Evaluation of the Retinal Vasculature in Hypertension and Chronic Kidney Disease in an elderly population of Irish nuns

Background: Chronic kidney disease (CKD) and hypertension are global public health problems associated with considerable morbidity, premature mortality and attendant healthcare costs. Previous studies have highlighted that non-invasive examination of the retinal microcirculation can detect microvascular pathology that is associated with systemic disorders of the circulatory system such as hypertension. We examined the associations between retinal vessel caliber (RVC) and fractal dimension (DF), with both hypertension and CKD in elderly Irish nuns.

Methods: Data from 1233 participants in the cross-sectional observational Irish Nun Eye Study (INES) were assessed from digital photographs with a standardized protocol using computer-assisted software. Multivariate regression analyses were used to assess associations with hypertension and CKD, with adjustment for age, body mass index (BMI), refraction, fellow eye RVC, smoking, alcohol consumption, ischemic heart disease (IHD), cerebrovascular accident (CVA), diabetes and medication use.

Results: In total, 1122 (91%) participants (mean age: 76.3 [range: 56-100] years) had gradable retinal images of sufficient quality for blood vessel assessment. Hypertension was significantly associated with a narrower central retinal arteriolar equivalent (CRAE) in a fully adjusted analysis (P = 0.002; effect size= -2.16 μm; 95% confidence intervals [CI]: -3.51, -0.81 μm). No significant associations between other retinal vascular parameters and hypertension or between any retinal vascular parameters and CKD were found.

Conclusions: Individuals with hypertension have significantly narrower retinal arterioles which may afford an earlier opportunity for tailored prevention and treatment options to optimize the structure and function of the microvasculature, providing additional clinical utility. No significant associations between retinal vascular parameters and CKD were detected.

WS02.7 Initial and chronic MRSA infection in cystic fibrosis

Objectives Chronic MRSA infection, which affects approximately 26% of CF patients in the USA, is associated with declining lung function and poor outcomes (Dasenbrook, 2010). Anaerobic niches have been described within the CF lung, potentially influencing the virulence of MRSA. This study aims to compare initial and chronic CF MRSA isolates, following aerobic and anaerobic culture. Methods Isolates, obtained from CF sputum at first isolation [“early” (n = 10)] or up to 5 years later, during chronic infection [“late” (n = 15)] were cultured in aerobic and anaerobic conditions. Differences in virulence were compared using the Galleria mellonella infection model. Biofilm formation of each isolate was assessed following staining with crystal violet. Production of Δ-haemolysin (Δ-hly), a surrogate marker for expression of the virulence regulator agr, was determined by haemolysis assay. Results MRSA grown in anaerobic conditions had significantly increased virulence in the G. mellonella model (p = 0.007), increased biofilm formation (p = 0.006) and increased Δ-hly production (p<0.0001). No significant difference between Δ-hly production or biofilm formation were observed between early and late isolates; however late isolates were found to be more virulent in the G. mellonella model (p = 0.0002). Conclusion These results suggest that an anaerobic environment, as found in the CF lung, may increase virulence of MRSA and aid in the establishment of chronic infection. Further clinical studies are required to determine how these phenotypic changes are associated with transition to chronic infection and patient outcome.

The vascular targeting agent combretastatin-A4 and a novel cis-Restricted {beta}-Lactam Analogue, CA-432, induce apoptosis in human chronic myeloid leukemia cells and ex vivo patient samples including those displaying multidrug resistance

