Tachyphylaxis and reproducibility of desmopressin response in perioperative persons with nonsevere hemophilia A: implications for clinical practice.

Background: Desmopressin is frequently used perioperatively in persons with nonsevere hemophilia A. However, increase in factor (F)VIII:C after desmopressin use is interindividually highly variable. Tachyphylaxis has only been reported in test setting for persons with hemophilia A, with a remaining response of approximately 70% after a second dose compared with that after a first dose. Objectives: To study tachyphylaxis of FVIII:C response after multiple administration(s) of desmopressin in perioperative persons with nonsevere hemophilia A. Methods: We studied FVIII:C levels after desmopressin before (day 0 [D0]) and on days 1 (D1) and 2 (D2) after surgery in 26 patients of the DAVID and Little DAVID studies. We studied tachyphylaxis by comparing the responses at D1 and D2 with that at D0. We also assessed the reproducibility of the D0 response in comparison to an earlier performed desmopressin test. Results: The median absolute FVIII:C increase was 0.50 IU/mL (0.35-0.74; n = 23) at D0, 0.21 IU/mL (0.14-0.28; n = 17) at D1, and 0.23 IU/mL (0.16-0.30; n = 11) at D2. The median percentage of FVIII increase after the second administration (D1) compared with the first (D0) was 42.9% (29.2%-52.5%; n = 17) and that of the third (D2) compared with the first (D0) was 36.4% (23.7%-46.9%; n = 11). The FVIII:C desmopressin response at D0 was comparable with the desmopressin test response in 74% of the patients. Conclusion: Tachyphylaxis in the surgical setting was considerably more pronounced than previously reported, with FVIII:C at D1 and D2 of 36% to 43% of the initial response. Our results may have important implications for monitoring repeated desmopressin treatment when used perioperatively.

Maternal and neonatal bleeding complications in relation to peripartum management in hemophilia carriers: A systematic review.

Currently, there is no consensus on the optimal management to prevent postpartum hemorrhage (PPH) in hemophilia carriers. We aimed to evaluate peripartum management strategies in relation to maternal and neonatal bleeding outcomes by performing an extensive database search up to August 2020. Seventeen case-reports/series and 11 cohort studies were identified of overall 'poor' quality describing 502 deliveries. The PPH incidence in the individual patient data was 63%; 44% for those women receiving prophylaxis to correct coagulation and 77% for those without (OR 0.23, CI 0.09-0.58) and in cohort data 20.3% (26.8% (11/41) vs. 19.4% (55/284) (OR: 1.53, 95% CI: 0.72-3.24), respectively. Peripartum management strategies mostly consisted of clotting factor concentrates, rarely of desmopressin or plasma. Tranexamic acid appears promising in preventing secondary PPH, but was not used consistently. Neonatal bleeding was described in 6 affected male neonates, mostly after instrumental delivery or emergency CS, but insufficient information was provided to reliably investigate neonatal outcome in relation to management. The high PPH risk seems apparent, at most mildly attenuated by prophylactic treatment. Prospective cohort studies are needed to determine the optimal perinatal management in hemophilia.

A feasibility study on two tailored interventions to improve adherence in adults with haemophilia.

INTRODUCTION: Haemophilia is a congenital bleeding disorder mainly affecting males. To prevent bleeding, patients need to perform regular intravenous injections (prophylaxis) throughout life. Non-adherence often occurs. Problems with acceptance or self-management appear to be the main reasons for non-adherence in haemophilia. The aim of this study was to test the feasibility and effects of two interventions focussed on acceptance (face-to-face) and self-management (online). METHODS: Patients with severe haemophilia and acceptance or self-management problems were eligible. The face-to-face group intervention was based on Acceptance and Commitment Therapy (ACT) (8 sessions/6 months, target N = 8 participants). The online intervention was based on a successful online programme in rheumatoid arthritis (5-8 modules/2 months, target N = 8). Both interventions were designed according to the MRC framework in collaboration with the patient society and experts. We compared adherence (VERITAS-Pro, optimum 0), quality of life (SF-36, optimum 100) and illness perception (BIPQ, optimum 0) before start (T0) and after 2 months (T2). Feasibility criteria were as follows: completion of training by > 50% of participants and ability to collect at least 80% of outcome parameters. RESULTS: The face-to-face intervention was feasible (89% enrolment and recruitment, 100% retention). One hundred percent of the outcome parameters was collected. Results were promising: although adherence (VERITAS-Pro) was stable (from 64 to 62 points), quality of life (SF-36) showed a clinically relevant improvement (> 5 points) in five of eight domains. Illness perception (BIPQ) showed a clinically relevant increase from 47 to 39 points. Patient evaluation was positive. The online intervention, however, was infeasible: enrolment was only 20% (6/30). Only three patients signed informed consent (recruitment 10%), and none completed more than one module (retention 0%). Consequently, the online intervention was terminated. CONCLUSION: The face-to-face acceptance intervention was considered feasible with promising results. Unfortunately, the online intervention was infeasible and therefore terminated. These findings suggest that adapting effective interventions to other settings does not guarantee success, despite the use of established methodology and patient participation. Population differences (only male participants, congenital disease) could be an explanation for failure of the online intervention in haemophilia despite success in rheumatoid arthritis. TRIAL REGISTRATION: NL55883.041.16.

