Search

Background: Harnessing mobile health (mHealth) solutions could improve the delivery of mental health services and mitigate their impact in Uganda and similar low-resource settings. However, successful adoption requires that mHealth solutions have good usability. We have previously implemented a telephone service to provide mental health information and advice in English and Luganda, utilizing an automated interactive voice response (IVR) system linked to live agents, including mental health care workers and peer support workers., Objective: This study aims to assess the usage and usability of this mental health telephone service., Methods: We obtained usage data from the system's call logs over 18 months to study call volumes and trends. We then surveyed callers to gather their characteristics and assess usability using the Telehealth Usability Questionnaire. Additionally, call recordings were evaluated for conversation quality by 3 independent health care professionals, using the Telephone Nursing Dialogue Process, and correlations between quality and usability aspects were investigated., Results: Over 18 months, the system received 2863 meaningful calls (ie, calls that went past the welcome message) from 1125 unique telephone numbers. Of these, 1153 calls (40.27%) stopped at the prerecorded IVR information, while 1710 calls (59.73%) opted to speak to an agent. Among those who chose to speak with an agent, 1292 calls (75.56%) were answered, 393 calls (22.98%) went to voicemail and were returned in the following working days, and 25 calls (1.46%) were not answered. Usage was generally sustained over time, with spikes in call volume corresponding to marketing events. The survey (n=240) revealed that most callers were caregivers of patients with mental health issues (n=144, 60.0%) or members of the general public (n=46, 19.2%), while a few were patients with mental health issues (n=44, 18.3%). Additionally, the majority were male (n=143, 59.6%), spoke English (n=180, 75.0%), had postsecondary education (n=164, 68.3%), lived within 1 hour or less from Butabika Hospital (n=187, 77.9%), and were aged 25-44 years (n=160, 66.7%). The overall usability score for the system was 4.12 on a 5-point scale, significantly higher than the recommended target usability score of 4 (P=.006). The mean scores for usability components ranged from 3.66 for reliability to 4.41 for ease of use, with all components, except reliability, scoring higher than 4 or falling within its CI. Usability scores were higher for Luganda speakers compared with English speakers, but there was no association with other participant characteristics such as sex, distance from the hospital, age, marital status, duration of symptoms, or treatment status. The quality of call conversations (n=50) was rated at 4.35 out of 5 and showed a significant correlation with usability (Pearson r=0.34, P=.02)., Conclusions: We found sustained usage of the mental health telephone service, along with a positive user experience and high satisfaction across various user characteristics. mHealth solutions like this should be embraced and replicated to enhance the delivery of health services in Uganda and similar low-resource settings., (©Johnblack K Kabukye, Juliet Nakku, Jackline Niwemuhwezi, James Nsereko, Rosemary Namagembe, Iris Dorothee Emilie Groen, Ritah Neumbe, Denis Mubiru, Caroline Kisakye, Roseline Nanyonga, Marie Sjölinder, Susanne Nilsson, Caroline Wamala-Larsson. Originally published in the Journal of Medical Internet Research (https://www.jmir.org), 21.10.2024.)

Background: Mental health conditions are a significant public health problem globally, responsible for >8 million deaths per year. In addition, they lead to lost productivity, exacerbate physical illness, and are associated with stigma and human rights violations. Uganda, like many low- and middle-income countries, faces a massive treatment gap for mental health conditions, and numerous sociocultural challenges exacerbate the burden of mental health conditions., Objective: This study aims to describe the development and formative evaluation of a digital health intervention for improving access to mental health care in Uganda., Methods: This qualitative study used user-centered design and design science research principles. Stakeholders, including patients, caregivers, mental health care providers, and implementation experts (N=65), participated in focus group discussions in which we explored participants' experience of mental illness and mental health care, experience with digital interventions, and opinions about a proposed digital mental health service. Data were analyzed using the Consolidated Framework for Implementation Research to derive requirements for the digital solution, which was iteratively cocreated with users and piloted., Results: Several challenges were identified, including a severe shortage of mental health facilities, unmet mental health information needs, heavy burden of caregiving, financial challenges, stigma, and negative beliefs related to mental health. Participants' enthusiasm about digital solutions as a feasible, acceptable, and convenient method for accessing mental health services was also revealed, along with recommendations to make the service user-friendly, affordable, and available 24×7 and to ensure anonymity. A hospital call center service was developed to provide mental health information and advice in 2 languages through interactive voice response and live calls with health care professionals and peer support workers (recovering patients). In the 4 months after launch, 456 calls, from 236 unique numbers, were made to the system, of which 99 (21.7%) calls went to voicemails (out-of-office hours). Of the remaining 357 calls, 80 (22.4%) calls stopped at the interactive voice response, 231 (64.7%) calls were answered by call agents, and 22 (6.2%) calls were not answered. User feedback was positive, with callers appreciating the inclusion of peer support workers who share their recovery journeys. However, some participant recommendations (eg, adding video call options) or individualized needs (eg, prescriptions) could not be accommodated due to resource limitations or technical feasibility., Conclusions: This study demonstrates a systematic and theory-driven approach to developing contextually appropriate digital solutions for improving mental health care in Uganda and similar contexts. The positive reception of the implemented service underscores its potential impact. Future research should address the identified limitations and evaluate clinical outcomes of long-term adoption., (©Johnblack K Kabukye, Rosemary Namagembe, Juliet Nakku, Vincent Kiberu, Marie Sjölinder, Susanne Nilsson, Caroline Wamala-Larsson. Originally published in JMIR Human Factors (https://humanfactors.jmir.org), 06.06.2024.)

