E-selectin ligands recognised by HECA452 induce drug resistance in myeloma, which is overcome by the E-selectin antagonist, GMI-1271.

Multiple myeloma (MM) is characterized by the clonal expansion and metastatic spread of malignant plasma cells to multiple sites in the bone marrow (BM). Recently, we implicated the sialyltransferase ST3Gal-6, an enzyme critical to the generation of E-selectin ligands, in MM BM homing and resistance to therapy. Since E-selectin is constitutively expressed in the BM microvasculature, we wished to establish the contribution of E-selectin ligands to MM biology. We report that functional E-selectin ligands are restricted to a minor subpopulation of MM cell lines which, upon expansion, demonstrate specific and robust interaction with recombinant E-selectin in vitro. Moreover, an increase in the mRNA levels of genes involved in the generation of E-selectin ligands was associated with inferior progression-free survival in the CoMMpass study. In vivo, E-selectin ligand-enriched cells induced a more aggressive disease and were completely insensitive to Bortezomib. Importantly, this resistance could be reverted by co-administration of GMI-1271, a specific glycomimetic antagonist of E-selectin. Finally, we report that E-selectin ligand-bearing cells are present in primary MM samples from BM and peripheral blood with a higher proportion seen in relapsed patients. This study provides a rationale for targeting E-selectin receptor/ligand interactions to overcome MM metastasis and chemoresistance.

Targeting the Pim kinases in multiple myeloma.

Multiple myeloma (MM) is a plasma cell malignancy that remains incurable. Novel treatment strategies to improve survival are urgently required. The Pims are a small family of serine/threonine kinases with increased expression across the hematological malignancies. Pim-2 shows highest expression in MM and constitutes a promising therapeutic target. It is upregulated by the bone marrow microenvironment to mediate proliferation and promote MM survival. Pim-2 also has a key role in the bone destruction typically seen in MM. Additional putative roles of the Pim kinases in MM include trafficking of malignant cells, promoting oncogenic signaling in the hypoxic bone marrow microenvironment and mediating resistance to therapy. A number of Pim inhibitors are now under development with lead compounds entering the clinic. The ATP-competitive Pim inhibitor LGH447 has recently been reported to have single agent activity in MM. It is anticipated that Pim inhibition will be of clinical benefit in combination with standard treatments and/or with novel drugs targeting other survival pathways in MM.

Management of parenteral nutrition associated hyperglycaemia: a comparison of subcutaneous and intravenous insulin regimen.

PN is associated with significant hyperglycaemia, which may be detrimental to clinical outcome. There are few data on the management of this phenomenon outside of intensive care units. In our unit, we studied the efficacy of protocol-based intravenous insulin delivery as compared to subcutaneous insulin prescribed individually outside of the critical care setting. In a retrospective review over a two-year period, we compared patients with PN-associated hyperglycaemia who had received both modes of insulin therapy. A total of 122 who developed PN-associated hyperglycaemia were identified. Those on the intravenous insulin regimen were within glycaemic target for more time than those on the subcutaneous regimen (62% Vs 43%, p = 0.008). We therefore conclude that outside of the critical care setting, intravenous insulin delivers better glycaemic control and should therefore be considered optimum therapy for patients with PN-associated hyperglycaemia.

Changes in leg ulcer management practice following training in an Irish community setting.

