Efficacy and safety of Diclofenac sodium plaster in patients with acute pain of the limbs: a randomized, placebo and active-controlled, double-blind, parallel-group trial.

OBJECTIVE: The aim of the present study was to assess the safety and efficacy of Diclofenac sodium (DS) 140 mg medicated plaster vs. Diclofenac epolamine (DIEP) 180 mg medicated plaster and placebo plaster, for the treatment of painful disease due to traumatic events of the limbs. PATIENTS AND METHODS: This was a multicenter, phase III study involving 214 patients, aged 18-65 years, affected by painful conditions due to soft tissue injuries. Patients were randomized to DS, DIEP or placebo arms and treated with once-daily application of the plaster for a total treatment period of 7 days. The primary objective was first to demonstrate the non-inferior efficacy of the DS treatment when compared to the reference DIEP treatment and second that both, test and reference treatments, were superior with respect to placebo. The secondary objectives included the evaluation of efficacy, adhesion, safety, and local tolerability of DS in comparison to both DIEP and placebo. RESULTS: The mean visual analog scale (VAS) score decrease for pain at rest was higher in the DS (-17.65 mm) and the DIEP group (-17.5 mm) than in the placebo (-11.3 mm). Both active formulation plasters were associated with a statistically significant pain reduction compared to placebo. No statistically significant differences were observed between DIEP and DS plasters efficacy in relieving pain. Secondary endpoint evaluations supported the primary efficacy results. No serious adverse events (SAEs) were registered, and the most commonly detected adverse events were skin reactions at the application site. CONCLUSIONS: The results showed that both the DS 140 mg plaster and the reference DIEP 180 mg plaster are effective in relieving pain and present a good safety profile.

Efficacy and tolerance of a comfrey root extract (Extr. Rad. Symphyti) in the treatment of ankle distorsions: results of a multicenter, randomized, placebo-controlled, double-blind study.

Comfrey (Symphytum officinale L.) is a medicinal plant with anti-inflammatory, analgesic and tissue regenerating properties. In a double-blind, multicenter, randomized, placebo-controlled, group comparison study on patients suffering from unilateral acute ankle sprains (n = 142, mean age 31.8 years, 78.9% male), the percutaneous efficacy of an ointment of comfrey extract (Kytta-Salbe f, four treatments per day for 8 days) was confirmed decisively. Compared to placebo, the active treatment was clearly superior regarding the reduction of pain (tonometric measurement, p<0.0001, as the primary efficacy variable) and ankle edema (figure-of-eight method, p = 0.0001). Statistically significant differences between active treatment and placebo could also be shown for ankle mobility (neutral zero method), and global efficacy. Under active treatment, no adverse drug reactions were reported. The good local and global tolerance of the trial medication could also be confirmed. The study results are consistent with the known pre-clinical and clinical data concerning comfrey.

Diclofenac patch for topical treatment of acute impact injuries: a randomised, double blind, placebo controlled, multicentre study.

OBJECTIVES: To investigate the clinical efficacy and safety of a newly developed diclofenac patch in the topical treatment of blunt impact injuries. METHODS: This was a randomised, placebo controlled, double blind, multicentre study in 120 patients with traumatic blunt soft tissue injury. Within 3 h of the injury participants of sport competitions and training camps were enrolled and treated twice daily with the diclofenac or a placebo patch over a period of 7 days. Patients were randomised (1:1) to two parallel groups. Tenderness produced by pressure was measured twice daily during the first 3 days after enrollment as well as at day 7. Tenderness was defined as the amount of pressure (measured by a calibrated caliper at the centre of the injury) that first produced a pain reaction as reported by the patient. RESULTS: The primary efficacy variable was the area under the curve for tenderness over the first 3 days. The diclofenac patch was significantly more effective than placebo (p<0.0001). The treatment effect was 64.7 kp h/cm2 (95% confidence interval 48.7 to 80.9) between diclofenac and placebo patches. These results were supported by all secondary efficacy variables. The diclofenac patch produced rapid pain relief as reflected by the time to reach resolution of pain at the injured site which was significantly shorter compared to placebo (p<0.0001). The diclofenac patch was well tolerated. The most frequently observed adverse events were local cutaneous adverse reactions (pruritus, rash) of minor severity occurring with the same frequency as in the placebo group. CONCLUSIONS: A newly developed diclofenac patch is effective and safe for the treatment of blunt impact injuries.