Thanks to its non-invasive nature and high-resolution imaging capabilities, magnetic resonance imaging (MRI) is a valuable diagnostic tool for pediatric patients. However, the fear and anxiety experienced by young children during MRI scans often result in suboptimal image quality and the need for sedation/anesthesia. This study aimed to evaluate the effect of a smartphone application called COSMO@home to prepare children for MRI scans to reduce the need for sedation or general anesthesia. The COSMO@home app was developed incorporating mini-games and an engaging storyline to prepare children for learning goals related to the MRI procedure. A multicenter study was conducted involving four hospitals in Belgium. Eligible children aged 4-10 years were prepared with the COSMO@home app at home. Baseline, pre-scan, and post-scan questionnaires measured anxiety evolution in two age groups (4-6 years and 7-10 years). Eighty-two children participated in the study, with 95% obtaining high-quality MRI images. The app was well-received by children and parents, with minimal technical difficulties reported. In the 4-6-year-old group (N = 33), there was a significant difference between baseline and pre-scan parent-reported anxiety scores, indicating an increase in anxiety levels prior to the scan. In the 7-10-year-old group (N = 49), no significant differences were observed between baseline and pre-scan parent-reported anxiety scores. Overall, the COSMO@home app proved to be useful in preparing children for MRI scans, with high satisfaction rates and successful image outcomes across different hospitals. The app, combined with minimal face-to-face guidance on the day of the scan, showed the potential to replace or assist traditional face-to-face training methods. This innovative approach has the potential to reduce the need for sedation or general anesthesia during pediatric MRI scans and its associated risks and improve patient experience.

Background: In this study an interactive distance solution (called the DISKO tool) was developed to enable home-based motor training after stroke., Objectives: The overall aim was to explore the feasibility and safety of using the DISKO-tool, customized for interactive stroke rehabilitation in the home setting, in different rehabilitation phases after stroke., Methods: Fifteen patients in three different stages in the continuum of rehabilitation after stroke participated in a home-based training program using the DISKO-tool. The program included 15 training sessions with recurrent follow-ups by the integrated application for video communication with a physiotherapist. Safety and feasibility were assessed from patients, physiotherapists, and a technician using logbooks, interviews, and a questionnaire. Qualitative content analysis and descriptive statistics were used in the analysis., Results: Fourteen out of 15 patients finalized the training period with a mean of 19.5 minutes spent on training at each session. The DISKO-tool was found to be useful and safe by patients and physiotherapists., Conclusions: This study demonstrates the feasibility and safety of the DISKO-tool and provides guidance in further development and testing of interactive distance technology for home rehabilitation, to be used by health care professionals and patients in different phases of rehabilitation after stroke.