OBJECTIVE: To identify regional changes in leg ulcer management following leg ulcer training for community-based nurses which incorporated Doppler ankle brachial pressure index (ABPI) assessment. METHODS: This was a two-part study conducted in the Irish Health Service Executive, Mid-Western Area. An initial audit in 2005 gathered details on all leg ulcer patients treated in the community in one week, including patient demographics, ulcer aetiology, assessment and treatment. The first audit was carried out before the introduction of a training course for community-based nurses in leg ulcer assessment and management. The training programme was delivered in 2005 and again in 2006. In total 30 public health nurses and community registered nurses from this region completed the course. The second part of the study involved repeating the audit in 2007. By comparing the results from 2005 with 2007 we were able to identify changes in leg ulcer assessment and management. RESULTS: A total of 426 and 449 leg ulcers were identified, with a prevalence of 0.12% and 0.1% in 2005 and 2007 respectively. Prevalence increased to 1.2% (2005) and 1.1% (2007) in those aged over 70 years. Most ulcers were venous in origin (63.3% in 2005 versus 68.8% in 2007). From 2005 to 2007 the number of venous leg ulcers treated with high compression increased significantly, by almost 16% (p < 0.0001). Once-weekly dressing changes increased by a significant 10%, reducing the number of dressings requiring more frequent changes (p = 0.002). CONCLUSION: Significant improvements in leg-ulcer practices were noted in the 18-month study period. The results show very significant increases in number of patients treated with high compression and a significant reduction in more than once-weekly dressing changes. These positive changes may be partly attributed to the enhanced knowledge and skills nurses gained by participating in training.

An exploration of current leg ulcer management practices in an Irish community setting.

OBJECTIVE: To establish the prevalence of leg ulceration in the Irish Health Service Executive (HSE) mid-western region and to determine the level of assessment and treatment patients have been receiving in the local community. METHOD: Before the introduction of a Doppler training programme, all public health and community health nurses working in the Irish HSE mid-western area were requested to complete an audit form on each patient being treated for leg ulceration during a predefined one-week period. This form recorded details on patient demographics, ulcer aetiology, assessment and treatment. Out of 97 nurses, 96 responded to this audit, giving a response rate of 98.9%. RESULTS: A total of 429 patients with 449 leg ulcers were identified. Mean age was 75.5 years (standard deviation 10.7). Overall prevalence was 0.12%, which increased to 1.2% in those aged 70 years and over. Women were almost twice as likely as men to be affected (ratio of 1.8:1). The main causes of ulceration were reported as venous incompetence accounting for 63.3% (284/449) and arterial insufficiency accounting for 8% (36/449) of all ulcers. Only 59.9% (269/449) of all ulcerated limbs had ABPI measurements performed. Of those reported as venous in origin, 71.8% (204/284) had ABPI measurements recorded. Evidence-based care was generally apparent in this group, with 47.5% (97/204) receiving high compression and 18.1% (37/204) receiving reduced compression. However, in venous leg ulcers where ABPIs were not recorded (n=80) care appeared haphazard and inappropriate. CONCLUSION: Our study has identified the benefit of ABPI Doppler assessment.This assessment could be done in local health centres by trained nurses who could provide more appropriate and timely care to patients, thereby improving outcomes and relieving pressure on acute hospital clinics.

High mineral and vitamin E intake by pregnant ewes lowers colostral immunoglobulin G absorption by the lamb.

A 2 x 2 factorial arrangement of treatments with 78 mature ewes was used to evaluate the effects of supplementing the pregnant ewe's diet with high levels of minerals and vitamin E on immunoglobulin G (IgG) absorption by the lamb and whether any altered efficacy of IgG absorption was due to the colostrum or to the lamb. The ewes were estrus-synchronized in October and housed in wk 10 of gestation. In the final 7 wk of gestation, a grass silage-based diet, offered ad libitum, was supplemented with 500 g of a 19% CP concentrate, and from 1 wk later until lambing, half the ewes was offered 48 g of a mineral/vitamin supplement containing 6.5 g of Ca, 4.9 g of P, 5.9 g of Mg, 4.0 g of Na, 790 mg of Zn, 3.5 mg of Se, 40 mg of I, 200 mg of Mn, 20 mg of Co, and 40 IU of vitamin E. At birth, the lambs were allocated to one of four treatments in a 2 x 2 factorial arrangement, with lamb origin and colostrum origin as the two factors. The lambs born to ewes not offered the mineral supplement were fed colostrum obtained from their own dams or from ewes in the mineral-supplemented treatment, whereas lambs born to ewes given supplemental minerals were fed colostrum obtained either from their dams or from ewes in the control treatment. The ewes were milked at 1, 10, and 18 h postpartum and the lambs were fed using a stomach tube. A 5-mL blood sample was taken from each lamb at 24 h postpartum for IgG analysis. The level of fecal adhesion to the upper tail/rump area of the lamb was subjectively scored at 72 h postpartum. There was no difference in gestation length, lamb birth weight, colostrum yield, or IgG production (P = 0.16 to 0.82). When ewes were fed supplemental minerals, the serum IgG content of the progeny was lower than in their control counterparts (6.8 vs. 16.1 g/L; P < 0.001), regardless of whether the lamb received colostrum from ewes with or without access to supplementary minerals. The difference in serum IgG concentrations at 24 h postpartum was a direct reflection of a compromised efficiency in IgG absorption. The progeny of ewes with access to minerals had higher (P < 0.05) levels of fecal adhesion, which was not related to the origin of the colostrum, indicating altered digestive function in these lambs. We conclude, using the sheep as a model, that high mineral intakes in late pregnancy not only lower serum IgG concentrations in the lamb, but also that high mineral intakes result in the neonate being preprogrammed at birth so that it is born with a compromised ability to absorb colostral IgG.