Efficacy and tolerability of escin/diethylamine salicylate combination gels in patients with blunt injuries of the extremities.

The aim of this confirmative, monocentre, double-blind, controlled clinical trial was to investigate whether different escin combinations show differences in comparison to placebo with regard to pain reactions in the topical treatment of sports injuries. A total of 126 patients with blunt injuries of the extremities were randomly allocated to four parallel groups: Reparil-Gel N (n = 32), Reparil-Gel (n = 31), Reparil-Sportgel (n = 32) and a placebo gel (n = 31). All patients were evaluated for efficacy (intention to treat) and tolerability. A per-protocol analysis was also carried out, in which 12 of the 126 patients were excluded due to protocol violations. The intention-to-treat and per-protocol analyses produced similar results. The patients had suffered contusions while participating in soccer, hockey, karate, tae-kwon-do, handball, American football, rugby or tennis. The measured variable was the pressure required at the centre of the lesion to elicit the first pain reaction (tenderness reaction) at measuring time 0 (baseline) and then 1, 2, 3, 4, 6 and 24 h after the injury. The primary variable was the area under the curve (AUC) for tenderness over a six-hour period. The mean AUC differed significantly in the four groups (Kruskal-Wallis test p = 0.0001). Then six pairwise comparisons of two treatment groups each were carried out using the Mann-Whitney test. To control the multiple significance level of 5%, the adjusted p-values according to the Holm-Shaffer method were used in these tests. The three active gels were significantly superior to the placebo gel (Mann-Whitney test, p = 0.0004 in each case) in terms of the AUC. There were no significant differences between the active test substances in terms of the primary variable. The intensity of the pain was also measured on a visual analogue scale (VAS). The pain diminished more rapidly with the Reparil gels than with the placebo. The tolerability of all test substances was good. No adverse events were observed in any of the 126 patients. Escin combination gels are more effective than a placebo and are also well tolerated. Therefore, they can be recommended for the treatment of blunt injuries caused during sports and leisure activities.

Bone marrow transplantation for T-B- severe combined immunodeficiency disease in Athabascan-speaking native Americans.

A distinct form of autosomal recessive T-B- severe combined immunodeficiency disease occurs with a high frequency among Athabascan-speaking Native Americans (SCIDA), including Navajo and Apache Indians from the southwestern US and Dene Indians from the Canadian Northwest Territories. The SCIDA gene has been linked to markers on chromosome 10p although its identity and role in the pathogenesis of this disease are unknown. We report our experience in treating 18 Navajo and Dene children with SCIDA between 1984 and 1999; 16 underwent bone marrow transplants (BMT). All children were symptomatic within 2 months of birth, had the T-B- NK(+)SCID phenotype and 67% presented with oral and/or genital ulcers. Three children had evidence of maternal engraftment prior to transplant. Two children died shortly after diagnosis. Three children required more than one BMT and 12 are alive with T cell reconstitution at a median follow-up of 7 years. Three children developed normal B cell immunity, two of whom received ablative conditioning therapy with either radiation or busulfan. Three of the four children who died received therapy with either radiation or busulfan and two of eight long-term survivors who were also recipients of cytotoxic chemotherapy have failed to develop secondary teeth. These results demonstrate the efficacy of BMT in treating infants with this distinct form of SCID, although B cell reconstitution remains a problem even with HLA-matched donors. Without conditioning, T cell engraftment is likely when closely HLA-matched donors are used. With T cell depletion of haplocompatible marrow, conditioning with immunosuppressive therapy may be necessary; however, children with SCIDA who were treated with intensive immunosuppressive and myeloablative therapy had a poor outcome.

Evidence for microvesicular storage and release of glycine in rodent pinealocytes.