CD46, a membrane cofactor expressed on all nucleated human cells, plays an essential role in suppressing autoimmune reactions and protecting host cells from complement-mediated attack. Human transgenic CD46 homozygous mice (CD46 +/+ ) are prone to lethal sepsis upon infection with Neisseria meningitidis (N. meningitidis). However, the underlying mechanisms are poorly understood. Here, we determined thatCD46 +/+ mice produce large numbers of M1 type macrophages with enhanced surface expression of MHC II and production of pro-inflammatory mediators such as IL-6, TNF, IL-12, and IL-1β In the presence of M-CSF or GM-CSF, CD46 signaling enhances monocyte-macrophage differentiation. Additionally, CD46 +/+ macrophages rapidly undergo apoptosis upon LPS challenge or meningococcal infection, which could contribute to uncontrolled bacterial dissemination in vivo. Adoptive transfer of CD46 +/+ peritoneal macrophages aggravated septic responses in wild-type mice, but the depletion of macrophages partially alleviated septic reactions in CD46 +/+ mice after N. meningitidis infection. Our findings reveal a novel role of CD46 in accelerating inflammatory responses upon meningococcal infection or LPS stimulation by regulating the functional polarization and survival of macrophages., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Unlabelled: Neisseria meningitidis colonizes the nasopharyngeal mucosa of healthy populations asymptomatically, although the bacterial surface is rich in motifs that activate the host innate immunity. What determines the tolerant host response to this bacterium in asymptomatic carriers is poorly understood. We demonstrated that the conserved meningococcal surface protein NhhA orchestrates monocyte (Mo) differentiation specifically into macrophage-like cells with a CD200R(hi) phenotype (NhhA-Mφ). In response to meningococcal stimulation, NhhA-Mφ failed to produce proinflammatory mediators. Instead, they upregulated interleukin-10 (IL-10) and Th2/regulatory T cell (Treg)-attracting chemokines, such as CCL17, CCL18, and CCL22. Moreover, NhhA-Mφ were highly efficient in eliminating bacteria. The in vivo validity of these findings was corroborated using a murine model challenged with N. meningitidis systematically or intranasally. The NhhA-modulated immune response protected mice from septic shock; Mo/Mφ depletion abolished this protective effect. Intranasal administration of NhhA induced an anti-inflammatory response, which was associated with N. meningitidis persistence at the nasopharynx. In vitro studies demonstrated that NhhA-triggered Mo differentiation occurred upon engaged Toll-like receptor 1 (TLR1)/TLR2 signaling and extracellular signal-regulated kinase (ERK) and Jun N-terminal protein kinase (JNK) activation and required endogenously produced IL-10 and tumor necrosis factor alpha (TNF-α). Our findings reveal a strategy that might be adopted by N. meningitidis to maintain asymptomatic nasopharyngeal colonization., Importance: Neisseria meningitidis is an opportunistic human-specific pathogen that colonizes the nasopharyngeal mucosa asymptomatically in approximately 10% of individuals. Very little is known about how this bacterium evades immune activation during the carriage stage. Here, we observed that N. meningitidis, via the conserved surface protein NhhA, skewed monocyte differentiation into macrophages with a CD200R(hi) phenotype. Both in vivo and in vitro data demonstrated that these macrophages, upon meningococcal infection, played an important role in forming a homeostatic immune microenvironment through their capacity to eliminate invading bacteria and to generate anti-inflammatory mediators. This work provides novel insight into the mechanisms underlying the commensal persistence of N. meningitidis., (Copyright © 2016 Wang et al.)

Neisseria meningitidis is an opportunistic human pathogen that usually colonizes the nasopharyngeal mucosa asymptomatically. Upon invasion into the blood and central nervous system, this bacterium triggers a fulminant inflammatory reaction with the manifestations of septicemia and meningitis, causing high morbidity and mortality. To reveal the bacterial adaptations to specific and dynamic host environments, we performed a comprehensive proteomic survey of N. meningitidis isolated from the nasal mucosa, CSF and blood of a mouse disease model. We could identify 51 proteins whose expression pattern has been changed during infection, many of which have not yet been characterized. The abundance of proteins was markedly lower in the bacteria isolated from the nasal mucosa compared to the bacteria from the blood and CSF, indicating that initiating adhesion is the harshest challenge for meningococci. The high abundance of the glutamate dehydrogenase (GdhA) and Opa1800 proteins in all bacterial isolates suggests their essential role in bacterial survival in vivo. To evaluate the biological relevance of our proteomic findings, four candidate proteins from representative functional groups, such as the bacterial chaperone GroEL, IMP dehydrogenase GuaB, and membrane proteins PilQ and NMC0101, were selected and their impact on bacterial fitness was investigated by mutagenesis assays. This study provides an integrated picture of bacterial niche-specific adaptations during consecutive infection processes., (Copyright © 2015 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

The aim of this work has been to develop a technical support enabling home-based motor training after stroke. The basis for the work plan has been to develop an interactive technical solution supporting three different groups of stroke patients: (1) patients with stroke discharged from hospital with support from neuro team; (2) patients with stroke whose support from neuro team will be phased out and (3) patients living with impaired motor functions long-term. The technology has been developed in close collaboration with end-users using a method earlier evaluated and described [12]. This paper describes the main functions of the developed technology. Further, results from early user-tests with end-users, performed to identify needs for improvements to be carried out during further technical development. The developed technology will be tested further in a pilot study of the safety and, usefulness of the technology when applied as a support for motor training in three different phases of the post-stroke rehabilitation process.