Maternal and neonatal bleeding complications in relation to peripartum management in women with Von Willebrand disease: A systematic review.

Women with Von Willebrand disease (VWD) have an increased risk of developing postpartum hemorrhage (PPH). Our aim is to evaluate peripartum management strategies in relation to maternal and neonatal bleeding complications in VWD. Electronic databases were searched up to January 2019. Seventy-one case-reports and -series and 16 cohort studies were selected, including 811 deliveries. Cohort studies reported primary PPH in 32% and secondary PPH in 13% of the women. The overall primary PPH incidence in the individual patient data was 34%, similar between women who received prophylactic treatment to prevent PPH and those who didn't. Neonatal bleeding events were reported in 4.6% of deliveries. Overall, the available evidence on peripartum management in women with VWD was of low quality. The ongoing high risk for PPH is evident, despite prophylactic treatment, as well as the need for higher quality evidence from larger prospective cohort studies to improve management strategies.

Population pharmacokinetics of factor IX in hemophilia B patients undergoing surgery.

Essentials Factor IX (FIX) dosing using body weight frequently results in under and overdosing during surgery. We aimed to establish a population pharmacokinetic (PK) model describing the perioperative FIX levels. Population PK parameter values for clearance and V1 were 284 mL h-170 kg-1 and 5450 mL70 kg-1. Perioperative PK parameters differ from those during non-surgical prophylactic treatment. SUMMARY: Background Hemophilia B is a bleeding disorder characterized by a deficiency of coagulation factor IX (FIX). In the perioperative setting, patients receive FIX concentrates to ensure hemostasis. Although FIX is usually dosed according to bodyweight, under- and overdosing occurs frequently during surgery. Aim The objective was to quantify and explain the interpatient variability of perioperatively administered plasma-derived (pd) and recombinant (r) FIX concentrates. Methods Data were collected from 118 patients (median age, 40 years [range, 0.2-90]; weight, 79 kg [range, 5.3-132]) with moderate (28%) or severe hemophilia B (72%), undergoing 255 surgical procedures. Population pharmacokinetic (PK) parameters were estimated using nonlinear mixed-effect modeling in NONMEM. Results Measured perioperative FIX level vs. time profiles were adequately described using a three-compartment PK model. For a typical 34-year-old patient receiving rFIX, clearance (CL), intercompartmental clearance (Q2, Q3), distribution volume of the central compartment (V1) and peripheral compartments (V2, V3) plus interpatient variability (%CV) were: CL, 284 mL h-170 kg-1 (18%); V1, 5450 mL70 kg-1 (19%); Q2, 110 mL h-170 kg-1; V2, 4800 mL70 kg-1; Q3, 1610 mL h-170 kg-1; V3, 2040 mL70 kg-1. From 0.2 years, CL and V1 decreased 0.89% and 1.15% per year, respectively, until the age of 34 years. Patients receiving pdFIX exhibited a lower CL (11%) and V1 (17%) than patients receiving rFIX. Interpatient variability was successfully quantified and explained. Conclusions The estimated perioperative PK parameters of both pdFIX and rFIX are different from those reported for prophylactic treatment. The developed model may be used to apply PK-guided dosing of FIX concentrates during surgery.

Perioperative replacement therapy in haemophilia B: An appeal to "B" more precise.