Prompted by previous studies suggesting a regulatory role for the inhibitory amino acid gamma-aminobutyric acid (GABA) within the mammalian pineal gland, we carried out a study of rat and gerbil pineal organs to elucidate whether there is evidence for a vesicular storage and release of GABA and/or glycine. Immunohistochemistry revealed the presence of the vesicular inhibitory amino acid transporter in pinealocytes. Moreover, we found that, in addition to glutamate and aspartate, cultured pinealocytes also released glycine upon stimulation by depolarizing concentrations of KCl, whereas the content of GABA in the culture medium did not exceed the detection limit either under control conditions or following KCl application. Therefore, we propose that glycine is a further component of the paracrine signaling system within the pineal organ which is based on the compartment of synaptic-like microvesicles (SLMVs) inside pinealocytes.

Defective NK cell activation in X-linked lymphoproliferative disease.

X-linked lymphoproliferative disease (XLP) is characterized by a selective immune deficiency to EBV. The molecular basis of XLP has been attributed to mutations of signaling lymphocytic activation molecule-associated protein, an intracellular molecule known to associate with the lymphocyte-activating surface receptors SLAM and 2B4. We have identified a single nucleotide mutation in SLAM-associated protein that affects the NK cell function of males carrying the mutated gene. In contrast to normal controls, both NK and lymphokine-activated killer cell cytotoxicity was significantly reduced in two XLP patients. In addition to decreased baseline cytotoxicity, ligation of 2B4 significantly augmented NK lytic function in normal controls but failed to enhance the cytotoxicity of NK cells from XLP patients. These findings suggest that association of SAP with 2B4 is necessary for optimal NK/lymphokine-activated killer cytotoxicity and imply that alterations in SAP/2B4 signaling contribute to the immune dysfunction observed in XLP.

Immunohistochemical study of the glutamate transporter proteins GLT-1 and GLAST in rat and gerbil pineal gland.

Several investigations performed during this decade have led to the hypothesis that small secretory vesicles of pinealocytes (generally referred to as synaptic-like microvesicles, SLMVs) are components of a system for intercellular paracrine communication between pineal cells, which shares many features with the process of synaptic neurotransmission. According to a recent study, one parallel that can be drawn to synaptic signal transduction seems to be the presence of pineal re-uptake systems for messenger molecules released from SLMVs, i.e. for neuroactive amino acids such as L-glutamate. In order to further characterize these uptake mechanisms, we have carried out an immunohistochemical study to explore the presence and cellular localization of the glutamate transporters GLT-1 and GLAST in rat and gerbil pineal glands. GLT-1 and GLAST were always detected in a subpopulation of pineal parenchymal cells in both species. Using immunostaining of serial semithin sections with antibodies against marker proteins of pineal cell types, GLT-1- and GLAST-positive cells were identified as interstitial glial cells. In addition, some pinealocytes also displayed immunoreactivity for GLT-1. In contrast to current thinking, our findings show that GLT-1 is not the only glutamate transporter subtype expressed in the pineal gland. Moreover, our observations point to a significant participation of interstitial cells in the process of pineal glutamatergic communication, reminiscent of the role of glial cells during glutamatergic neurotransmission in the central nervous system.

Interstitial glial cells of the gerbil pineal gland display immunoreactivity for the metabotropic glutamate receptors mGluR2/3 and mGluR5.

Recent studies have strengthened the hypothesis that neuroactive amino acids such as L-glutamate play an important role in the physiology of the mammalian pineal gland. In particular, there is now considerable evidence that L-glutamate is liberated from electron-lucent microvesicles of pinealocytes for a paracrine modulation of melatonin synthesis and release which may at least partially be mediated by the metabotropic glutamate receptor mGluR3. In order to expand our incomplete knowledge of possible pineal target cells and signal transduction mechanisms which are involved in glutamate-dependent intercellular communication, we have performed an immunohistochemical study of the gerbil pineal gland with antibodies directed against the metabotropic glutamate receptors mGluR2/3 and mGluR5. Using microwave irradiation of cryostat sections prior to immunostaining, strong immunoreactivity for both receptor subtypes was constantly observed in a subpopulation of pineal cells. Interestingly, these mGluR-positive cells could be identified as interstitial glial cells since they were labeled by antibodies against the intermediate filament protein vimentin in double immunofluorescence histochemistry. This indicates that interstitial glial cells in the gerbil possess the capacity to express at least two metabotropic glutamate receptors coupled to different intracellular signal transduction pathways. Therefore, it can be concluded that the glutamatergic communication system of the pineal gland may not only enable paracrine crosstalk among pinealocytes but probably also relies on interactions between pinealocytes and interstitial cells analogous to neuronal-glial signaling.