The elderly population is increasing and there is a need to provide care and safety at a high level with limited resources. New social alarm solutions may contribute to safety and independence for many elderly. However, it is important to understand the needs within the user group. This work studied social alarms in a broad sense and from several user perspectives. In the first study, social alarm use and its aspects were investigated. To understand where there may be problems and weaknesses, users, caregivers, managers of municipalities, and personnel at alarm centers were interviewed. The interviews helped identify a number of problems. For municipalities, the processes of procuring new alarms and managing their organization were found to be complex. The effect of this was that the same social alarm systems had been ordered over and over again without taking into account new user needs or new technical solutions. For alarm users, one large problem was that the alarms had very limited reach and were designed for indoor use only. This has resulted in users hesitating to leave their homes, which in turn has negative effects due to lack of physical activity and fewer social contacts. One important result from the first study was the need for a social alarm solution that worked outdoors. In a second study, needs regarding outdoor social alarms were investigated. The results from this study showed that wearable outdoor alarms must be easy to use, provide communication, and be well designed. Finally, these alarms must work both indoors and outdoors, and the user should not have to worry about where he/she is or who is acting on an alarm.

Background: There is an increasing social isolation among the elderly today. This will be an even larger issue in the future with growing numbers of elderly and less resources, for example, in terms of economy and staff. Loneliness and social isolation can, however, be addressed in several ways using different interactive eHealth services., Objective: This case study investigated novel eHealth services for the elderly, and their usage of a social interactive device designed especially for them., Methods: In this work, we used an innovative mobile communication device connected to the television (TV), which worked as a remotely controlled large interactive screen. The device was tested by 8 volunteers who visited a senior center. They were between 65 and 80 years of age and lived in their own homes. Throughout the 1.5 year-long project, 7 design workshops were held with the seniors and the staff at the center. During these workshops, demands and preferences regarding existing and new services were gathered. At the end of the project the participants' experience of the device and of the services was elaborated in 3 workshops to get ideas for improved or new meaningful services. During the data analyses and development process, what seniors thought would be useful in relation to what was feasible was prioritized by the development company., Results: Regarding daily usage, the seniors reported that they mainly used the service for receiving information from the senior center and for communication with other participants in the group or with younger relatives. They also read information about events at the senior center and they liked to perform a weekly sent out workout exercise. Further, they played games such as Memory and Sudoku using the device. The service development focused on three categories of services: cognitive activities, social activities, and physical activities. A cognitive activity service that would be meaningful to develop was a game for practicing working memory. In the social activities category, the seniors wanted different quizzes and multi-player games. For physical activities, the seniors desired more workout exercises and suggestions for guided walking routes. A new category, "information and news", was suggested since they lacked services like senior-customized global and local news., Conclusions: This study showed the importance of input from a group of seniors when designing new services for elderly citizens. Besides input to interactive eHealth service development for seniors, this study showed the importance of a social context around such work. The seniors were very engaged throughout the project and workshops were frequently visited and the seniors became friends. The high amount of input from the seniors could be explained in terms of social inclusion; they belonged to a group and each member was considered important for the work. The friendly workshop atmosphere facilitated new ideas and redesign of the services.

Activation of macrophages by Toll-like receptors (TLRs) and functionally related proteins is essential for host defense and innate immunity. TLRs recognize a wide variety of pathogen-associated molecules. Here, we demonstrate that the meningococcal outer membrane protein NhhA has immunostimulatory functions and triggers release of proinflammatory cytokines from macrophages. NhhA-induced cytokine release was found to proceed via two distinct pathways in RAW 264.7 macrophages. Interleukin-6 (IL-6) secretion was dependent on activation of TLR4 and required the TLR signaling adaptor protein MyD88. In contrast, release of tumor necrosis factor (TNF) was TLR4 and MyD88 independent. Both pathways involved NF-κB-dependent gene regulation. Using a PCR-based screen, we could identify additional targets of NhhA-dependent gene activation such as the cytokines and growth factors IL-1α, IL-1β, granulocyte colony-stimulating factor (G-CSF), and granulocyte-macrophage colony-stimulating factor (GM-CSF). In human monocyte-derived macrophages, G-CSF, GM-CSF, and IL-6 were found to be major targets of NhhA-dependent gene regulation. NhhA induced transcription of IL-6 and G-CSF mRNA via TLR4-dependent pathways, whereas GM-CSF transcription was induced via TLR4-independent pathways. These data provide new insights into the role of NhhA in host-pathogen interaction.

It is generally assumed that CTCF-binding sites are synonymous with the demarcation of expression domains by promoting the formation of chromatin loops. We have proposed earlier, however, that such features may be context-dependent. In support of this notion, we show here that chromatin loop structures, impinging on CTCF-binding sites 1/2 and 3/4 at the 5' and 3'-ends, respectively, within the maternal allele of the H19 imprinting control region (ICR), differ significantly. Although abrogation of CTCF binding to the maternal H19 ICR allele results in loss of chromatin loops in the 3'-region, there is a dramatic gain of long-range chromatin loops impinging on the 5'-region. As the degree of occupancy of its four CTCF-binding sites discriminates between the chromatin insulator and replication timing functions, we submit that the CTCF-binding sites within the H19 ICR are functionally diverse and organize context-dependent higher order chromatin conformations.

