins, and have monitored the modulation of BMP ligands during the inflammatory response. Inflamed bronchial epithelial cells and a few scattered alveolar cells expressed levels of phosphorylated Smad1 (pSmad1/5), indicative of active BMP/Smad signaling. This was in contrast to healthy epithelium, which was devoid of immunoreactivity. A mechanistic explanation for increased pSmad1/5 staining during inflammation was provided by the upregulated expression of all the BMP type I receptors, i.e., activin receptor-like kinase (ALK)2, ALK3, and ALK6, in the inflamed bronchial epithelial cells. Furthermore, the mRNA and protein profiles for BMP ligands were significantly altered during airway inflammation with induction of BMP2, BMP4, and BMP6, and downregulation of BMP5 and BMP7. Collectively, our data demonstrate for the first time active BMP/Smad signaling during airway inflammation in bronchial epithelial cells and thus raise the possibility that BMPs could play a determining role in respiratory pathophysiology.

Bruton's tyrosine kinase (Btk) is a nonreceptor tyrosine kinase, critical for B-cell development and function. Mutations that inactivate this kinase were found in families with X-linked agammaglobulinaemia (XLA). In this study the Btk gene was analyzed in 13 registered Greek patients with XLA phenotype originated from 12 unrelated families, in order to provide a definite diagnosis of the XLA. The structure of Btk was analyzed at the cDNA level using the recently developed method, NIRCA (Non-Isotopic-Rnase-Cleavage-Assay). Alterations were detected in all patients and sequencing analysis confirmed the results and defined six novel XLA-associated Btk mutations (three missense mutations: C337G, L346R, L452P; one nonsense mutation: Y392X, and two frameshift alterations: c1211-1212delA, c1306-1307insA). Having defined the genetic alteration in the affected males of these families, the information was used to design polymerase chain reaction (PCR) primers and the Btk segments containing the mutated sequences were amplified from peripheral blood derived genomic DNA of potential female carriers. The PCR products were directly sequenced and carrier status was determined in 12 out of 16 phenotypically normal females analyzed. This protocol can be used once the nature of the Btk mutation has been defined in one of the affected males and provides a convenient, simple and reliable way to determine the carrier status of other female family members. Molecular genetic analysis constitutes a determinative tool for the definitive diagnosis of XLA and may allow accurate carrier and prenatal diagnosis for genetic counselling.

Background and Objectives: N-ras mutations are the most commonly detected molecular abnormalities in hematologic malignancies, especially in those of myeloid origin. Different techniques have been used to detect N-ras mutations; however, most of them are either labor intensive or provide sequence data for only a limited number of codons. Consequently, study of the N-ras oncogene has not been convenient in every day clinical practice being restricted, as a rule, to retrospective analysis of patients., Design and Methods: In this study we used a recently developed method that enables rapid and reliable detection of mutations at the cDNA level, namely, the non-isotopic RNase cleavage assay (NIRCA). Using this method we were able to screen the N-ras oncogene rapidly and determine the incidence and prognostic significance of N-ras mutations in 77 Greek patients with acute leukemia, myelodysplastic syndromes and chronic myeloproliferative disorders, both at the presentation and during relapse or progression of the disease., Results: Activating N-ras mutations were detected in 7 patients and our results were confirmed by direct sequencing. Interestingly, two novel alterations were identified, a mutation at codon 8 (characterized by a substitution of valine by leucine) in a patient with chronic myeloid leukemia during hematologic relapse of the disease and a polymorphism at codon 92 (1002T-->C, without amino acid substitution) in a patient with chronic myelomonocytic leukemia., Interpretation and Conclusions: A rapid and easy protocol that allows the analyses of N-ras sequences has been developed. This reverse transcription-polymerase chain reaction (RT-PCR)/NIRCA protocol can allow the study of this proto-oncogene in every day clinical practice, rapidly facilitating the validation of the diagnostic and prognostic value of N-ras mutational analyses in patients with hematologic malignancies.