INTRODUCTION: Haemophilia B is caused by a deficiency of coagulation factor IX (FIX) and characterized by bleeding in muscles and joints. In the perioperative setting, patients are treated with FIX replacement therapy to secure haemostasis. Targeting of specified FIX levels is challenging and requires frequent monitoring and adjustment of therapy. AIM: To evaluate perioperative management in haemophilia B, including monitoring of FIX infusions and observed FIX levels, whereby predictors of low and high FIX levels were assessed. METHODS: In this international multicentre study, haemophilia B patients with FIX < 0.05 IU mL-1 undergoing elective, minor or major surgical procedures between 2000 and 2015 were included. Data were collected on patient, surgical and treatment characteristics. Observed FIX levels were compared to target levels as recommended by guidelines. RESULTS: A total of 255 surgical procedures were performed in 118 patients (median age 40 years, median body weight 79 kg). Sixty percent of FIX levels within 24 hours of surgery were below target with a median difference of 0.22 IU mL-1 [IQR 0.12-0.36]; while >6 days after surgery, 59% of FIX levels were above target with a median difference of 0.19 IU mL-1 [IQR 0.10-0.39]. Clinically relevant bleeding complications (necessity of a second surgical intervention or red blood cell transfusion) occurred in 7 procedures (2.7%). CONCLUSION: This study demonstrates that targeting of FIX levels in the perioperative setting is complex and suboptimal, but although this bleeding is minimal. Alternative dosing strategies taking patient and surgical characteristics as well as pharmacokinetic principles into account may help to optimize and individualize treatment.

Facilitating the implementation of pharmacokinetic-guided dosing of prophylaxis in haemophilia care by discrete choice experiment.

INTRODUCTION: Patients', parents' and providers' preferences with regard to medical innovations may have a major impact on their implementation. AIM: To evaluate barriers and facilitators for individualized pharmacokinetic (PK)-guided dosing of prophylaxis in haemophilia patients, parents of young patients, and treating professionals by discrete choice experiment (DCE) questionnaire. PATIENTS/METHODS: The study population consisted of patients with haemophilia currently or previously on prophylactic treatment with factor concentrate (n = 114), parents of patients aged 12-18 years (n = 19) and haemophilia professionals (n = 91). DCE data analysis was performed, taking preference heterogeneity into account. RESULTS: Overall, patients and parents, and especially professionals were inclined to opt for PK-guided dosing of prophylaxis. In addition, if bleeding was consequently reduced, more frequent infusions were acceptable. However, daily dosing remained an important barrier for all involved. 'Reduction of costs for society' was a facilitator for implementation in all groups. CONCLUSIONS: To achieve implementation of individualized PK-guided dosing of prophylaxis in haemophilia, reduction of bleeding risk and reduction of costs for society should be actively discussed as they are motivating for implementation; daily dosing is still reported to be a barrier for all groups. The knowledge of these preferences will enlarge support for this innovation, and aid in the drafting of implementable guidelines and information brochures for patients, parents and professionals.

Perioperative treatment of hemophilia A patients: blood group O patients are at risk of bleeding complications.

UNLABELLED: ESSENTIALS: Targeting of factor VIII values is a challenge during perioperative replacement therapy in hemophilia. This study aims to identify the extent and predictors of factor VIII underdosing and overdosing. Blood group O predicts underdosing and is associated with perioperative bleeding. To increase quality of care and cost-effectiveness of treatment, refining of dosing is obligatory. BACKGROUND: Perioperative administration of factor VIII (FVIII) concentrate in hemophilia A may result in both underdosing and overdosing, leading to respectively a risk of bleeding complications and unnecessary costs. OBJECTIVES: This retrospective observational study aims to identify the extent and predictors of underdosing and overdosing in perioperative hemophilia A patients (FVIII levels < 0.05 IU mL(-1)). PATIENTS AND METHODS: One hundred nineteen patients undergoing 198 elective, minor, or major surgical procedures were included (median age 40 years, median body weight 75 kg). Perioperative management was evaluated by quantification of perioperative infusion of FVIII concentrate and achieved FVIII levels. Predictors of underdosing and (excessive) overdosing were analyzed by logistic regression analysis. Excessive overdosing was defined as upper target level plus ≥ 0.20 IU mL(-1). RESULTS: Depending on postoperative day, 7-45% of achieved FVIII levels were under and 33-75% were above predefined target ranges as stated by national guidelines. A potential reduction of FVIII consumption of 44% would have been attained if FVIII levels had been maintained within target ranges. Blood group O and major surgery were predictive of underdosing (odds ratio [OR] 6.3, 95% confidence interval [CI] 2.7-14.9; OR 3.3, 95% CI 1.4-7.9). Blood group O patients had more bleeding complications in comparison to patients with blood group non-O (OR 2.02, 95% CI 1.00-4.09). Patients with blood group non-O were at higher risk of overdosing (OR 1.5, 95% CI 1.1-1.9). Additionally, patients treated with bolus infusions were at higher risk of excessive overdosing (OR 1.8, 95% CI 1.3-2.4). CONCLUSION: Quality of care and cost-effectiveness can be improved by refining of dosing strategies based on individual patient characteristics such as blood group and mode of infusion.