Phagocytotic cells play a fundamental role in the defense against bacterial pathogens. One mechanism whereby bacteria evade phagocytosis is to produce factors that trigger apoptosis. Here we identify for the first time a meningococcal protein capable of inducing macrophage apoptosis. The conserved meningococcal outer membrane protein NhhA (Neisseria hia/hsf homologue A, also known as Hsf) mediates bacterial adhesion and interacts with extracellular matrix components heparan sulphate and laminin. Meningococci lacking NhhA fail to colonise nasal mucosa in a mouse model of meningococcal disease. We found that exposure of macrophages to NhhA resulted in a highly increased rate of apoptosis that proceeded through caspase activation. Exposure of macrophages to NhhA also led to iNOS induction and nitric oxide production. However, neither nitric oxide production nor TNF-α signaling was found to be a prerequisite for NhhA-induced apoptosis. Macrophages exposed to wildtype NhhA-expressing meningococci were also found to undergo apoptosis whereas NhhA-deficient meningococci had a markedly decreased capacity to induce macrophage apoptosis. These data provide new insights on the role of NhhA in meningococcal disease. NhhA-induced macrophage apoptosis could be a mechanism whereby meningococci evade immunoregulatory and phagocytotic actions of macrophages.

Euthyroid sick syndrome characterized by reduced levels of thyroid hormones (THs) is observed in patients with meningococcal shock. It has been found that the level of THs reflects disease severity and is predictive for mortality. The present study was conducted to investigate the impact of THs on host defense during meningococcal infection. We found that supplementation of thyroxine to mice infected with Neisseria meningitidis enhanced bacterial clearance, attenuated the inflammatory responses and promoted survival. In vitro studies with macrophages revealed that THs enhanced bacteria-cell interaction and intracellular killing of meningococci by stimulating inducible nitric oxide synthase (iNos)-mediated NO production. TH treatment did not activate expression of TH receptors in macrophages. Instead, the observed TH-directed actions were mediated through nongenomic pathways involving the protein kinases PI3K and ERK1/2 and initiated at the membrane receptor integrin αvβ3. Inhibition of nongenomic TH signaling prevented iNos induction, NO production and subsequent intracellular bacterial killing by macrophages. These data demonstrate a beneficial role of THs in macrophage-mediated N. meningitidis clearance. TH replacement might be a novel option to control meningococcal septicemia.

Recent observations highlight that the mammalian genome extensively communicates with itself via long-range chromatin interactions. The causal link between such chromatin cross-talk and epigenetic states is, however, poorly understood. We identify here a network of physically juxtaposed regions from the entire genome with the common denominator of being genomically imprinted. Moreover, CTCF-binding sites within the H19 imprinting control region (ICR) not only determine the physical proximity among imprinted domains, but also transvect allele-specific epigenetic states, identified by replication timing patterns, to interacting, nonallelic imprinted regions during germline development. We conclude that one locus can directly or indirectly pleiotropically influence epigenetic states of multiple regions on other chromosomes with which it interacts.

Accumulating evidence converges on the possibility that chromosomes interact with each other to regulate transcription in trans. To systematically explore the epigenetic dimension of such interactions, we devised a strategy termed circular chromosome conformation capture (4C). This approach involves a circularization step that enables high-throughput screening of physical interactions between chromosomes without a preconceived idea of the interacting partners. Here we identify 114 unique sequences from all autosomes, several of which interact primarily with the maternally inherited H19 imprinting control region. Imprinted domains were strongly overrepresented in the library of 4C sequences, further highlighting the epigenetic nature of these interactions. Moreover, we found that the direct interaction between differentially methylated regions was linked to epigenetic regulation of transcription in trans. Finally, the patterns of interactions specific to the maternal H19 imprinting control region underwent reprogramming during in vitro maturation of embryonic stem cells. These observations shed new light on development, cancer epigenetics and the evolution of imprinting.

In the dipteran Chironomus tentans, actin binds to hrp65, a nuclear protein associated with mRNP complexes. Disruption of the actin-hrp65 interaction in vivo by the competing peptide 65-2CTS reduces transcription drastically, which suggests that the actin-hrp65 interaction is required for transcription. We show that the inhibitory effect of the 65-2CTS peptide on transcription is counteracted by trichostatin A, a drug that inhibits histone deacetylation. We also show that actin and hrp65 are associated in vivo with p2D10, an evolutionarily conserved protein with histone acetyltransferase activity that acts on histone H3. p2D10 is recruited to class II genes in a transcription-dependent manner. We show, using the Balbiani ring genes of C. tentans as a model system, that p2D10 is cotranscriptionally associated with the growing pre-mRNA. We also show that experimental disruption of the actin-hrp65 interaction by the 65-2CTS peptide in vivo results in the release of p2D10 from the transcribed genes, reduced histone H3 acetylation, and a lower level of transcription activity. Furthermore, antibodies against p2D10 inhibit run-on elongation. Our results suggest that actin, hrp65, and p2D10 are parts of a positive feedback mechanism that contributes to maintaining the active transcription state of a gene by recruiting HATs at the RNA level.

