Some factor VIII inhibitor antibodies recognize a common epitope corresponding to C2 domain amino acids 2248 through 2312, which overlap a phospholipid-binding site

The finding that human factor VIII (fVIII) inhibitor antibodies with C2 domain epitopes interfere with the binding of fVIII to phosphatidylserine (PS) suggested that this is the mechanism by which they inactivate fVIII. We constructed a recombinant C2 domain polypeptide and demonstrated that it bound to all six human inhibitors with fVIII light chain specificity. Thus, some antibodies within the polyclonal anti-light chain population require only amino acids within C2 for binding. Recombinant C2 also partially or completely neutralized the inhibitor titer of these plasmas, demonstrating that anti-C2 antibodies inhibit fVIII activity. Immunoblotting of a series of C2 deletion polypeptides, expressed in Escherichia coli, with inhibitor plasmas showed that the epitopes for human inhibitors consist of a common core of amino acid residues 2248 through 2312 with differing extensions for individual inhibitors. The epitope of inhibitory monoclonal antibody (MoAb) ESH8 was localized to residues 2248 through 2285. Three human antibodies and anti-C2 MoAb NMC-VIII/5 bound to a synthetic peptide consisting of amino acids 2303 through 2332, a PS- binding site, but MoAb ESH8 did not. These antibodies also inhibited the binding of fVIII to synthetic phospholipid membranes of PS and phosphatidylcholine, confirming that the blocked epitopes contribute to membrane binding as well as binding to PS. In contrast, MoAb ESH8 did not inhibit binding. As the maximal function of activated fVIII in the intrinsic factor Xase complex requires its binding to a phospholipid membrane, we propose that fVIII inhibition by anti-C2 antibodies is related to the overlap of their epitopes with the PS-binding site. MoAb ESH8 did not inhibit fVIII binding to PS-containing membranes, suggesting the existence of a second mechanism of fVIII inhibition by anti-C2 antibodies.     

Autoantibodies against the platelet glycoproteins (GP) IIb/IIIa, Ia/IIa, and IV and partial deficiency in GPIV in a patient with a bleeding disorder and a defective platelet collagen interaction

To evaluate the physiologic importance of the different collagen receptors on platelets, we screened 806 patients admitted to the hospital because of hemorrhagic diathesis for eventual laboratory evidence of a pathologic platelet collagen interaction, and found 5 patients with an isolated deficiency in collagen-induced platelet aggregation. Four of these five patients had a partial defect, one had a complete defect. The structural and functional analysis of the platelets from the patient with a complete defect showed a deficiency in glycoprotein (GP) IV and autoantibodies against GPIIb/IIIa, GPIa/IIa, and GPIV. Patient plasma had only a minimal effect on normal control platelets and Naka-negative platelets. The analyses of the defect in the patient and of the data in the literature suggest that a single defect may not result in clinical bleeding (GPIV-deficient patients do not bleed), but may become symptomatic in combination with another defect such as the autoantibodies against GPIa/IIa, GPIV, and/or GPIIb/IIIa, all of which are involved in platelet collagen interactions (three of four of our immune thrombocytopenic purpura patients with anti-GPIV and anti-GPIIb/IIIa autoantibodies had a bleeding disorder). We hypothesize that it is the synergism of two abnormalities that results in the defective function, a mechanism that is in agreement with earlier studies on platelet collagen interaction that suggests that a double defect in platelet collagen interactions is required to become clinically apparent.     

Characterization of a factor IX variant with a glycine207 to glutamic acid mutation

Factor IXTaipei9 is a factor IX variant from a hemophilia B patient with reduced levels of circulating protein molecules (cross-reacting material reduced, CRM). This variant contained a glycine (Gly) to glutamic acid (Glu) substitution at the 207th codon of mature factor IX. The functional consequences of the Gly-->Glu mutation in factor IXTaipei9 (IXG207E) were characterized in this study. Plasma-derived IXG207E exhibited a mobility similar to that of normal factor IX on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its specific activity was estimated to be 3.5% that of the purified normal factor IX in a one-stage partial thromboplastin time assay (aPTT). Cleavage of factor IXG207E by factor XIa or factor VIIa-tissue factor complex appeared to be normal. When the calcium-dependent conformational change was examined by monitoring quenching of intrinsic fluorescence, both normal factor IX and IXG207E exhibited equivalent intrinsic fluorescence quenching. Activated factor IXG207E (IXaG207E) also binds antithrombin III equally as well as normal factor IXa. However, aberrant binding of the active site probe p-aminobenzamidine was observed for factor XIa-activated factor IXG207E, indicating that the active site pocket of the heavy chain of factor IXaG207E was abnormal. Moreover, the rate of activation of factor X by factor IXaG207E, as measured in a purified system using chromogenic substrates, was estimated to be 1/40 of that of normal factor IXa. A computer-modeled heavy-chain structure of factor IXa predicts a hydrophobic environment surrounding Gly-207 and this Gly forms a hydrogen bound to the active site serine-365. The molecular mechanism of the Gly-->Glu mutation in factor IXTaipei9 might result in the alteration of the microenvironment of the active site pocket which renders the active site serine-365 inaccessible to its substrate.     