Pseudodystrophy. A conversion disorder mimicking reflex sympathetic dystrophy.

The authors suggest some criteria by which pseudodystrophy and reflex sympathetic dystrophy, although sharing some similar clinical features, can be distinguished as two different conditions, each requiring its own approach and management. The most important distinction is found on bone scintigraphy. In reflex sympathetic dystrophy the bone scan shows a typical increased tracer uptake (at least during stages I and II); in pseudodystrophy there is a normal or decreased tracer uptake in the affected region. Moreover the vascularization is increased in reflex sympathetic dystrophy stage I, whereas in pseudodystrophy hypovascularization is found from the beginning. The clinical features, as well as the results of technical investigations, psychological evaluation and treatment of 4 patients with pseudodystrophy are presented. The importance of distinguishing this condition from reflex sympathetic dystrophy is stressed.

Cdc42 induces filopodia by promoting the formation of an IRSp53:Mena complex.

BACKGROUND: The Rho GTPases Rho, Rac, and Cdc42 regulate the organization of the actin cytoskeleton by interacting with multiple, distinct downstream effector proteins. Cdc42 controls the formation of actin bundle-containing filopodia at the cellular periphery. The molecular mechanism for this remains as yet unclear. RESULTS: We report here that Cdc42 interacts with IRSp53/BAP2 alpha, an SH3 domain-containing scaffold protein, at a partial CRIB motif and that an N-terminal fragment of IRSp53 binds, via an intramolecular interaction, to the CRIB motif-containing central region. Overexpression of IRSp53 in fibroblasts leads to the formation of filopodia, and both this and Cdc42-induced filopodia are inhibited by expression of the N-terminal IRSp53 fragment. Using affinity chromatography, we have identified Mena, an Ena/VASP family member, as interacting with the SH3 domain of IRSp53. Mena and IRSp53 act synergistically to promote filopodia formation. CONCLUSION: We conclude that the interaction of Cdc42 with the partial CRIB motif of IRSp53 relieves an intramolecular, autoinhibitory interaction with the N terminus, allowing the recruitment of Mena to the IRSp53 SH3 domain. This IRSp53:Mena complex initiates actin filament assembly into filopodia.

Plexin-B semaphorin receptors interact directly with active Rac and regulate the actin cytoskeleton by activating Rho.

Semaphorins and their receptors, plexins, are widely expressed in embryonic and adult tissues. In general, their functions are poorly characterized, but in neurons they provide essential attractive and repulsive cues that are necessary for axon guidance [1-3]. The Rho family GTPases Rho, Rac, and Cdc42 control signal transduction pathways that link plasma membrane receptors to the actin cytoskeleton and thus regulate many actin-driven processes, including cell migration and axon guidance [4-7]. Using yeast two-hybrid screening and in vitro interaction assays, we show that Rac in its active, GTP bound state interacts directly with the cytoplasmic domain of mammalian and Drosophila B plexins. Plexin-B1 clustering in fibroblasts does not cause the formation of lamellipodia, which suggests that Rac is not activated. Instead, it results in the assembly of actin:myosin filaments and cell contraction, which indicates Rho activation. Surprisingly, these cytoskeletal changes are both Rac and Rho dependent. Clustering of a mutant plexin, lacking the Rac binding region, induced similar cytoskeletal changes, and this finding indicates that the physical interaction of plexin-B1 with Rac is not required for Rho activation. Our findings that plexin-B signaling to the cytoskeleton is both Rac and Rho dependent form a starting point for unraveling the mechanism by which semaphorins and plexins control axon guidance and cell migration.