Restoration of CD4 T-cell responses to cytomegalovirus is short-lived in severely immunodeficient HIV-infected patients responding to highly active antiretroviral therapy.

OBJECTIVES: To define the level of pathogen-specific immune reconstitution persisting over 3 to 5 years of highly active antiretroviral therapy (HAART) in HIV-infected patients who began therapy with CD4 T-cell counts below 50 cells/microL. METHODS: Cytomegalovirus (CMV)-specific T-cell responses were analysed in adult HIV-1-infected patients with nadir CD4 T-cell counts below 50 cells/microL before HAART. CMV-specific CD4 T-cell responses were measured by interferon-gamma enzyme-linked immunospot assay (ELISpot assay), lymphoproliferation and interferon-gamma levels in cell culture supernatants. RESULTS: CD4 T-cell responses to CMV were low in untreated patients and remained low during the first year on HAART, but increased progressively to levels similar to those found in HIV-seronegative CMV-seropositive controls at 3 years. Responses then declined markedly and at 5 years were lower than controls. This could not be explained by changes in CD4 or CD8 T-cell counts or plasma HIV RNA levels. Interferon-gamma and interleukin-5 responses to a mitogen were maintained or elevated. CONCLUSIONS: CMV-specific CD4 T-cell responses were found to decline after 3-5 years on HAART and may provide inadequate long-term protection against CMV disease in patients who are severely immunodeficient prior to treatment.

Levels of IL-6 and soluble IL-6 receptor are increased in HIV patients with a history of immune restoration disease after HAART.

OBJECTIVES: We have previously described immune restoration diseases (IRD) associated with asymptomatic opportunistic infections presenting in immunodeficient HIV patients responding to highly active antiretroviral therapy (HAART). Here we address the question of whether patients with a history of IRD exhibit persistent immune activation, shown by elevated levels of interleukin-(IL)-6 and soluble IL-6 receptor (sIL-6R). METHODS: Peripheral blood mononuclear cells (PBMCs) and plasma were collected from HIV patients with nadir CD4 T cell counts of < 80/microL and who had achieved immune reconstitution after HAART with (n=14) or without (n=15) experiencing IRD, severely immunodeficient (SID) patients with < 80 CD4 T cells/microL (n=8) and HIV seronegative controls (n=15). PBMC production and plasma levels of IL-6, sIL-6R and interferon (IFN)-gamma (PBMC only) were measured by enzyme linked immunosorbent assay (ELISA). Intracellular flow cytometry was used to determine the predominant cellular source of IL-6 in HIV patients and controls. RESULTS: Unstimulated PBMC from IRD patients produced significantly higher amounts of IL-6 and sIL-6R than non-IRD patients and HIV seronegative controls. The sIL-6R concentration was also significantly higher in supernatants from mitogen-stimulated PBMC from IRD patients compared to non-IRD patients. The production of IFN-gamma did not differ between IRD and non-IRD patients. IRD patients had significantly higher plasma levels of IL-6 compared to non-IRD patients, SID patients and controls. Monocytes were the predominant source of IL-6 in both HIV patients and controls. CONCLUSIONS: Patients with a history of IRD after HAART have elevated levels of IL-6 and sIL-6R.