CD44v6 expression in patients with stage II or stage III sporadic colorectal cancer is superior to CD44 expression for predicting progression

Background: Currently, it is difficult to predict the prognosis of patients exhibiting stage II or stage III colorectal cancer (CRC) and to identify those patients most likely to benefit from aggressive treatment. The current study was performed to examine the clinicopathological significance of CD44 and CD44v6 protein expression in these patients. Study design: We retrospectively investigated 187 consecutive patients who underwent surgery with curative intent for stage II to III CRC from 2007 to 2013 in the Beijing Civil Aviation Hospital. CD44 and CD44v6 protein expression levels were determined using immunohistochemistry and compared to the clinicopathological data. Results: Using immunohistochemical detection, CD44 expression was observed in 108 (57.75%) of the CRC patients; and its detection was significantly associated with greater invasion depth, lymph node metastasis, angiolymphatic invasion, and a more advanced pathological tumor-lymph node-metastasis (TNM) stage. CD44v6 expression was observed in 135 (72.19%) of the CRC patients; and its expression was significantly associated with a poorly differentiated histology, greater invasion depth, lymph node metastasis, angiolymphatic invasion, and a more advanced pathological TNM stage. Expression of CD44v6 was higher than that of CD44 in stage II and stage III sporadic CRC. Conclusion: CD44v6 is a more useful marker for predicting a poor prognosis in stage II and stage III sporadic CRC as compared to CD44.     

Protein kinases potentially capable of catalyzing the phosphorylation of p47-phox in normal neutrophils and neutrophils of patients with chronic granulomatous disease

A procedure for uncovering novel protein kinases was used to search for enzymes in neutrophils that may catalyze the phosphorylation of the 47- Kd subunit of the NADPH oxidase system (p47-phox). This component of the oxidase can undergo phosphorylation on multiple sites. The method is based on the ability of renatured kinases to recognize exogenous substrates fixed in gels. We report that neutrophils contain several uncharacterized protein kinases that catalyze the phosphorylation of a peptide substrate that corresponds to amino acid residues 297 through 331 of p47-phox. Some of these enzymes are strongly activated on stimulation of the cells with phorbol 12-myristate 13-acetate (PMA). The results indicate that the phosphorylation of p47-phox in neutrophils may be more complicated than previously appreciated and may involve multiple protein kinases. In addition, we have examined both the renaturable protein kinases and the properties of protein kinase C (PKC) in neutrophils from patients with chronic granulomatous disease (CGD) who are deficient in cytochrome b558. Previous studies have shown that these cells exhibit incomplete phosphorylation of p47-phox on stimulation. In this study, we were unable to detect any alterations in the renaturable protein kinases or PKC in CGD neutrophils that could explain these defects in the phosphorylation of p47-phox.     

Quantitative and cell-cycle differences in progenitor cells mobilized by recombinant human interleukin-7 and recombinant human granulocyte colony-stimulating factor

Administration of recombinant human interleukin-7 (rhIL-7) to mice increases the exportation of myeloid progenitors (colony-forming unit [CFU]-c and CFU-granulocyte erythroid megakaryocyte macrophage [CFU- GEMM]) from the bone marrow (BM) to peripheral organs, including blood, and also increases the number of primitive progenitor and stem cells in the peripheral blood (PB). We now report that combined treatment of mice with rhIL-7 and recombinant human granulocyte-colony stimulating factor (rhG-CSF) stimulates a twofold to 10-fold increase in the total number of PB CFU-c, and a twofold to fivefold increase in the total number of PB CFU-spleen at day 8 (CFU-S8) over the increase stimulated by rhIL-7 or rhG-CSF alone. In addition, the quality of mobilized cells with trilineage, long-term marrow-repopulating activity is maintained or increased in mice treated with rhIL-7 and rhG-CSF compared with rhIL- 7 or rhG-CSF alone. These differences in mobilizing efficiency suggest qualitative differences in the mechanisms by which rhIL-7 and rhG-CSF mobilize progenitor cells, in fact, the functional status of progenitor cells mobilized by rhIL-7 differs from that of cells mobilized by rhG- CSF in that the incidence of actively cycling (S-phase) progenitors obtained from the PB is about 20-fold higher for rhIL-7-treated mice than for mice treated with rhG-CSF. These results suggest the use of rhIL-7-mobilized progenitor/stem cells for gene-modification and tracking studies, and highlight different functions and rates of repopulation after reconstitution with PB leukocytes obtained from mice treated with rhIL-7 versus rhG-CSF.     