Objectives: Extrapulmonary tuberculosis (TB) constitutes the main cause of classic fever of unknown origin (FUO) in many populations. The aim of this study was to improve the diagnostic field of the disease using a nested polymerase chain reaction (PCR) assay, specific for the IS6110 insertion element of Mycobacterium tuberculosis complex, in order to achieve a more timely diagnosis and treatment., Setting: Twenty-four, HIV-negative classic FUO patients who were admitted to the Regional Hospital of Alexandroupolis between April 1997 and July 1999., Subjects and Design: The above patients were considered as putative extrapulmonary TB after 3 weeks of in-patient investigation and underwent exhaustive examination for diagnosis of the disease. For this purpose, specimens were obtained from peripheral blood and bone marrow from these patients, as well as from damaged tissues, and analysed by both PCR and conventional methods. Anti-tuberculous treatment was initiated in 16 out of 24 patients and the response to this regimen was considered as the final criterion for diagnosis of tuberculosis., Results: Extrapulmonary TB was established in 11 patients. The PCR-based methodology, when applied to samples derived from bone marrow aspirations and suspected damaged tissues, was able to diagnose 10 of them, whereas the conventional methods were able to detect only two., Conclusions: Our results confirm the diagnostic value of molecular detection of M. tuberculosis in cases of FUO, thus supporting the application of PCR in tissue samples suspected of bacillus infection. Furthermore, our studies demonstrate that bone marrow aspiration specimens constitute an alternative, easy, safe and reliable source for such PCR analysis.

Background: The diagnosis of X-linked agammaglobulinemia (XLA) is not always clearcut. Not all XLA conform to the classic phenotype and less than 50% of affected boys have a family history of immunodeficiency. Mutations in the gene for Bruton's tyrosine kinase (BTK) are responsible for the majority of agammaglobulinemia cases. However, a certain proportion of patients may have mutations involving other genes, although they show with an XLA phenotype. We performed BTK gene mutation analysis in 37 males with presumed XLA and analyzed the pattern of X-chromosome inactivation (XCI) in 31 mothers to evaluate the relevance of these approaches to diagnosis and genetic counseling., Materials and Methods: Twenty affected males with a sporadic occurrence and 17 familial cases belonging to nine families were enrolled within the framework of the Italian Multicenter Clinical Study on XLA. We used non-isotopic RNase cleavage assay (NIRCA), followed by cDNA sequence determination to screen for BTK mutations and X-chromosome inactivation analysis for carrier detection., Results: Using the cDNA-based approach, the identification of BTK gene abnormalities confirmed the clinical diagnosis of XLA in 31 of 37 affected infants. Missense was the most frequent mutational event and the kinase domain was mostly involved. In addition, nine novel mutations were identified. In sporadic cases, BTK gene abnormalities were identified in 9 out of 10 patients whose mothers had a nonrandom pattern of XCI and in 5 out of 6 patients whose mother had a random pattern of XCI. With the exception of one family, all patients with a familial occurrence and born to mothers with a nonrandom pattern of XCI had mutations of the BTK gene., Conclusions: Our findings indicate that in sporadic cases BTK gene sequencing is the only reliable tool for a definitive diagnosis of XLA and support XCI as the first diagnostic tool in the mothers of affected males in multiple generations. Furthermore, our molecular analysis confirms that 10-20% of BTK-unaltered patients have disorders caused by defects in other genes.

Mutations in the Bruton's tyrosine kinase (BTK ) gene are responsible for X-linked Agammaglobulinemia (XLA), an immunodeficiency caused by a block in B cell differentiation. Non Isotopic RNAse Cleavage Assay (NIRCA), followed by sequencing was used to screen for BTK mutations in 11 Italian XLA patients. Nine novel mutations were identified: 6 missense (Y39S, L512P, L512Q, R544G, S578Y, E589K), one non-sense (Q260X), one frameshift (1599-1602del GCGC) and one in-frame insertion (2037-2038insTTTTAG), that represents the first case of premature stop codon introduction in the BTK coding frame. These data support the high molecular heterogeneity of BTK gene in XLA disease and provide new insight to the diagnosis and to the role of BTK domain in XLA and in B cell signal transduction and development. Hum Mutat 15:117, 2000., (Copyright 2000 Wiley-Liss, Inc.)