Combretastatin-A4 (CA-4) is a natural derivative of the African willow tree Combretum caffrum. CA-4 is one of the most potent antimitotic components of natural origin, but it is, however, intrinsically unstable. A novel series of CA-4 analogs incorporating a 3,4-diaryl-2-azetidinone (β-lactam) ring were designed and synthesized with the objective to prevent cis -trans isomerization and improve the intrinsic stability without altering the biological activity of CA-4. Evaluation of selected β-lactam CA-4 analogs demonstrated potent antitubulin, antiproliferative, and antimitotic effects in human leukemia cells. A lead β-lactam analog, CA-432, displayed comparable antiproliferative activities with CA-4. CA-432 induced rapid apoptosis in HL-60 acute myeloid leukemia cells, which was accompanied by depolymerization of the microtubular network, poly(ADP-ribose) polymerase cleavage, caspase-3 activation, and Bcl-2 cleavage. A prolonged G(2)M cell cycle arrest accompanied by a sustained phosphorylation of mitotic spindle checkpoint protein, BubR1, and the antiapoptotic proteins Bcl-2 and Bcl-x(L) preceded apoptotic events in K562 chronic myeloid leukemia (CML) cells. Molecular docking studies in conjunction with comprehensive cell line data rule out CA-4 and β-lactam derivatives as P-glycoprotein substrates. Furthermore, both CA-4 and CA-432 induced significantly more apoptosis compared with imatinib mesylate in ex vivo samples from patients with CML, including those positive for the T315I mutation displaying resistance to imatinib mesylate and dasatinib. In summary, synthetic intrinsically stable analogs of CA-4 that display significant clinical potential as antileukemic agents have been designed and synthesized.

Sequential treatment with flavopiridol synergistically enhances pyrrolo-1,5-benzoxazepine-induced apoptosis in human chronic myeloid leukaemia cells including those resistant to imatinib treatment

The Bcr-Abl kinase inhibitor, imatinib mesylate, is the front line treatment for chronic myeloid leukaemia (CML), but the emergence of imatinib resistance has led to the search for alternative drug treatments and the examination of combination therapies to overcome imatinib resistance. The pro-apoptotic PBOX compounds are a recently developed novel series of microtubule targeting agents (MTAs) that depolymerise tubulin. Recent data demonstrating enhanced MTA-induced tumour cell apoptosis upon combination with the cyclin dependent kinase (CDK)-1 inhibitor flavopiridol prompted us to examine whether this compound could similarly enhance the effect of the PBOX compounds. We thus characterised the apoptotic and cell cycle events associated with combination therapy of the PBOX compounds and flavopiridol and results showed a sequence dependent, synergistic enhancement of apoptosis in CML cells including those expressing the imatinib-resistant T315I mutant. Flavopiridol reduced the number of polyploid cells formed in response to PBOX treatment but only to a small extent, suggesting that inhibition of endoreplication was unlikely to play a major role in the mechanism by which flavopiridol synergistically enhanced PBOX-induced apoptosis. The addition of flavopiridol following PBOX-6 treatment did however result in an accelerated exit from the G2/M transition accompanied by an enhanced downregulation and deactivation of the CDK1/cyclin B1 complex and an enhanced degradation of the inhibitor of apoptosis protein (IAP) survivin. In conclusion, results from this study highlight the potential of these novel series of PBOX compounds, alone or in sequential combination with flavopiridol, as an effective therapy against CML.

Hypoxia response element-driven cytosine deaminase/5-fluorocytosine gene therapy system:a highly effective approach to overcome the dynamics of tumour hypoxia and enhance the radiosensitivity of prostate cancer cells in vitro

BACKGROUND: We proposed to exploit hypoxia-inducible factor (HIF)-1alpha overexpression in prostate tumours and use this transcriptional machinery to control the expression of the suicide gene cytosine deaminase (CD) through binding of HIF-1alpha to arrangements of hypoxia response elements. CD is a prodrug activation enzyme, which converts inactive 5-fluorocytosine to active 5-fluorouracil (5-FU), allowing selective killing of vector containing cells.

METHODS: We developed a pair of vectors, containing either five or eight copies of the hypoxia response element (HRE) isolated from the vascular endothelial growth factor (pH5VCD) or glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (pH8GCD) gene, respectively. The kinetics of the hypoxic induction of the vectors and sensitization effects were evaluated in 22Rv1 and DU145 cells in vitro.

RESULTS: The CD protein as selectively detected in lysates of transiently transfected 22Rv1 and DU145 cells following hypoxic exposure. This is the first evidence of GAPDH HREs being used to control a suicide gene therapy strategy. Detectable CD levels were sustained upon reoxygenation and prolonged hypoxic exposures. Hypoxia-induced chemoresistance to 5-FU was overcome in both cell lines treated with this suicide gene therapy approach. Hypoxic transfectants were sensitized to prodrug concentrations that were ten-fold lower than those that are clinically relevant. Moreover, the surviving fraction of reoxygenated transfectants could be further reduced with the concomitant delivery of clinically relevant single radiation doses.