Cell-mediated and antibody immune responses to AIK-C and Connaught monovalent measles vaccine given to 6 month old infants.

Measles vaccination of infants younger than 1 year of age should be successful in populations with a high proportion of measles vaccinated mothers. Infants whose mothers were vaccinated are born with less maternal antibody which can interfere with vaccination compared with infants whose mothers had measles. AIK-C or Connaught (CLL) measles vaccine was given to 300 6 month infants born to mothers who had measles (group 1) or who were vaccinated against measles (group 2). Pre- and post-vaccination measles antibody was measured by EIA and PRN and cell mediated immunity (CMI) by blast transformation and production of interferon-gamma and interleukin-10. After vaccination, mean antibody level, seroconversion and blastogenesis were significantly lower for group 1 than group 2 (p < 0.05). Post-vaccination measles IgG was significantly higher for group 2 CLL vaccinees compared with group 2 AIK-C (p < 0.05); seroconversion rates were 73 and 63%, respectively. More than 93% of group 2 infants had elevated measles IgG after vaccination. About 89% of all children had some evidence of a blastogenic response. Lymphoproliferation correlated strongly with cytokine production and weakly with IgG. Not all seroresponders had a CMI response and vice versa. AIK-C and CLL vaccines induce strong measles specific T and B immunity in most 6 month infants of vaccinated mothers.

A comparison of alternate immunization regimes for measles in vaccinated populations.

Infants today lose maternal measles antibody sooner than in the past. This is related to demographic changes in maternal immunization. Data for rates of decay of maternal antibody and seroconversion after measles vaccination for infants born to naturally immune (Group 1) or vaccinated (Group 2) mothers have been used to evaluate two vaccination schedules: Regime 1, measles-mumps-rubella (MMR) at 1 year of age and Regime 2, monovalent measles at 6 months followed by MMR at 15 months of age. Regime 2 costs less because MMR can be administered at 15 months with the last pentavalent booster. Months of protection/1000 children aged 0-15 months (child-months of protection) were estimated for infant populations ranging from 0 to 100% Group 1 for Regimes 1 and 2. Regime 1 provides more child-months of protection only for 100% Group 1 populations. For the study population Regime 2 provided at least 17% more child-months of protection than Regime 1. Regime 2 provides increased medical and financial benefits in proportion to the number of Group 2 infants in the population and thus is ever more advantageous for today's increasingly vaccinated populations.

Mutations of the CD40 ligand gene and its effect on CD40 ligand expression in patients with X-linked hyper IgM syndrome.

X-linked hyper IgM syndrome (XHIM) is a primary immunodeficiency disorder caused by mutations of the gene encoding CD40 ligand (CD40L). We correlated mutations of the CD40L gene, CD40L expression, and the clinical manifestations observed in XHIM patients from 30 families. The 28 unique mutations identified included 9 missense, 5 nonsense, 9 splice site mutations, and 5 deletions/insertions. In 4 of 9 splice site mutations, normally spliced and mutated mRNA transcripts were simultaneously expressed. RNase protection assay demonstrated that 5 of 17 mutations tested resulted in decreased levels of transcript. The effect of the mutations on CD40L expression by activated peripheral blood mononuclear cells (PBMC) and T-cell lines or clones was assessed using one polyclonal and four monoclonal antibodies and a CD40-Ig fusion protein. In most patients, the binding of at least one antibody but not of CD40-Ig was observed, suggesting nonfunctional CD40L. However, activated PBMC from three patients and activated T-cell lines from two additional patients, each with different genotype, bound CD40-Ig at low intensity, suggesting functional CD40L. Thus, failure of activated PBMC to bind CD40-Ig is not an absolute diagnostic hallmark of XHIM and molecular analysis of the CD40L gene may be required for the correct diagnosis. Patients with genotypes resulting in diminished expression of wild-type CD40L or mutant CD40L that can still bind CD40-Ig appear to have milder clinical consequences.