Bruton's tyrosine kinase (Btk) is a non-receptor protein tyrosine kinase (PTK) that is expressed in all haemopoietic lineages except mature T cells and plasma cells. Despite the broad range of expression. mutations that inactivate this molecule affect primarily the development of the B-cell lineage. As a PTK, Btk could potentially be involved directly or indirectly in the processes that relate to the malignant transformation of all the cell lineages where this molecule is expressed. Previous studies have failed to demonstrate mutations in patients with B-cell origin acute lymphoblastic leukaemia (ALL). We have utilized a recently developed method that enables the rapid and convenient detection of mutations at the cDNA level, namely, the non-isotopic RNase cleavage assay (NIRCA) to analyse Btk sequences from 27 patients with different types of acute myeloid leukaemia (AML). The only alteration that we observed was a polymorphism at position 2031. This polymorphism has already been seen in previous studies. Furthermore, using the same methodology, we identified the Btk mutations in six XLA (X-linked agammaglobulinaemia) patients. Our results, although they do not exclude the involvement of Btk mutations in the development or progression of some type of AML, nevertheless suggest that such mutations do not constitute a major co-factor in the development of myeloid malignancies.

Genes were isolated using the suppression subtractive hybridization method by stimulation of pro/pre B cells with anti-CD40 and interleukin (IL)-4 to mature S mu-Sepsilon-switched cells. One of the strongly upregulated genes encodes a novel murine CC chemokine we have named ABCD-1. The ABCD-1 gene has three exons separated by 1. 2- and 2.7-kb introns. It gives rise to a 2.2-kb transcript containing an open reading frame of 276 nucleotides. Two polyadenylation sites are used, giving rise to cDNAs with either 1550 or 1850 bp of 3' untranslated regions. The open reading frame encodes a 24 amino acid-long leader peptide and a 68 amino acid-long mature protein with a predicted molecular mass of 7.8 kD. ABCD-1 mRNA is found in highest quantities in activated splenic B lymphocytes and dendritic cells. Little chemokine mRNA is present in lung, in unstimulated splenic cells, in thymocytes, and in lymph node cells. No ABCD-1 mRNA is detected in bone marrow, liver, kidney, or brain, in peritoneal exudate cells as well as in the majority of all unstimulated B lineage cells tested. It is also undetectable in Concanavalin A-activated/IL-2-restimulated splenic T cells, and in bone marrow-derived IL-2-induced natural killer cells and IL-3-activated macrophages. Recombinant ABCD-1 revealed a concentration-dependent and specific migration of activated splenic T lymphoblasts in chemotaxis assays. FACS(R) analyses of migrated cells showed no preferential difference in migration of CD4(+) versus CD8(+) T cell blasts. Murine as well as human T cells responded to ABCD-1. Freshly isolated cells from bone marrow, thymus, spleen, and lymph node, IL-2-activated NK cells, and LPS-stimulated splenic cells, all did not show any chemotactic response. Thus, ABCD-1 is the first chemokine produced in large amounts by activated B cells and acting selectively on activated T lymphocytes. Therefore, ABCD-1 is expected to play an important role in the collaboration of dendritic cells and B lymphocytes with T cells in immune responses.

Several groups have used high doses of interleukin-2 (IL-2), achieving a significant rate of responses in patients with acute myelogenous leukemia (AML). These results have mainly been observed in AML patients with limited disease (bone marrow blasts < 25%), but this therapy is associated with significant toxicity which is dose-related. In this report we describe a patient with AML in whom conventional chemotherapy had achieved partial remission. This patient received subcutaneously intermediate doses of IL-2 (12 x 10(6)/m2/week) and achieved complete remission which was maintained for eleven months. The side effects of IL-2 were mild. The results of this report document the antileukemic effect of IL-2 in AML with limited disease as well as the efficacy of subcutaneous maintenance treatment for prolonged periods.