Towards a combined prognostic index for survival in HIV infection: the role of ‘non‐HIV’ biomarkers

Background

As those with HIV infection live longer, ‘non‐AIDS’ condition associated with immunodeficiency and chronic inflammation are more common. We ask whether ‘non‐HIV’ biomarkers improve differentiation of mortality risk among individuals initiating combination antiretroviral therapy (cART).

Methods

Using Poisson models, we analysed data from the Veterans Aging Cohort Study (VACS) on HIV‐infected veterans initiating cART between 1 January 1997 and 1 August 2002. Measurements included: HIV biomarkers (CD4 cell count, HIV RNA and AIDS‐defining conditions); ‘non‐HIV’ biomarkers (haemoglobin, transaminases, platelets, creatinine, and hepatitis B and C serology); substance abuse or dependence (alcohol or drug); and age. Outcome was all cause mortality. We tested the discrimination (C statistics) of each biomarker group alone and in combination in development and validation data sets, over a range of survival intervals, and adjusting for missing data.

Results

Of veterans initiating cART, 9784 (72%) had complete data. Of these, 2566 died. Subjects were middle‐aged (median age 45 years), mainly male (98%) and predominantly black (51%). HIV and ‘non‐HIV’ markers were associated with each other (P<0.0001) and discriminated mortality (C statistics 0.68–0.73); when combined, discrimination improved (P<0.0001). Discrimination for the VACS Index was greater for shorter survival intervals [30‐day C statistic 0.86, 95% confidence interval (CI) 0.80–0.91], but good for intervals of up to 8 years (C statistic 0.73, 95% CI 0.72–0.74). Results were robust to adjustment for missing data.

Conclusions

When added to HIV biomarkers, ‘non‐HIV’ biomarkers improve differentiation of mortality. When evaluated over similar intervals, the VACS Index discriminates as well as other established indices. After further validation, the VACS Index may provide a useful, integrated risk assessment for management and research.     

Release of ATP from rat urinary bladder mucosa: role of acid,vanilloids and stretch

Background and purpose:

ATP, released from urothelial cells, modulates afferent nerve firing from the urinary bladder. Here, we have characterized ATP release from the rat bladder mucosa in response to acid, capsaicin, electrical field stimulation (EFS) and stretch, using agonists and antagonists at transient receptor potential vanilloid receptor 1 (TRPV1) and acid-sensing ion channels (ASICs).

Experimental approach:

Rat mucosal strips (containing urothelium and lamina propria) in Perspex microbaths were superfused with Krebs solution. ATP was measured after exposure of matched strips to acid (pH 6.6–5.0), capsaicin (0.1–10 µM), EFS or stretch (150% of original length).

Key results:

Median basal ATP release was 3.46 nmol·g⁻¹. The mucosal strips responded to stimuli with potency order (median, IQR): acid (pH 5.6–6.0) 286 (103–555) > 10 µM capsaicin 188 (117–431) > 10 Hz EFS 63.0 (13.3–96.4) > stretch 24.4 (6.73–55.1) nmol ATP g⁻¹. ATP release in response to acid was pH dependent (P < 0.05). Responses to capsaicin did not desensitize nor were they concentration dependent. TRPV1 antagonist, capsazepine (10 µM) abolished capsaicin-evoked ATP release, and reduced acid-evoked (pH 6.5) release to 30% (P < 0.001). The ASIC channel antagonists gadolinium (0.1 mM) and amiloride (0.3 µM) reduced (P < 0.05) the acid-evoked (pH 6.5) release to 40 and 6.5% respectively. ASIC (ASIC1, ASIC2a, ASIC2b, ASIC3) and two TRPV1 gene products were detected in mucosal and detrusor extracts.