The mammalian homologue of the Caenorhabditis elegans polarity protein PAR-6 is a binding partner for the Rho GTPases Cdc42 and Rac1.

A mammalian homologue of the PDZ domain containing Caenorhabditis elegans protein PAR-6 was found in a yeast two-hybrid system screen as binding to the Rho family member Cdc42. PAR-6 contains a PDZ domain and in C. elegans it has been shown to be crucial for the asymmetric cleavage and establishment of cell polarity during the first cell divisions in the growing embryo. Mammalian PAR-6 interacted with Cdc42 and Rac1 both in the yeast two-hybrid system and in in vitro binding assays. Co-immunoprecipitation experiments, employing transiently transfected Cos-1 cells, further confirmed that Cdc42 and Rac1 are physiological binding partners for PAR-6. We found that, in epithelial Madin-Darby canine kidney cells (MDCK), endogenous PAR-6 was present in the tight junctions, as judged from its co-localisation with the tight junction protein ZO-1, however, PAR-6 was also detected in the cell nucleus. Stimulation of MDCK cells with scatter factor/hepatocyte growth factor induced a loss of PAR-6 from the areas of cell-cell contacts in conformity with their progressive breakdown. In C. elegans PAR-6 co-localises with PAR-3 and has been suggested to form a direct complex. In agreement with earlier studies, mammalian PAR-3 was found to be present in tight junctions of MDCK cells but, in contrast to PAR-6, the protein could not be detected in the nucleus. Furthermore, co-immunoprecipitation experiments, employing Cos-1 cells, demonstrated that mammalian PAR-6 and PAR-3 formed a direct complex. These findings, together with the reported roles of PAR-6 and PAR-3 in C. elegans, suggest that Cdc42 and Rac1 and PAR-6/PAR-3 are involved in the establishment of cell polarity in epithelial cells.

Phosphatidylinositol 4,5-bisphosphate induces actin-based movement of raft-enriched vesicles through WASP-Arp2/3.

BACKGROUND: Phosphatidylinositol 4,5-bisphosphate (PIP(2)) has been implicated in the regulation of the actin cytoskeleton and vesicle trafficking. It stimulates de novo actin polymerization by activating the pathway involving the Wiskott-Aldrich syndrome protein (WASP) and the actin-related protein complex Arp2/3. Other studies show that actin polymerizes from cholesterol-sphingolipid-rich membrane microdomains called 'rafts', in a manner dependent on tyrosine phosphorylation. Although actin has been implicated in vesicle trafficking, and rafts are sites of active phosphoinositide and tyrosine kinase signaling that mediate apically directed vesicle trafficking, it is not known whether phosphoinositide regulation of actin dynamics occurs in rafts, or if it is linked to vesicle movements. RESULTS: Overexpression of type I phosphatidylinositol phosphate 5-kinase (PIP5KI), which synthesizes PIP(2), promoted actin polymerization from membrane-bound vesicles to form motile actin comets. Pervanadate (PV), a tyrosine phosphatase inhibitor, induced comets even in the absence of PIP5KI overexpression. PV increased PIP(2) levels, suggesting that it induces comets by changing PIP(2) homeostasis and by increasing tyrosine phosphorylation. Platelet-derived growth factor (PDGF) enhanced PV-induced comet formation, and these stimuli together potentiated the PIP5KI effect. The vesicles at the heads of comets were enriched in PIP5KIs and tyrosine phosphoproteins. WASP-Arp2/3 involvement was established using dominant-negative WASP constructs. Endocytic and exocytic markers identified vesicles enriched in lipid rafts as preferential sites of comet generation. Extraction of cholesterol with methyl-beta-cyclodextrin reduced comets, establishing that rafts promote comet formation. CONCLUSIONS: Sphingolipid-cholesterol rafts are preferred platforms for membrane-linked actin polymerization. This is mediated by in situ PIP(2) synthesis and tyrosine kinase signaling through the WASP-Arp2/3 pathway. Actin comets may provide a novel mechanism for raft-dependent vesicle transport and apical membrane trafficking.

Effect of physical therapy on limited joint mobility in the diabetic foot. A pilot study.