Despite the considerable progress molecular genetics of filamentous fungi has made during the past decade, there is still an urgent need for efficiently working selectable markers for fungal transformation. Using Pichia pastoris as a host, we describe the development of a new dominant selectable marker of prokaryotic origin. This system, termed sor(R), is based upon the resistance of the bacterial enzyme acetyl-CoA carboxylase (ACCase) to the macrocyclic polyketide soraphen A, a potent inhibitor of fungal ACCase produced by the myxobacterium Sorangium cellulosum. In this study, we firstly demonstrate that the integration of a single sor(R) cassette into the P. pastoris genome confers resistance to elevated concentrations of soraphen A. Furthermore, it has been shown that the versatility of this marker can be considerably increased by splitting the sor(R) cassette, especially when successive transformations are performed on the same strain. As pronounced sensitivity to soraphen A is the rule among filamentous fungi, we expect the sor(R) marker to be a widely applicable tool for fungal transformation.

A human cDNA library was screened for proteins interacting with the deafness locus putative guanine nucleotide exchange factor (DelGEF) using a yeast two-hybrid system. A protein with a predicted size of 9 kDa was identified as a binding partner, this protein was designated DelGEF interacting protein 1 (DelGIP1). The interaction between DelGEF and DelGIP1 was verified by co-immunoprecipitation of a DelGEF-DelGIP1 complex from cell lysates. Highly conserved homologues of DelGIP1 were identified in higher and lower eukaryotes by database searching. The human DelGIP1 gene is ubiquitously expressed as judged by human multiple tissue Northern blot analysis. DelGEF was recently shown to interact with Sec5, a protein involved in secretion, and to regulate secretion of proteoglycans. Downregulation of endogenous DelGIP1 in HeLa cells induced increased extracellular secretion of proteoglycans indicating a possible role for DelGIP1 in the secretion process.

Neutrophils from patients with chronic myeloid leukaemia (CML) have an aberrant expression of leukotriene (LT)C4 synthase. In order to learn more about the regulation of this abnormality, LTC4 synthase mRNA expression was determined by reverse transcription polymerase chain reaction. A digoxigenin (DIG)-labelled LTC4 synthase RNA was synthesized and incubated in cytosolic extracts from CML neutrophils, normal neutrophils and eosinophils. LTC4 synthase mRNA was detected in total but not cytoplasmic RNA from normal neutrophils. In contrast, LTC4 synthase mRNA was found in the cytoplasm of CML neutrophils and in normal eosinophils, which also express the enzyme. The DIG-labelled LTC4 synthase RNA was, as opposed to normal neutrophils, degraded in cytosolic extracts from CML neutrophils. The degradation was time dependent and cell concentration dependent. Degradation was also seen in eosinophils, indicating that degradation of LTC4 synthase RNA was correlated to the expression of the protein. This study showed that the difference in expression of LTC4 synthase in normal and CML neutrophils was not because of a total lack of LTC4 synthase mRNA in normal neutrophils. However normal neutrophils lack, in contrast to CML neutrophils, LTC4 synthase mRNA in the cytoplasm. This discrepancy is not caused by a stabilized LTC4 synthase RNA in the cytosol of CML neutrophils. Instead an abnormal degradation of LTC4 synthase RNA was found in the cytosol of CML neutrophils.