An evaluation of serum soluble CD30 levels and serum CD26 (DPPIV) enzyme activity as markers of type 2 and type 1 cytokines in HIV patients receiving highly active antiretroviral therapy.

This study evaluates serum CD26 (dipeptidyl peptidase IV, DPPIV) enzyme activity and serum levels of soluble CD30 as markers of T1 and T2 cytokine environments in HIV patients who achieved immune reconstitution after highly active antiretroviral therapy (HAART). Patients who had experienced inflammatory disease associated with pre-existent opportunistic infections after HAART (immune restoration diseases, IRD) were considered separately. Serum sCD30 levels and CD26 (DPPIV) enzyme activity were compared with IFN-gamma production by PBMC cultured with cytomegalovirus (CMV) antigen in controls and patient groups. High sCD30 levels were associated with low IFN-gamma production after antigenic stimulation in control subjects and, to a lesser extent, in immune reconstituted HIV patients. There was no association between serum CD26 (DPPIV) enzyme activity and IFN-gamma production or sCD30 levels. Serum sCD30 levels and CD26 (DPPIV) enzyme activity were significantly increased in immune reconstituted patients with high HIV viral loads. Patients who had experienced CMV retinitis as an IRD had significantly higher sCD30 levels than all other patient groups. Hence, high sCD30 levels may be a marker of a T2 cytokine environment in HIV patients with immune reconstitution and are associated with higher HIV viral loads and a history of CMV associated IRD.

Immune dysfunction and immune restoration disease in HIV patients given highly active antiretroviral therapy.

BACKGROUND: Some immune defects caused by HIV infection resolve following treatment with highly active antiretroviral therapy (HAART), but residual immune dysfunction may cause disease. Problems with the regulation of the restored immune system in the first six months of treatment can lead to atypical presentations of mycobacterial, cytomegalovirus (CMV), hepatitis B virus or hepatitis C virus (HCV) disease. We defined these conditions as immune restoration diseases (IRD) and showed that they occur in 30-40% of individuals who begin HAART from low CD4 T cell counts. OBJECTIVES: Analysis of immune dysregulation in patients who have responded to HAART. STUDY DESIGN: Patients with successful immune reconstitution following HAART were selected from a database containing details of all patients managed at Royal Perth Hospital (Western Australia) on the basis a CD4 T cell count <100/microl before HAART and an increase of >4-fold or to >200 CD4 T cells/microl. RESULTS: Patients who had experienced an IRD demonstrated increased levels of bioavailable IL-6 and increased expression of CCR5 and CCR3 on monocytes and granulocytes, but numbers of gammadeltaT-cells were similar to patients with similar CD4 T cell counts without an IRD. Carriage of HLA-A2, -B44 was associated with a history of CMV retinitis and/or encephalomyelitis as an IRD, but not with IRD initiated by Mycobacterium sp., cutaneous varicella zoster or herpes simplex infections or HCV. We also identified a patient with Graves' thyrotoxicosis and pronounced lymphadenopathy after HAART, and demonstrated that thyroid stimulating hormone receptor antibody production was associated with an increase in serum soluble CD30, suggesting acquired immune dysregulation. CONCLUSIONS: IRD are associated with persistent immune activation, where differences in genetic profiles suggest that distinct pathological mechanisms are responsible for retinitis/encephalomyelitis IRD. Further studies are important as dysregulated T-cell responses may cause disease later in the course of immune reconstitution.

MHC haplotypes affect the expression of opportunistic infections in HIV patients.