[Clinical relevance of I-123/I-131-MIBG scintigraphy in intestinal carcinoid tumors]. / Klinische Relevanz der I-123/I-131-MIBG-Szintigraphie bei intestinalen Karzinoiden.

AIM: Of this retrospective study was to determine the value of MIBG-scintigraphy in patients with intestinal carcinoids dependent on histological, clinical, and biochemical parameters. METHODS: In 15 patients uptake in carcinoid tumors and metastasis was correlated with location of the primary tumor, intra- and extrahepatic tumor masses, histology, immunhistochemistry, neuroendocrinological markers, and clinical symptoms. RESULTS: High uptake was to be seen almost only in tumor masses of primary tumors located in the terminal ileum. There also was a positive correlation with clinical symptoms for carcinoids and urinary 5-HIAA level. No correlation between MIBG uptake and tumor masses, histology, and most of the immunhistochemical and neuroendocrinological markers could be found. CONCLUSION: There is a limited indication for MIBG-scintigraphy in follow up of intestinal carcinoids. In patient with proven uptake MIBG scintigraphy is suitable for long-term follow up and therapy monitoring.

Munc-18-1 and cysteine string protein (csp) in pinealocytes of the gerbil pineal gland.

Mammalian pinealocytes contain several synaptic membrane proteins which probably play a role in the targeting and exocytosis of secretory vesicles, in particular of synaptic-like microvesicles (SLMVs). The latter are considered as the endocrine equivalent of neuronal synaptic vesicles. By means of immunocytochemical techniques and immunoblot analyses, we now show that two further key components of the molecular apparatus regulating neurotransmitter release are present in the gerbil pineal gland, i.e., munc-18-1 and cysteine string protein (csp). In addition to varicosities of nerve fibres, munc-18-1 and csp could be localized to pinealocytes where both proteins were markedly enriched in process swellings. When using antibodies against csp for an immunogold electron-microscopic study of pinealocytes, gold particles consistently decorated profiles of pleomorphic SLMVs. Interestingly, we found that also the cytosolic protein munc-18, which is partially recruited to the plasmalemma in neurons, was associated to a significant extent with SLMVs of pinealocytes and synaptic vesicles of neurons, respectively. This localization implies that munc-18 at least partially exerts its regulatory functions while being bound to secretory vesicle membranes. Our results indicate that in endocrine cells such as pinealocytes the synaptic proteins munc-18-1 and csp play essential roles during the life cycle of SLMVs.

The gene for severe combined immunodeficiency disease in Athabascan-speaking Native Americans is located on chromosome 10p.

Severe combined immunodeficiency disease (SCID) consists of a group of heterogeneous genetic disorders. The most severe phenotype, T-B- SCID, is inherited as an autosomal recessive trait and is characterized by a profound deficiency of both T cell and B cell immunity. There is a uniquely high frequency of T-B- SCID among Athabascan-speaking Native Americans (A-SCID). To localize the A-SCID gene, we conducted a genomewide search, using linkage analysis of approximately 300 microsatellite markers in 14 affected Athabascan-speaking Native American families. We obtained conclusive evidence for linkage of the A-SCID locus to markers on chromosome 10p. The maximum pairwise LOD scores 4.53 and 4.60 were obtained from two adjacent markers, D10S191 and D10S1653, respectively, at a recombination fraction of straight theta=.00. Recombination events placed the gene in an interval of approximately 6.5 cM flanked by D10S1664 and D10S674. Multipoint analysis positioned the gene for the A-SCID phenotype between D10S191 and D10S1653, with a peak LOD score of 5.10 at D10S191. Strong linkage disequilibrium was found in five linked markers spanning approximately 6.5 cM in the candidate region, suggesting a founder effect with an ancestral mutation that occurred sometime before 1300 A.D.

Cerebellar hypoplasia and frontal lobe cognitive deficits in disorders of early childhood.