Objective: Leukotriene (LT) C(4) synthase (LTC(4)S) is the key enzyme in the biosynthesis of LTC(4), which has been reported to stimulate the growth of human myeloid progenitor cells and is specifically overproduced in chronic myeloid leukemia (CML). The aim of this study was to clarify the expression of LTC(4)S during normal and leukemic myelopoiesis and to investigate the correlation between abnormal LTC(4)S expression in CML myeloid cells and the activity of the disease-specific tyrosine kinase p210 BCR-ABL., Materials and Methods: Immature and mature myeloid cell subpopulations were isolated with magnetic cell sorting from healthy volunteer bone marrow (n = 11) and CML patient peripheral blood (n = 8), respectively. The cells were subjected to analysis of LTC(4)S protein expression and activity. Expression of LTC(4)S was investigated in CD16(+) neutrophils from CML patients before and after 1 month of medication with imatinib mesylate (STI571), which is a specific inhibitor of p210 BCR-ABL., Results: Among normal cells, the highest enzyme activity was observed in the most immature, CD34(+) progenitor cell-enriched and CD15(+) myelocyte-enriched fractions. Subsequently, LTC(4)S activity decreased with increasing maturity, with only negligible amounts of LTC(4) produced in CD16(+) neutrophils. LTC(4)S was expressed at the protein level in the immature myeloid cell fractions but not in CD16(+) cells. In CML cells, LTC(4)S activity and expression were consistently elevated. Thus, the CML CD34(+) and CD15(+) cell fractions, as well as the CD11b(+) myelocyte/metamyelocyte-enriched fractions, produced 6 to 10 times as much LTC(4) as the corresponding normal cells. Again, enzyme expression was highest in the most immature cells, although evident LTC(4)S expression and activity remained in CML CD16(+) neutrophils. Interestingly, treatment of five CML patients with imatinib mesylate down-regulated the abnormal neutrophil LTC(4)S expression and activity., Conclusions: Expression of LTC(4)S in immature myelopoid cells is in line with a role for this enzyme in myelopoiesis. In addition, consistent overexpression of LTC(4)S in CML and the correlation to p210 BCR-ABL activity suggests that LTC(4)S may be involved in leukemic pathogenesis.

In order to identify the function of deafness locus putative guanine nucleotide exchange factor (DelGEF), a protein homologous to the nucleotide exchange factor for the small GTPase Ran, a cDNA library was screened for interacting proteins using a yeast two-hybrid system. The human homologue of Sec5, a protein involved in vesicle transport and secretion, was identified as a binding partner. The interaction between DelGEF and Sec5 was found to be dependent on Mg2+ and stimulated by guanosine triphosphate (GTP) or deoxycytidine triphosphate (dCTP). Downregulation of endogenous DelGEF in HeLa cells induced increased extracellular secretion of proteoglycans indicating a possible role for DelGEF in the secretion process.

Elevated leukotriene (LT)C(4) synthase activity was observed in peripheral blood granulocyte suspensions from patients with chronic myeloid leukemia (CML). Magnetic cell sorting (MACS) with CD16 monoclonal antibodies (mAbs), which were used to fractionate granulocytes from CML patients and healthy individuals, yielded highly purified suspensions of CD16(+) neutrophils. The purity of these cell fractions was verified by extensive morphologic examination. Reverse transcriptase-polymerase chain reaction (RT-PCR) analyses, demonstrating the absence of interleukin-4 messenger RNA (IL-4 mRNA), further confirmed the negligible contamination of eosinophils in these fractions. Notably, purified CML CD16(+) neutrophils from all tested patients transformed exogenous LTA(4) to LTC(4). These cells also produced LTC(4 )after activation with ionophore A23187 or the chemotactic peptide fMet-LeuPhe (N-formylmethionyl-leucyl-phenylalanine). Subcellular fractionation revealed that the enzyme activity was exclusively distributed to the microsomal fraction. Expression of LTC(4) synthase mRNA in CML CD16(+) neutrophils was confirmed by RT-PCR. Furthermore, Western blot analyses consistently demonstrated expression of LTC(4) synthase at the protein level in CML CD16(+) neutrophils, whereas expression of microsomal glutathione S-transferase 2 occurred occasionally. Expectedly, LTC(4) synthase activity or expression of the protein could not be demonstrated in CD16(+) neutrophil suspensions from any of the healthy individuals. Instead, these cells, as well as CML CD16(+) neutrophils, transformed LTA(4) to LTB(4). The results indicate that aberrant expression of LTC(4) synthase is a regular feature of morphologically mature CML CD16(+) neutrophils. This abnormality, possibly associated with malignant transformation, can lead to increased LTC(4) synthesis in vivo. Such overproduction may be of pathophysiological relevance because LTC(4 )has been demonstrated to stimulate proliferation of human bone marrow-derived myeloid progenitor cells. (Blood. 2000;95:1456-1464)

The expression of neurotrophin and neurotrophin receptor mRNAs in human granulocytes and bone marrow cells was examined using ribonuclease protection assay and reverse transcription-polymerase chain reaction. The granulocytes expressed mRNA coding for nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-4 (NT-4), but not neurotrophin-3 (NT-3). Moreover, the inflammatory mediator leukotriene B4 (LTB4) up-regulated the expression of NT-4 mRNA in granulocytes, but did not affect the expression of other neurotrophin mRNAs. Granulocytes generally lacked expression of mRNA coding for neurotrophin receptors. In contrast, human bone marrow cells consistently expressed mRNA for trkB (the BDNF and NT-4 receptor) and displayed variable expression of mRNA coding for trkA (the tyrosine kinase NGF receptor) and LNGFR (the low-affinity NGF receptor), whereas mRNA for trkC (the NT-3 receptor) was not expressed. Contrary to granulocytes, normal bone marrow cells generally expressed only low levels of mRNA encoding BDNF and NT-4. Expression of mRNA encoding NGF and NT-3 was not detected. However, significantly increased expression of BDNF mRNA was observed when bone marrow cells from patients with chronic myeloproliferative disorders (MPD) were analyzed. The results suggest that neurotrophins may act as granulocyte-derived effector molecules and that human bone marrow cells may be targets for these compounds, in particular BDNF and NT-4.