Eleven patients with limited joint mobility and neuropathy were enrolled in a physical therapy program of passive joint mobilization at a rate of two sessions per week. Treatment resulted in a significant improvement in joint mobility after 10 sessions. Further improvement after 20 sessions did not reach the level of statistical significance, although near-normal joint mobility was attained. After completion of therapy, there was a progressive deterioration in joint mobility. No serious adverse effects were noted during treatment. This study provides some evidence that use of physical therapy may result in significant, although temporary, improvement in the mobility of the ankle and foot joints in diabetic patients with limited joint mobility and neuropathy. As limited joint mobility has been associated with the development of abnormally high pressures under the feet, which in turn may contribute to plantar ulceration in the susceptible neuropathic foot, the results indicate that physical therapy may be useful in the prevention of plantar ulceration in diabetic patients with limited joint mobility and neuropathy, although this must be verified by additional research.

LFA-1 to LFA-1 signals involve zeta-associated protein-70 (ZAP-70) tyrosine kinase: relevance for invasion and migration of a T cell hybridoma.

We previously showed that LFA-1-dependent in vitro invasion and in vivo migration of a T cell hybridoma was blocked in cells overexpressing a truncated dominant-negative zeta-associated protein (ZAP)-70. The truncated ZAP-70 also blocked LFA-1-dependent chemotaxis through ICAM-1-coated filters induced by 1 ng/ml stromal cell-derived factor-1, but not LFA-1-independent chemotaxis induced by 100 ng/ml stromal cell-derived factor-1. This suggested that LFA-1 engagement triggers a signal that amplifies a weak chemokine signal and that dominant-negative ZAP-70 blocks this LFA-1 signal. Here we show that cross-linking of part of the LFA-1 molecules with Abs causes activation of free LFA-1 molecules (not occupied by the Ab) on the same cell, which then bind to ICAM-2 on other cells. This causes cell aggregation that was also blocked by dominant-negative ZAP-70. Thus, an LFA-1 signal involving ZAP-70 activates other LFA-1 molecules, suggesting that the chemokine signal can be amplified by multiple cycles of LFA-1 activation. The chemokine and the LFA-1 signal were both blocked by a phospholipase C inhibitor and a calpain inhibitor, suggesting that one of the amplified signals is the phospholipase C-dependent activation of calpain. Finally, we show that both Src-homology 2 domains are required for inhibition of invasion, chemotaxis, and aggregation by the truncated ZAP-70, suggesting that ZAP-70 interacts with a phosphorylated immunoreceptor tyrosine-based activation motif (ITAM) sequence. Remarkably, this is not an ITAM in the TCR/CD3 complex because this is not expressed by this T cell hybridoma.

What is reflex sympathetic dystrophy?

In the literature there is no unanimity with respect to the diagnosis of reflex sympathetic dystrophy (RSD). Frequently, the diagnosis is established on mere clinical grounds. In our opinion, however, bone scintigraphy is of major importance for the diagnosis. Using this examination, true RSD can be clearly differentiated from other conditions which are incorrectly diagnosed and treated as RSD. If the bone scan is not suggestive of RSD, the clinical picture, radiological examination and vascular scan may lead to the correct diagnosis. This may be a pseudodystrophy, in which a hypovascularization is found right from the start, while in true RSD there is initially a hypervascularization. Other conditions which may be confused with RSD are causalgia, neurotic compulsive postures, hysterical conversion, malingering and even self-mutilation. In the spontaneous course of RSD three phases can be distinguished. Stage I is the warm or hypertrophic phase, stage II the cold or atrophic phase. Per definition the third phase corresponds to stabilization or, in rare instances, to healing. By means of the vascular scan the correct stage can be determined, and the results of treatment evaluated. Finally it should be noted that in children the condition is completely different from true RSD, as it concerns a pseudodystrophy or disuse-related dystrophy. This condition may also be seen in adults and adolescents, usually females. The bone scan is always negative. In this way bone scintigraphy constitutes the means to answer the question as to what RSD is and what it is not. An algorithm for the differential diagnosis is presented.

Activation of G1 progression, JNK mitogen-activated protein kinase, and actin filament assembly by the exchange factor FGD1.