A developmental chronometry hypothesis of early brain damage is suggested in which regions of the brain with a protracted course of postnatal development will be more vulnerable than earlier maturing areas to deleterious effects of early insult and, therefore, may become common sites of abnormality across many disorders originating in early childhood. Initial investigations of the cerebellum and frontal lobes are presented using MRI and neuropsychological measures. Planimetric measures of the cerebellar vermis (lobuli I-V and VI-VII) and pons, and neuropsychological frontal lobe measures were obtained from high functioning individuals with autism (A), survivors of acute lymphoblastic leukemia (ALL) with brain sequelae following radiation and chemotherapy, and from rigorously selected healthy controls (C). The neuropsychological results were clustered according to functions commonly related to frontal brain, posterior brain, and left and right hemispheres. The A and ALL groups, as compared to C, yielded modest but consistently reduced MRI measures for vermal lobuli I-V and VI-VII. Hypoplasia of lobuli VI-VII was more marked than I-V. Performance on neuropsychological tests for frontal lobe functions was generally depressed in both groups, with more severe deficits in A. Between-group differences in verbal, visual-spatial, and emotional-social skills are discussed. The cerebellar and frontal brain deficits that are present in both clinical groups (A and ALL) may be common to other developmental and acquired disorders of early childhood. Such joint manifestation of cerebellar and frontal lobe abnormalities is in agreement with the concept of cerebellar significance for the development of higher cognitive functions.

Expression of synaptic membrane proteins in gerbil pinealocytes in primary culture.

Pinealocytes of various mammalian species contain abundant synaptic-like microvesicles (SLMVs) which are considered the endocrine equivalent of neuronal synaptic vesicles. Although the pinealocytes may thus be a suitable cellular model for experimental in vitro studies of SLMVs, nothing is known about the presence of SLMVs in isolated pinealocytes maintained under tissue culture conditions. In the present investigation, we prepared dissociated primary cultures of gerbil pinealocytes to study the expression and distribution of protein components of synaptic vesicles/SLMVs and the presynaptic plasmalemma in pinealocytes kept in vitro. Using immunofluorescence microscopy, we found that cultured pinealocytes readily expressed all synaptic membrane proteins investigated, i.e., synaptophysin, synaptotagmin I, synaptobrevin II, syntaxin I and SNAP-25. Punctuate immunoreactivity for the vesicle-associated proteins could be detected throughout the cell bodies of pinealocytes and was also distributed into all of their processes which began to develop within the first days in culture. Outgrowing processes exhibited growth cone-like structures which were enriched in synaptic vesicle-associated proteins. After 1 week in vitro, pinealocytes had frequently formed an elaborate network of long interwoven processes. Accumulations of synaptic vesicle-associated proteins were observed in varicosities and terminal swellings of the processes. The vesicle-rich process swellings often established synaptic-like process swellings often established synaptic-like contacts with somata and processes of other pinealocytes. Some of the pinealocyte processes possessed additional axon-like properties as demonstrated by their lack of immunoreactivity for the somato-dendritic marker MAP2 and the transferrin receptor. The comparison of the staining patterns for synaptophysin and the endocytotic marker transferrin receptor by confocal laser scanning microscopy revealed a largely differential intracellular distribution of the two proteins. This may indicate that a substantial fraction of pinealocyte SLMVs by-passes the early endosomal-related recycling pathway of SLMVs. Herewith, we have shown that isolated gerbil pinealocytes maintained in primary culture can acquire morphological and neurochemical traits which closely mimick those observed in vivo. In particular, these cultures permit experimental studies of the compartment of pinealocyte SLMVs which seem to make up a major secretory pathway for paracrine intrapineal communication.

Kinetics of immunologic responses after primary MMR vaccination.

To study the kinetics of humoral as well as cellular immunity to measles and to test for associated immunosuppression 124 12 month old children were studied twice, before routine MMR and either 14, 22, 30, or 38 days after vaccination. Plaque reduction neutralization (PRN) titres were determined at these time points and lymphocytes were evaluated to identify changes in proportions of phenotype, their capacity to generate cytokines and to respond to blast transformation (BT) to measles hemagglutinin (HA), tetanus toxoid and Candida antigen. The PRN titre and BT to HA plateaued at 30 days and CD8+ and NK cells increased after immunization. Interleukin 2, 4, and 10 showed no significant changes. There was mild suppression of BT at 14 and 22 days post-immunization Interferon-gamma was the principal cytokine produced after primary measles immunization, suggesting primary measles immunization induces predominantly a TH1 type response.