Leukotrienes (LT) are inflammatory mediators which can also exert regulatory effects on human myelopoiesis. We have studied the LT-producing capacity of freshly isolated leucocyte suspensions (containing blast cells in variable proportions) from 41 patients with acute myeloid leukaemia (AML) or chronic myeloid leukaemia (CML) in blast crisis (CMLbc) at diagnosis or relapse/resistant disease. Leucocyte suspensions from 19/29 AML patients (66%), and 2/12 CMLbc patients (17%; P = 0.012) demonstrated deficient capacity to synthesize LT from endogenous substrate after ionophore A23187 stimulation. Thus, these cells produced < 8 pmol LTB4+LTC4/10(6) cells (< 20% of mean LT formation in leucocyte suspensions from 18 healthy subjects). Addition of exogenous arachidonic acid did not normalize the LT synthesis in poor-producing cell suspensions. Purified, morphologically mature granulocytes from two AML patients also failed to produce normal amounts of LT. In leucocyte suspensions from the remaining 20 AML/CMLbc patients A23187 provoked LT biosynthesis, with markedly increased production of LTC4, but decreased LTB4 formation. Furthermore, elevated conversion of exogenous LTA4 to LTC4 was noted in the patient samples, independent of their capacity to produce LT after A23187 stimulation. The percentage of blast cells in patient white blood cell differential counts correlated inversely with ionophore-induced LT synthesis, but positively with the conversion of exogenous LTA4 to LTC4. The results suggest elevated LTC4 synthase activity and suppressed 5-lipoxygenase activity as novel enzymatic features of myeloid leukaemia patients with immature phenotype.

Human platelets have been demonstrated to possess leukotriene (LT)-C4 synthase activity and may thus be involved in transcellular 5(S)-hydroxy-6(R)-S-glutathionyl-7,9-trans-11,14-cis-eicosatetraenoic acid (LTC4) synthesis. In this study, platelets from seven different species were screened for LTC4 synthase activity. Very high enzyme activity was observed in suspensions of bovine platelets, with approximately 70% conversion of 5(S)-trans-5,6-oxido-7,9-trans-11,14-cis-eicosatetraenoic acid (LTA4) to LTC4. The capacity of equine platelets to produce LTC4 was similar to that of human platelets. In addition, ovine, rabbit, and rat platelets also produced LTC4 after incubation with LTA4. The results demonstrate that LTC4 synthase activity is a common feature among platelets from various species. In contrast, porcine platelets failed to transform LTA4 to LTC4. Instead, these cells produced 5(S),12(R)-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid (LTB4), indicating the presence of LTA4 hydrolase in porcine platelets. A protein with a molecular mass of approximately 18 kDa and LTC4 synthase activity was solubilised from lyophilised bovine platelet concentrates and purified to near homogeneity by affinity chromatography and gel filtration. The N-terminal amino acid sequence of this protein was analysed and found to be almost identical to the corresponding sequence of human LTC4 synthase (17 of 18 amino acid residues identical). Kinetic analysis of partially purified bovine platelet LTC4 synthase revealed Km (for LTA4) and Vmax values of 3.3 microM and 521 nmol x mg protein(-1) x min(-1), respectively. In addition, the presence of a mRNA transcript encoding LTC4 synthase was demonstrated in equine platelets by reverse transcription (RT) PCR using primers derived from the human LTC4 synthase cDNA sequence. Cloning and sequencing of the PCR fragment corresponding to a region near the N-terminus demonstrated very high identity between equine and human leukotriene-C4 synthase in this region. In summary, the present study establishes that platelets contain LTC4 synthase and indicates that this enzyme is widely distributed among platelets from various species.

The effect of the protein kinase C activator, phorbol 12-myristate 13-acetate (PMA), on the metabolism of exogenous leukotriene (LT)A4 in human granulocytes was investigated. After incubation with LTA4 decreased levels of LTC4 but not LTB4 were observed in granulocyte suspensions pretreated with PMA. This finding could in part be ascribed to oxidative metabolism of LTC4, since PMA induced a rapid degradation of exogenously added LTC4. After blocking of LTC4 metabolism with the H2O2 scavenger catalase, a PMA-provoked suppression of the conversion of LTA4 to LTC4 was observed, indicating PKC-dependent regulation of LTC4 synthase activity. This effect, as well as PMA-induced degradation of LTC4 was prevented by specific protein kinase C inhibitors.