This study explores whether MHC genes affect manifestations of opportunistic infections in HIV patients not treated with highly active antiretroviral therapy (HAART) and immunopathologic responses to pre-existing infections in patients who achieved immune reconstitution following HAART (i.e., "immune restoration diseases" or IRD). HLA-B27 and B17 were relatively rare in all HIV patients, but no HLA-B alleles significantly affected cytomegalovirus (CMV) or Mycobacterium avium complex (MAC) disease in patients who had not received HAART. However coexpression of alleles previously defined as the 44.1 ancestral haplotype (HLA-A2, -B44, and -DR4) was more common in the MAC and CMV patients. After HAART, HLA-B44 and (HLA-A2, -B44, -DR4) were found in 66% and 33%, respectively, of patients who experienced an IRD manifested as CMV retinitis and/or encephalomyelitis. This was confirmed by examination of microsatellite alleles, where the C1_2_5 locus in the class I region was most concordant with the 44.1 haplotype in the patients. HLA-B44 was not associated with IRD initiated by Mycobacterium sp, cutaneous VZV or HSV, or HCV infections, suggesting distinct pathologic mechanisms are responsible. CMV retinitis/encephalomyelitis IRD patients had marginally lower pretreatment CD4 T-cell counts, but indices of immune reconstitution were similar in all groups and independent of HLA-B44.

Assessment of immune function by lymphoproliferation underestimates lymphocyte functional capacity in HIV patients treated with highly active antiretroviral therapy.

The objective of this study was to evaluate T cell responses in HIV-infected patients after highly active antiretroviral therapy (HAART), using four assays of immune function, and to determine which best reflects the presence of CD4(+) T cells able to respond to CMV antigen. Peripheral blood mononuclear cells from 41 HIVinfected patients and 31 healthy HIV-seronegative controls were cultured with mitogen (PMA/Ca(2+) ionophore) or antigen (CMV). Production of interferon gamma (IFN-gamma) determined by ELISpot assay was compared with lymphoproliferation, IFN-gamma production assessed by ELISA, and CD69 expression and intracellular IFN-gamma assessed by flow cytometry. Cells from patients whose CD4(+) T cells counts increased 4-fold or to >200 cells/microl after HAART responded as well as control cells when assessed by IFN-gamma production and CD69 expression after mitogenic stimulation, but lymphoproliferation responses were depressed by about 52%. Patients who did not meet these criteria for immune reconstitution had lymphoproliferative responses up to 30-fold lower than control subjects, while intracellular IFN-gamma and CD69 expression and ELISpot counts were less than 3-fold lower. Responses to CMV antigen could not be detected by flow cytometry, but were readily detected by ELISpot in CMV-seropositive patients whose CD4(+) T cell counts had increased after HAART. This included patients with low responses assessed by lymphoproliferation. Moreover, ELISpot responses measured with fresh and frozen cells were comparable, while lymphoproliferation assays required fresh cells. In conclusion, the ELISpot assay is a sensitive and efficient technique for detecting CMV-specific IFN-gamma responses in samples that display poor responses when assessed by lymphoproliferation assays.

Structure-activity studies of the regulatory interaction of the 10 kilodalton C-terminal fragment of caldesmon with actin and the effect of mutation of caldesmon residues 691-696.

We have used isotope-edited nuclear magnetic resonance spectroscopy, binding studies, and ATPase activity assays to investigate the interaction with F-actin of the 10 kDa C-terminal 658C fragment of chicken gizzard caldesmon and two site-directed mutants of this fragment. Simultaneous dual-sited contacts with F-actin are observed for the segments of the 658C sequence flanking tryptophan residues 692 and 722. Competition experiments showed that both 658C contacts with actin are displaced by substoichiometric concentrations of the short inhibitory region of troponin-I indicative of different binding sites on actin for these regions of troponin-I and caldesmon. Substitution of caldesmon serine-702 by aspartic acid within the spacer region linking the two actin contacts of 658C led to weaker binding but with retention of equivalent affinity for each interaction site. Differential binding affinity of the two sites was achieved by replacement of the sequence Glu691-Trp-Leu-Thr-Lys-Thr696 by Pro-Gly-His-Tyr-Asn-Asn. Consistent with these data, the concentration of this Cg1 mutant required to achieve 50% inhibition of actin-tropomyosin-activated myosin ATPase was 4-fold greater than found for the 658C fragment. Although calmodulin binding to Cg1 was observed, calmodulin proved ineffective in relieving the inhibition induced by the binding of this mutant to actin. These results are discussed in light of the actin contacts which are involved in the inhibitory activity possessed by different regions of the C-terminus of caldesmon.

