Activation of MAP kinase-activated protein kinase 2 in human neutrophils after phorbol ester or fMLP peptide stimulation

In response to extracellular stimulation, one of the earliest events in human neutrophils is protein phosphorylation, which mediates signal transduction and leads to the regulation of cellular functions. Mitogen- activated protein (MAP) kinases are rapidly activated by a variety of mitogens, cytokines, and stresses. The activated MAP kinases in turn regulate their substrate molecules by phosphorylation. MAP kinase- activated protein (MAPKAP) kinase 2, a Ser/Thr kinase, has been shown to be phosphorylated by p38 MAP kinase both in vivo and in vitro. Phosphorylation of the Thr-334 site of MAPKAP kinase 2 results in a conformational change with subsequent activation of the enzyme. To better define the role of MAPKAP kinase 2 in the activation of human neutrophils, its enzymatic activity was measured after stimulation by either a phorbol ester (phorbol myristate acetate [PMA]), a potent protein kinase C activator, or the tripeptide fMLP, which is a chemotactic factor. The in vitro kinase assays indicate that both PMA and fMLP stimulated a transient increase in the enzymatic activity of cellular MAPKAP kinase 2. The induced kinase activation was concentration-dependent and reached a maximum at 5 minutes for PMA and 1 minute for fMLP. To identify potential substrate molecules for MAPKAP kinase 2, a highly active kinase mutant was generated by mutating the MAP kinase phosphorylation site in the C-terminal region. The replacement of threonine 334 with alanine resulted in a marked augmentation of catalytic activity. Analysis of in vitro protein phosphorylation in the presence of the active kinase indicates that a 60-kD cytosolic protein (p60) was markedly phosphorylated and served as the major substrate for MAPKAP kinase 2 in human neutrophils. Based on the MAPKAP kinase 2 phosphorylation site of Hsp27, a competitive inhibitory peptide was synthesized. This competitive inhibitory peptide specifically inhibited MAPKAP kinase 2 enzymatic activity, as well as the in vitro and in vivo kinase-induced p60 phosphorylation. To assess the contribution of MAPKAP kinase 2 in neutrophil function, the oxidative burst response after manipulation of endogenous kinase activity was measured. Intracellular delivery of the competitive inhibitory peptide into human neutrophils reduced both PMA- and fMLP- stimulated superoxide anion production. Thus, the results strongly suggest that MAPKAP kinase 2 is involved in the activation of human neutrophils.     

Carrier detection in hemophilia A: a cooperative international study. I. The carrier phenotype

Eight laboratories in six countries cooperated to clarify several issues concerning the phenotypes of heterozygous carriers of hemophilia "A." Plasma levels of factor VIII (F.VIII:C, formerly VIII:C) and von Willebrand factor (VWF:Ag, formerly VIIIR:Ag) of carriers and normal women were determined by various "in-house" methods; a single lyophilized plasma standard was used for all assays. Analysis of the collated data from 336 carriers (296 obligatory carriers and 40 sporadic carriers) and 137 normal women showed that there was no difference in the F.VIII:C levels of "paternal" carriers (women who had obtained the abnormal gene from their fathers) and "maternal" carriers. Neither was there a difference in the VWF:Ag levels of normal women and either type of carrier. Age was found to have a significant effect on both F.VIII:C and VWF:Ag, values being higher at very young and very old ages, the minima occurring in the 25- to 30-year range. ABO blood type had a striking effect. Women of types A, B, and AB (designated non- O in the study), both normals and carriers, had significantly higher levels of both factors than did women of type O. Analysis by laboratories showed that differences in mean levels of both factors between laboratories were highly significant. It was concluded that age, ABO blood type, and laboratory variation should be taken into account in carrier detection.     

The heterogeneity of type IIA von Willebrand's disease: studies with protease inhibitors

The absence of large von Willebrand factor (vWF) multimers from plasma is a characteristic of Type IIA von Willebrand's disease (vWD) and is thought to contribute to the clinical expression of this disorder. Recently, three IIA patients have been reported in whom intermediate and large multimers could be restored if blood were collected in 5 mm EDTA, 6 mmol/L N-ethylmaleimide, and 1 mmol/L leupeptin. This suggested that absence of large multimers resulted from in vitro proteolysis. We have now collected blood from ten Type IIA vWD patients in these inhibitors but were not able to detect large multimers in the plasma of any of them. In addition, intermediate-sized multimers were reduced or completely absent in all. The inclusion of inhibitors in the citrate anticoagulant, as compared to citrate alone, was found to increase the relative proportion of intermediate multimers in some patients but had no effect in others, and in none did it restore large multimers to plasma. The results with platelet vWF were more varied. Four patients showed an absence or decrease of large multimers, whereas in seven patients large multimers were present. When compared with citrate anticoagulant alone, the inclusion of inhibitors in the anticoagulant had little or no effect on the platelet multimeric pattern. 1-Deamino-8- D-Arginine Vasopressin (DDAVP) was administered to six patients from five families. Two patients from one family showed complete correction and a third patient showed almost complete correction of her bleeding time. Two patients showed minimal correction and one showed no detectable correction. An increase in multimer size after DDAVP tended to be associated with correction of the bleeding time. However, in no case did the largest multimers appear in plasma even in patients with complete bleeding time correction. The presence or absence of inhibitors in the anticoagulant had little or no effect on the multimeric pattern after DDAVP. These results indicate that Type IIA vWD is a heterogeneous disorder in which absence of largest and intermediate multimers is an in vivo phenomenon.     