Conclusions and implications:

Capsaicin (at TRPV1) and acid (at both TRPV1 and ASIC) induce ATP release from the rat bladder mucosa. This ATP appears to be principally of urothelial origin. This study highlights the importance of ATP and acid as signalling molecules in modulating bladder function.     

Preclinical evaluation of a monoclonal antibody targeting the epidermal growth factor receptor as a radioimmunodiagnostic and radioimmunotherapeutic agent

Background and purpose:

The studies described here are the first to evaluate the in vitro and in vivo properties of ¹¹¹In-CHX-A″-panitumumab for radioimmunotherapy (α- and β^--emitters) and radioimmunoimaging (single photon emission computed tomography and positron emission tomography).

Experimental approach:

Twenty-seven human carcinoma cell lines were analysed for expression of epidermal growth factor receptors by flow cytometry. Panitumumab was conjugated with CHX-A″-DTPA (diethylenetriamine-pentaacetic acid) and radiolabelled with ¹¹¹In. Immunoreactivity of the CHX-A″-DTPA-panitumumab and ¹¹¹In-CHX-A″-DTPA-panitumumab was evaluated by radioimmunoassays. Tumour targeting was determined in vivo by direct quantitation of tumour and normal tissues and by γ-scintigraphy.

Key results:

For 26 of 27 human tumour cell lines, 95% of the cells expressed epidermal growth factor receptors over a range of intensity. Immunoreactivity of panitumumab was retained after modification with CHX-A″-DTPA. Radiolabelling of the immunoconjugate with ¹¹¹In was efficient with a specific activity of 19.5 ± 8.9 mCi·mg⁻¹ obtained. Immunoreactivity and specificity of binding of the ¹¹¹In-panitumumab was shown with A431 cells. Tumour targeting by ¹¹¹In-panitumumab was demonstrated in athymic mice bearing A431, HT-29, LS-174T, SHAW or SKOV-3 s.c. xenografts with little uptake observed in normal tissues. The ¹¹¹In-panitumumab was also evaluated in non-tumour-bearing mice. Pharmacokinetic studies compared the plasma retention time of the ¹¹¹In-panitumumab in both non-tumour-bearing and A431 tumour-bearing mice. Tumour targeting was also visualized by γ-scintigraphy.

Conclusions and implications:

Panitumumab can be efficiently radiolabelled with ¹¹¹In with high labelling yields. Based on the efficiency in tumour targeting and low normal tissue uptake, panitumumab may be an effective targeting component for radioimmunodiagnostic and radioimmunotherapeutic applications.     

Induction of trefoil factor (TFF)1, TFF2 and TFF3 by hypoxia is mediated by hypoxia inducible factor-1: implications for gastric mucosal healing

Background and purpose:

Mucosal microcirculation is compromised during gastric damage induced by non-steroidal anti-inflammatory drugs, such as aspirin. Consequently, oxygen supply to epithelial cells is decreased. The trefoil factor (TFF) peptides are involved in mechanisms of defence and repair in the gastrointestinal tract but their regulation at sites of gastric injury is unknown.

Experimental approach:

Hypoxia and expression of TFF genes and peptides were measured in the damaged stomach of aspirin-treated rats. In a human gastric cell line (AGS cells), the effects of hypoxia and of hypoxia inducible factor (HIF)-1 (through transient transfection of HIF-1α siRNA or over-expression of HIF-1α) on TFF gene expression were evaluated.

Key results:

Hypoxyprobe immunostaining, up-regulation of TFF2 (1.9-fold) and TFF3 (1.8-fold) and a non-significant increase of TFF1 (1.5-fold) mRNA were observed in the damaged stomach of aspirin-treated rats, compared with control animals. Hypoxia (3% O₂, 16 h) induced mRNA for TFF1 (5.8-fold), TTF2 (9.1-fold) and TFF3 (9.3-fold) in AGS cells, an effect mediated by HIF-1, as transient transfection of HIF-1α siRNA reduced the effects of hypoxia. Over-expression of HIF-1α by transfection in non-hypoxic epithelial cells produced a similar pattern of TFF induction to that observed with hypoxia and transactivated a TFF1 reporter construct.

Conclusions and implications:

Hypoxia inducible factor-1 mediated the induction of TFF gene expression by hypoxia in gastric epithelial cells. Low oxygen levels and up-regulation of TFF gene expression in the damaged stomach of aspirin-treated rats suggest that hypoxia induced expression of TFF genes at sites of gastric injury.