CONCLUSIONS: This strategy thus has the potential to sensitize the hypoxic compartment of prostate tumours and improve the outcome of current therapies.

The novel pyrrolo-1,5-benzoxazepine, PBOX-21, potentiates the apoptotic efficacy of STI571 (imatinib mesylate) in human chronic myeloid leukaemia cells

The Bcr-Abl kinase inhibitor, STI571, is the first line treatment for chronic myeloid leukaemia (CML), but the recent emergence of STI571 resistance has led to the examination of combination therapies. In this report, we describe how a novel non-toxic G1-arresting compound, pyrrolo-1,5-benzoxazepine (PBOX)-21, potentiates the apoptotic ability of STI571 in Bcr-Abl-positive CML cells. Co-treatment of CML cells with PBOX-21 and STI571 induced more apoptosis than either drug alone in parental (K562S and LAMA84) and STI571-resistant cells lines (K562R). This potentiation of apoptosis was specific to Bcr-Abl-positive leukaemia cells with no effect observed on Bcr-Abl-negative HL-60 acute myeloid leukaemia cells. Apoptosis induced by PBOX-21/STI571 resulted in activation of caspase-8, cleavage of PARP and Bcl-2, upregulation of the pro-apoptotic protein Bim and a downregulation of Bcr-Abl. Repression of proteins involved in Bcr-Abl transformation, the anti-apoptotic proteins Mcl-1 and Bcl-(XL) was also observed. The combined lack of an early change in mitochondrial membrane potential, release of cytochrome c and cleavage of pro-caspase-9 suggests that this pathway is not involved in the initiation of apoptosis by PBOX-21/STI571. Apoptosis was significantly reduced following pre-treatment with either the general caspase inhibitor Boc-FMK or the chymotrypsin-like serine protease inhibitor TPCK, but was completely abrogated following pre-treatment with a combination of these inhibitors. This demonstrates the important role for each of these protease families in this apoptotic pathway. In conclusion, our data highlights the potential of PBOX-21 in combination with STI571 as an effective therapy against CML.

STI-571 (imatinib mesylate) enhances the apoptotic efficacy of pyrrolo-1,5-benzoxazepine-6, a novel microtubule-targeting agent, in both STI-571-sensitive and -resistant Bcr-Abl-positive human chronic myeloid leukemia cells

Interactions between the Bcr-Abl kinase inhibitor STI-571 (imatinib mesylate) and a novel microtubule-targeting agent (MTA), pyrrolo-1,5-benzoxazepine (PBOX)-6, were investigated in STI-571-sensitive and -resistant human chronic myeloid leukemia (CML) cells. Cotreatment of PBOX-6 with STI-571 induced significantly more apoptosis in Bcr-Abl-positive CML cell lines (K562 and LAMA-84) than either drug alone (P < 0.01). Cell cycle analysis of propidium iodide-stained cells showed that STI-571 significantly reduced PBOX-6-induced G2M arrest and polyploid formation with a concomitant increase in apoptosis. Similar results were obtained in K562 CML cells using lead MTAs (paclitaxel and nocodazole) in combination with STI-571. Potentiation of PBOX-6-induced apoptosis by STI-571 was specific to Bcr-Abl-positive leukemia cells with no cytoxic effects observed on normal peripheral blood cells. The combined treatment of STI-571 and PBOX-6 was associated with the down-regulation of Bcr-Abl and repression of proteins involved in Bcr-Abl transformation, namely the antiapoptotic proteins Bcl-x(L) and Mcl-1. Importantly, PBOX-6/STI-571 combinations were also effective in STI-571-resistant cells. Together, these findings highlight the potential clinical benefits in simultaneously targeting the microtubules and the Bcr-Abl oncoprotein in STI-571-sensitive and -resistant CML cells.