The ordered phosphorylation of cardiac troponin I by the cAMP-dependent protein kinase--structural consequences and functional implications.

The pattern of phosphorylation of adjacent serine residues in several peptides based on the N-terminal region of human cardiac troponin I has been analysed by PAGE and 1H NMR spectroscopy to identify the products. With cAMP-dependent protein kinase, Ser24 is rapidly phosphorylated, and subsequent much slower phosphorylation of Ser23 occurs only after phosphorylation of Ser24 is almost complete. Monophosphorylation of the peptide at Ser23 was not detected at any time. On replacement of Arg22 with Ala or Met the sole phosphorylation target was Ser23, phosphorylation being considerably slower than for Ser24 in the wild-type peptide, while diphosphorylation could not be detected after prolonged incubation. The results emphasise the importance of the N-terminal sequence RRRSS for the function of cardiac troponin I and imply that in human cardiac muscle unstimulated by adrenaline, troponin I is phosphorylated on Ser24. Comparative two-dimensional NOESY data indicate that in the diphosphorylated form at physiological pH values, specific structural constraints are imposed on the N-terminal peptide region. These constraints result in the effective screening of the two phosphate groups from each other by the arginine residues N-terminal to the serine pair and stabilisation of the structure in the region of residues 25-29, which is adjacent to a site of interaction between troponin I and troponin C. These conformational changes presumably underlie the decrease in calcium sensitivity of the myofibrillar ATPase that occurs after adrenaline intervention.

Structural determinants of substrate selection by the human insulin-receptor protein-tyrosine kinase.

Using NMR spectroscopy to visualise tyrosine phosphorylation kinetics in real time, we have investigated the sequence-dependent determinants of the selectivity of the human insulin receptor protein-tyrosine kinase for different tyrosine residues. The peptides used encompass the multiple-tyrosine-containing autophosphorylation site sequences from the insulin receptor kinase core domain (Tyr1158, Tyr1162 and Tyr1163) and from its specific C-terminal tail domain (Tyr1328 and Tyr1334). Comparison of the phosphorylation kinetics with those found for the tyrosine residues on a peptide comprising the regulatory tyrosine phosphorylation site of cdc2 points to the role of the primary sequence context of the phosphate acceptor. The particularly deleterious influence of a basic residue immediately C-terminal to the tyrosine is discussed in relation to the autophosphorylation properties of the regulatory loop regions of the insulin and epidermal growth factor receptor kinases. The data further suggest that receptor tyrosine kinase active sites and their substrate targets act in concert to ensure that specific downstream effects are activated.

Phosphorylation effects on flanking charged residues. Structural implications for signal transduction in protein kinases.

1H-NMR and 31P-NMR spectroscopy were employed to assess the electrostatic consequences of phosphorylation of single and multiple tyrosine residues in peptides derived from the core and tail autophosphorylation regions of the human insulin receptor tyrosine-kinase domain. In both peptides, phosphorylation was accompanied by changes in the resonances from basic side-chains; those from acidic residues were unaffected. Tyrosine phosphorylation caused increases of up to one in the pKa values of histidine residues situated up to eight residues away in the primary sequence. Titration curve analysis by Hill plots suggested some cooperativity of histidine and phosphate ionizations. Behaviour closely analogous to that of the insulin receptor tail peptide was observed during changes in phosphorylation of the intact insulin receptor kinase domain, suggesting that the electrostatic dissemination effects seen for the isolated peptide are retained by the peptide sequence in the context of the much larger protein. Similar changes in the behaviour of basic residues were also observed upon tyrosine phosphorylation of a cdc2-derived peptide, suggesting that this potential of phosphorylation events to propagate directed structural changes may find a widespread utility in the activation of protein kinases and in the transduction of phosphorylation-based signalling.