Therapy of patients with human T-cell lymphotrophic virus I-induced adult T-cell leukemia with anti-Tac, a monoclonal antibody to the receptor for interleukin-2

Human T-cell lymphotropic virus I (HTLV-I)-induced adult T-cell leukemia (ATL) cells constitutively express interleukin-2 (IL-2) receptors identified by the anti-Tac monoclonal antibody (MoAb), whereas normal resting cells do not. This observation provided the scientific basis for a trial of intravenous anti-Tac in the treatment of nine patients with ATL. The patients did not suffer untoward reactions and did not have a reduction in the normal formed elements of the blood, and only one of the nine produced antibodies to the anti-Tac MoAb. Three patients had transient mixed, partial, or complete remissions lasting from 1 to more than 8 months after anti-Tac therapy, as assessed by routine hematologic tests, immunofluorescence analysis of circulating cells, and molecular genetic analysis of HTLV-I provirus integration and of the T-cell receptor gene rearrangement. The precise mechanism of the antitumor effects is unclear; however, the use of a MoAb that prevents the interaction of IL-2 with its receptor on ATL cells provides a rational approach for the treatment of this malignancy.     

Oculocutaneous albinism

Oculocutaneous albinism represents a group of inherited skin disorders characterized by a generalized reduction of cutaneous, ocular and pilar pigmentation from the time of birth. Oculocutaneous albinism types 1 and 2 are the most common, but several other types have been described. A defect in the melanin synthesis pathway, resulting in reduced formation of melanin, is responsible for oculocutaneous albinism. Aetiology, clinical manifestations, diagnosis and management are discussed.     

Genetic analysis of the cloned genome of phage Mu

Randomly sheared fragments of Mu cts62 DNA have been cloned into the EcoRI cleavage site of plasmid p0P203(UV-5)-3, a derivative of pMB9 containing the lactose operator promoter, using poly (dA · dT) tailing with terminal transferase. The extent of Mu DNA carried by the recombinant plasmids was determined genetically by crosses with known markers in essential genes of Mu in rec⁺ backgrounds. Plasmids which rescued essential genes were transformed to rec^? backgrounds to check for expression of the Mu genes. Plasmids were found which rescued and complemented 23 of the 24 essential genes of Mu. Many of these plasmids carried overlapping segments of the Mu genome. The plasmid library was also screened for clones containing the nonessential genes gin and mom. Clones carrying gene A were not found in the initial screening, however, several clones which were resistant to infection by Mu due to expression of the Mu repressor were able to rescue and complement a D108cts-MuAts hybrid phage at 43°.     

Sensitivity to change and association of three-dimensional meniscal measures with radiographic joint space width loss in rapid clinical progression of knee osteoarthritis

Objective

To determine whether 3D meniscal measures had similar sensitivity to longitudinal change as cartilage thickness; to what extent these measures are associated with longitudinal joint space width (JSW) change; and whether the latter associations differ between minimum (mJSW) and fixed-location JSW.

Methods

Two-year changes in medial meniscal position and morphology, cartilage thickness (MRI) and minimum and fixed-location JSW (radiography) were determined in 35 Osteoarthritis Initiative knees [12 men, age: 67 (51-77) years; 23 women, age: 65 (54-78) years], progressing from baseline Kellgren-Lawrence grade ≤2 to knee replacement within 3-5 years. Multiple linear regression assessed the features contributing to JSW change.

Results

Meniscal measures, cartilage thickness and JSW displayed similar sensitivity to change (standardised response mean≤|0.76|). Meniscal changes were strongly associated with JSW change (r≤|0.66|), adding ≤20% to its variance in addition to cartilage thickness change. Fixed-location JSW change (multiple r²=72%) was more strongly related to cartilage and meniscal change than mJSW (61%). Meniscal morphology explained more of fixed-location JSW and meniscal position more of mJSW.