CD38 expression level and pattern of expression remains a reliable and robust marker of progressive disease in chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) follows a variable clinical course which is difficult to predict at diagnosis. We assessed somatic mutation (SHM) status, CD38 and ZAP-70 expression in 87 patients (49 male, 38 female) with stage A CLL and known cytogenetic profile to compare their role in predicting disease progression, which was assessed by the treatment free interval (TFI) from diagnosis. Sixty (69%) patients were SHM+, 24 (28%) were CD38+ and ten (12%) were ZAP-70+. The median TFI for: (i) SHM + versus SHM- patients was 124 versus 26 months; hazard ratio (HR) = 3.6 [95% confidence interval (CI) = 1.8 - 7.3; P = 0.001]: (ii) CD38- versus CD38+ patients was 120 versus 34 months; HR = 2.4 (95% CI = 1.4 - 5.3; P = 0.02); and (iii) ZAP70- versus ZAP70+ was 120 versus 16 months; HR = 3.4 (95% CI = 1.4 - 8.7; P = 0.01). SHM status and CD38 retained prognostic significance on multivariate analysis whereas ZAP-70 did not. We conclude that ZAP-70 analysis does not provide additional prognostic information in this group of patients.

Favorable effect on acute and chronic graft-versus-host disease with cyclophosphamide and in vivo anti-CD52 monoclonal antibodies for marrow transplantation from HLA-identical sibling donors for acquired aplastic anemia

Between August 1989 and November 2003, 33 patients at our center with acquired aplastic anemia underwent bone marrow transplantation (BMT) from HLA-identical sibling donors with cyclophosphamide and in vivo anti-CD52 monoclonal antibodies (MoAb) for conditioning. The median age at BMT was 17 years (range, 4-46 years). Before BMT, 58% were heavily transfused (>50 transfusions), and 42% had previously experienced treatment failure with antithymocyte globulin-based immunosuppressive therapy. Unmanipulated bone marrow was used as the source of stem cells in all patients except 1. Graft-versus-host disease (GVHD) prophylaxis was with cyclosporine alone in 19 (58%) patients; 14 received anti-CD52 MoAb in addition to cyclosporine. The conditioning regimen was well tolerated without significant acute toxicity. Graft failure was seen in 8 patients (primary, n = 4; secondary, n = 4). Of those whose grafts failed, 4 survived long-term (complete autologous recovery, n = 2; rescue with previously stored marrow, n = 1; second allograft, n = 1). The cumulative incidence of graft failure and grade II to IV acute and chronic GVHD was 24%, 14%, and 4%, respectively. None developed extensive chronic GVHD. With a median follow-up of 59 months, the 5-year survival was 81% (95% confidence interval, 68%-96%). No unexpected early or late infectious or noninfectious complications were observed. We conclude that the conditioning regimen containing cyclophosphamide and anti-CD52 MoAb is well tolerated and effective for acquired aplastic anemia with HLA-matched sibling donors. The favorable effect on the incidence and severity of GVHD is noteworthy in this study and warrants further investigation.

Activation of the c-Jun N-terminal kinase (JNK) signaling pathway is essential during PBOX-6-induced apoptosis in chronic myelogenous leukemia (CML) cells

The mitogen-activated protein (MAP) kinase family is activated in response to a wide variety of external stress signals such as UV irradiation, heat shock, and many chemotherapeutic drugs and leads to the induction of apoptosis. A novel series of pyrrolo-1,5-benzoxazepines have been shown to potently induce apoptosis in chronic myelogenous leukemia (CML) cells, which are resistant to many chemotherapeutic agents. In this study we have delineated part of the mechanism by which a representative compound known as PBOX-6 induces apoptosis. We have investigated whether PBOX-6 induces activation of MAP kinase signaling pathways in CML cells. Treatment of K562 cells with PBOX-6 resulted in the transient activation of two JNK isoforms, JNK1 and JNK2. In contrast, PBOX-6 did not activate the extracellular signal-regulated kinase (ERK) or p38. Apoptosis was found to occur independently of the small GTPases Ras, Rac, and Cdc42 but involved phosphorylation of the JNK substrates, c-Jun and ATF-2. Pretreatment of K562 cells with the JNK inhibitor, dicoumarol, abolished PBOX-6-induced phosphorylation of c-Jun and ATF-2 and inhibited the induced apoptosis, suggesting that JNK activation is an essential component of the apoptotic pathway induced by PBOX-6. Consistent with this finding, transfection of K562 cells with the JNK scaffold protein, JIP-1, inhibited JNK activity and apoptosis induced by PBOX-6. JIP-1 specifically scaffolds JNK, MKK7, and members of the mixed-lineage kinase (MLK) family, implicating these kinases upstream of JNK in the apoptotic pathway induced by PBOX-6 in K562 cells.