Cdc42 has been shown to control bifurcating pathways leading to filopodia formation/G1 cell cycle progression and to JNK mitogen-activated protein kinase activation. To dissect these pathways further, the cellular effects induced by a Cdc42 guanine nucleotide exchange factor, FGD1, have been examined. All exchange factors acting on the Rho GTPase family have juxtaposed Dbl homology (DH) and pleckstrin homology (PH) domains. We report here that FGD1 triggers G1 cell cycle progression and filopodia formation in Swiss 3T3 fibroblasts as well as JNK mitogen-activated protein kinase activation in COS cell transfection assays. FGD1-induced filopodia formation is Cdc42-dependent, and both the DH and PH domains are essential. Although expression of the FGD1 DH domain alone does not activate Cdc42 and induce filopodia, it does trigger both the JNK cascade in COS cells and G1 progression in quiescent Swiss 3T3 cells. We conclude that FGD1 can trigger G1 progression independently of actin polymerization or integrin adhesion complex assembly. Furthermore, since FGD1 activates JNK and G1 progression in a Cdc42-independent manner, it must have additional, as yet unidentified, targets.

Infrequent presentations of reflex sympathetic dystrophy and pseudodystrophy.

Pseudodystrophy is a condition that in the past was regarded incorrectly as a kind of reflex sympathetic dystrophy. The difference between both diseases can be ascertained easily by a bone scintigraphy. Other clinical features of pseudodystrophy are discussed. Infrequent presentations of reflex sympathetic dystrophy are described: radial, parcellar, and zonal cases; vertebral localization; and other misleading conditions.

Activation of G-proteins with AIF-4 induces LFA-1-mediated adhesion of T-cell hybridoma cells to ICAM-1 by signal pathways that differ from phorbol ester- and manganese-induced adhesion.

T-cell hybridomas metastasize widely, and the extent of dissemination correlates with invasiveness in fibroblast cultures. Previously, we provided evidence that both metastasis and in vitro invasion require activation of LFA-1, induced by G-protein-transduced signals triggered by as yet unidentified factors. We show here that LFA-1-mediated adhesion of TAM2D2 T-cell hybridoma cells to ICAM-1 can in fact be induced by direct activation of G-proteins using AIF-4, to the same extent as by using PMA or Mn2+. We assessed effects of protein kinase C (PKC), tyrosine kinase (TK), PI3-kinase (PI3K), and phospholipase C (PLC) inhibitors. Both AIF-4-induced adhesion and invasion were completely blocked by the TK inhibitor genistein and partially blocked by the PI3K inhibitor wortmannin, but not influenced by PKC inhibitor GF109203X. Downregulation of PKC did not affect invasion or adhesion induced by AIF-4 either. In contrast, GF109203X and PKC downregulation blocked PMA-induced adhesion, but genistein and wortmannin had no effect. Invasion and both AIF-4- and PMA-induced adhesion were completely blocked by the PLC inhibitor U73122. Mn(2+)-induced adhesion, which was not or was only partially blocked by the other inhibitors, was delayed by U73122, and spreading of Mn(2+)-treated cells was completely prevented by U73122. However, PLC activity during adhesion was not detected. We conclude that signals required for invasion and G-protein-induced adhesion are similar and are distinct from PKC-induced adhesion, and that in all cases PLC is likely to be activated, but is probably too local and/or transient to be detected.

Inhibition and stimulation of LFA-1 and Mac-1 functions by antibodies against murine CD18. Evidence that the LFA-1 binding sites for ICAM-1, -2, and -3 are distinct.

The murine CD18 monoclonal antibody (mAb) M18/2 was reported to inhibit lymphoma metastasis [Zahalka, M. A. et al. (1993) J. Immunol. 150, 4466]. To identify the pathways potentially blocked, we studied the effects of M18/2 compared with two new mAb against murine CD18, GAME-46, and -245. Whereas the GAME mAb blocked most Mac-1-mediated interactions, M18/2 had no effect, or even stimulated. The same was true for adhesion of LFA-1 to ICAM-1. To test effects on interactions with different ICAMs, we used L cells transfected with human ICAM-1, -2, and -3. As previously described, mouse LFA-1 does not bind to human ICAM-1 but we show here that mouse LFA-1 does bind to human ICAM-2 and -3. Again, the GAME mAb blocked completely, but M18/2 did not. These results indicate that the LFA-1 binding sites for ICAM-1 and ICAM-2 and -3, although in close vicinity, are distinct. Furthermore, effects of M18/2 on metastasis cannot be ascribed to blocking of any known beta2-integrin activity.