Conclusion

Meniscal measures provide independent information in explaining the variance of radiographic JSW change. Fixed-location JSW appears to be more reflective of structural change than mJSW and, hence, a potentially superior measure of structural progression.

Key Points

? 3D positional/morphological meniscal measures change in rapidly progressing knees. ? Similar sensitivity to 2-year change of quantitative meniscal/cartilage measures in rapid progression. ? Changes in meniscal measures are strongly associated with radiographic JSW change. ? Meniscal change provides information to explain JSW variance independent of cartilage. ? Fixed-location JSW reflects structural disease stage more closely than minimum JSW.

     

Ultrastructural observations on beaded α-motoneuron dendrites

Beaded dendrites of 1α-motoneurons intracellularly labelled with horseradish peroxidase (HRP) were studied ultrastructurally in eight adult cats. For comparison, adjacent unlabelled beaded dendrites of unknown origin were also included in the study. Electron microscopy revealed no signs of degeneration or poor fixation according to common criteria. With the exception of the HRP-reaction product no difference in structure was observed between labelled and unlabelled beaded dendrites. Both the beads and their interconnecting segments were postsynaptic to boutons of normal appearance containing spherical (S-type boutons) or flattened vesicles (F-type boutons). The values for synaptic covering and synaptic packing density of the beaded dendritic regions, which usually were located in the periphery of the dendritic trees, were clearly lower than values obtained previously for cell bodies and proximal dendrites of a-motoneurons.     

A phase I trial of c-Raf kinase antisense oligonucleotide ISIS 5132 administered as a continuous intravenous infusion in patients with advanced cancer.

Raf proteins play a central role in the mitogen-activated protein kinase signaling pathway and hence are involved in oncogenic transformation and tumor cell proliferation. ISIS 5132 is a 20-base antisense phosphorothioate oligodeoxyribonucleotide that specifically down-regulates c-raf expression. We report here an initial study of the safety and tolerability of an i.v. infusion of ISIS 5132 in patients with advanced cancer. A continuous i.v. infusion of ISIS 5132 was administered for 21 days every 4 weeks to 34 patients with a variety of solid tumors refractory to standard therapy. The dose of ISIS 5132 was increased in sequential cohorts of patients, as toxicity allowed, until a final dose of 5.0 mg/kg body weight was reached. Toxicity was scored by common toxicity criteria, and tumor response was monitored. Pharmacokinetic studies were performed for 30 patients treated at doses of < or =4.0 mg/kg/day. The initial dose of ISIS 5132 was 0.5 mg/kg body weight and was successfully increased incrementally to 5.0 mg/kg body weight. Toxicities through the 4.0 mg/kg dose level were not dose limiting. Side effects were minimal and could not be specifically related to ISIS 5132. Two patients had prolonged stabilization of their disease, and one patient with ovarian carcinoma had a significant response with a 97% reduction in CA-125 levels. ISIS 5132, an antisense oligonucleotide against c-raf, was well tolerated at doses up to and including 4.0 mg/kg/day by 21-day continuous i.v. infusion and demonstrated antitumor activity at the doses tested.     

Unique changes in interstitial extracellular matrix composition are associated with rejection and cyclosporine toxicity in human renal allograft biopsies

Renal allograft loss from chronic rejection or cyclosporine toxicity (CsAT) is characterized by progressive interstitial fibrosis, yet the protein composition of these lesions is unknown. The normal tubular basement membrane (TBM) contains laminin (LM), collagen IV (containing collagen IV alpha chain 1 [COL4A1] and COL4A2), thrombospondin (TSP), and fibronectin (FN). Only TSP and FN extend beyond the TBM into the interstitial space. Very scanty amounts of interstitial collagens (I and III) are detected in the interstitium. In a pilot study of human renal allograft biopsy specimens, three patterns of extracellular matrix (ECM) composition were identified. Pattern 1 showed no change in ECM composition; pattern 2 showed generalized accumulation of collagens I and III in the interstitium; and pattern 3 showed new expression of COL4A3 and LM-beta2 in the proximal TBM. Criteria were established for the clinicopathological diagnosis of CsAT and rejection. These diagnoses were correlated with the ECM composition in 22 renal allograft biopsy specimens. Control groups were examined in a similar manner and included native kidney biopsy specimens from patients with other allografts (n = 7), renal biopsy specimens from patients with glomerular disease (n = 9), and renal allograft biopsy specimens from patients without clinicopathological evidence of renal disease. These data show that rejection is associated with pattern 3 and CsAT is associated with pattern 2. Thus, detection of ECM composition may be a useful adjunct to standard microscopy in distinguishing rejection from CsAT in renal allograft biopsy specimens. These data suggest that interstitial fibrosis associated with rejection and CsAT result from different pathogenic mechanisms.