Incidence and significance of the JAK2 V617F mutation in patients with chronic myeloproliferative disorders

BACKGROUND: The chronic myeloproliferative disorders (MPD) are clonal haemopoietic stem cell disorders.

AIMS: The incidence of JAK2 V617F mutation was sought in a population of patients with MPD.

METHODS: The JAK2 V617 mutation status was determined in 79 patients with known MPD and 59 patients with features suggestive of MPD.

RESULTS: The mutation was found in patients with polycythaemia vera, essential thrombocythaemia, idiopathic myelofibrosis and in patients with other chronic myeloproliferative disorders. Eight JAK2 V617F positive cases were identified amongst those patients with features suggestive of MPD.

CONCLUSIONS: The incidence of the JAK2 V617F mutation in MPD patients is similar to that reported by other groups. The assay confirmed and refined the diagnosis of several patients with features indicative of MPD. We suggest screening for this mutation in all patients with known and suspected MPD as identification is valuable in classification and is a potential target for signal transduction therapy.

Marrow aplasia developing 13 years after HLA-identical sibling allogeneic transplantation for chronic myeloid leukaemia:successful treatment with antithymocyte globulin and peripheral blood stem cell infusion from the original donor

Secondary or late graft failure has been defined as the development of inadequate marrow function after initial engraftment has been achieved. We describe a case of profound marrow aplasia occurring 13 years after sibling allogeneic bone marrow transplantation for chronic myeloid leukaemia (CML) in first chronic phase. Although the patient remained a complete donor chimera, thereby suggesting that an unselected infusion of donor peripheral blood stem cells (PBSC) or bone marrow might be indicated, the newly acquired aplasia was thought to be immune in aetiology and some immunosuppression was therefore considered appropriate. Rapid haematological recovery was achieved after the infusion of unselected PBSC from the original donor following conditioning with anti-thymocyte globulin (ATG).

Achieving hypoxia-inducible gene expression in tumors

Hypoxia is an inevitable feature of solid tumors and a common cause of treatment failure. Hypoxia acts as a trigger to genetic instability, apoptosis and possibly metastases. The adaptive response to cellular hypoxia involves the modulation of the synthesis of multiple proteins controlling processes such as glucose homeostasis, angiogenesis, vascular permeability and inflammation. The hypoxia responsive element (HRE) sequences isolated from oxygen-responsive genes have been shown to selectively induce gene expression in response to hypoxia when placed upstream of a promoter. The levels of induced gene expression were dependent on the number of HRE copies and the oxygen tension. Hypoxia-mediated cancer gene therapy strategies may represent a promising mean to significantly improve the efficacy of standard radiation therapy and chemotherapy approaches.

Fludarabine, Cyclophosphamide and Rituximab:an effective chemoimmunotherapy combination with high remission rates for chronic lymphocytic leukaemia

BACKGROUND: Combined Fludarabine and Cyclophosphamide is now standard first-line therapy in chronic lymphocytic leukaemia (CLL) and the addition of Rituximab improves outcome.

METHODS: We adopted a modified Fludarabine, Cyclophosphamide and Rituximab (FCR) protocol in treating 39 patients (median age 57 years) with progressive or advanced CLL. Depending on CR, treatment was given for four or six cycles.

RESULT: Twenty-six patients were treatment naïve and 13 were pre-treated. Twelve patients had progressive Binet stage A, 16 stage B and 11 stage C disease. The overall response rate (ORR) was 100%, with 75% achieving CR. Neutropenia was the major toxicity in 71/187 (38%) of the cycles. There were five deaths, two from infection and three from progressive disease. Twenty-six of 31 patients have maintained their post-treatment disease status for a median of 17 months (2-41).

CONCLUSION: We conclude that FCR is a feasible, well-tolerated and effective treatment for patients with CLL.