Anxiety and Depression as Comorbidities of Multiple Sclerosis.

Multiple Sclerosis (MS), a chronic inflammatory neurodegenerative disease, is accompanied by a number of comorbidities. Among the psychiatric ones, depression and anxiety occupy a special place. It is estimated that the prevalence of anxiety in the MS population is 22.1% verus 13% in the general population; whereas the prevalence of anxiety levels, as determined by various questionnaires, reaches even 34.2%. Systematic literature reviews (SPL) show considerable data variations due to differences in study design, sample size, diagnostic criteria and extremely high heterogeneity (I2). Among the more conspicuous factors associated with anxiety disorder in MS are demographic factors (age and gender), nonsomatic depressive symptoms, higher levels of disability, immunotherapy treatments, MS type, and unemployment. Depression is the most common psychiatric commorbidity in MS and the lifetime risk of developing depression in MS patients is >50%. According to some research, the prevalence of depression in MS vary between 4.98% and 58.9%, with an average of 23.7% (I2=97.3%). Brain versus spinal cord lesions, as well as temporal lobe, fasciculus arcuatus, superior frontal and superior parietal lobe lesions in addition to the cerebral atrophy have been shown to be the anatomical predictors of depressive disorder in MS. Hyperactivity of the hypothalamic-pituitary-adrenal axis (HPA) and the consequent dexamethasone-insupressible hypercortisolemia, in addition to cytokine storm (IL-6, TNF-α, TGFß1, IFNγ/IL-4) present the endocrine and inflammatory basis for development of depression. Fatigue, insomnia, cognitive dysfunction, spasticity, neurogenic bladder, pain, and sexual dysfunction have shown to be additional precipitating factors in development of anxiety and depression in MS patients.

Comorbidities in Patients with Multiple Sclerosis in Croatia.

BACKGROUND: Comorbidities in multiple sclerosis (MS) have a big role in management of this chronic demyelinating neurodegenerative disorder. The aim of this study was to evaluate comorbidities in patients with MS in Croatia. SUBJECTS AND METHODS: This was a prospective cross-sectional study carried out in an out-patient setting at a tertiary healthcare centre over 10 months, which included 101 consecutive patients with MS (mean age 42.09 (range 19-77) years, 75 female, 26 male, EDSS score 3.1 (range 0.0-7.0)). The average duration of the disease was 13.5±7.487 (range 1-42) years. Thirty-six patients were treated with disease modifying therapies (DMTs). Information on comorbidities was obtained during the medical interview. Data was analysed using software package IBM Corp. Released 2015. IBM SPSS Statistics for Windows, Version 23.0. Armonk, NY: IBM Corp. RESULTS: 33% (n=34) patients did not have any comorbidities, and there is an equal number of patients (n=34, 33%) that just had one comorbidity. 17.6% (n=18) of patients had two comorbidities, and 15.7% (n=16) three or more comorbidities. The most frequent comorbidity was depression found in 25 (24.75%) patients (19 (18.8%) women, 6 (5.9%) men), followed by the hypertension in 12.87% (n=13). Hyperlipidemia and migraine were each found in 6.93% (n=7), and hypothyreosis and arrhythmia each in 3.96% (n=4). The number of the comorbidities was found to significantly increase with the duration of MS (r=0.232, p=0.037). Women were found to have significantly bigger numbers of comorbidities than men (t=-2.59, df=74, p<0.05). Older patients with MS were found to have significantly more comorbidities (r=0.335, p<0.01). CONCLUSIONS: This study gives insight into the presence of comorbidities in Croatian patients with MS. Connection with comorbidities must be considered when managing patients with MS. Any other comorbidity in MS may also affect the condition of the patient in general, and also their quality of life, and requires a tailored approach in management.

Vitamin D and Neurotrophin Levels and Their Impact on the Symptoms of Schizophrenia.

INTRODUCTION: Vitamin D is involved in brain development and functioning, as well as in regulation of neurotrophic factors. Changes in the expression of those factors are possibly responsible for morphologic abnormalities and symptoms in patients suffering from schizophrenia. OBJECTIVE: The main goal of this research was to investigate the interrelationship between vitamin D, nerve growth factors (NGF, brain-derived neurotrophic factor [BDNF], and neuregulin-1 [NRG1]), and schizophrenia symptom domains. METHODS: This research included 97 inpatients diagnosed with schizophrenia. Schizophrenia symptoms were assessed using the Positive and Negative Syndrome Scale (PANSS). Blood samples were taken in order to analyze concentrations of vitamin D, BDNF, NRG1, and NGF growth factors. The obtained results were used in a multiple regression analysis. RESULTS: The vitamin D concentration positively affected the concentration of NRG1 (F = 8.583, p = 0.005) but not the concentration of other investigated growth factors (BDNF and NGF). The clinical characteristics and symptom domains of schizophrenia seemed to be unaffected by the concentrations of vitamin D, BDNF, and NGF, while the NRG1 concentration significantly affected positive symptom domains of schizophrenia (F = 4.927, p = 0.030). CONCLUSION: The vitamin D concentration positively affected NRG1 levels but not schizophrenia symptomatology as measured by PANSS. The as-sociation between the two could be intermediated via NRG1.

Lower Urinary Tract Symptoms and Depression in Patients with Multiple Sclerosis.

BACKGROUND: Both depression and lower urinary tract symptoms (LUTS) may be present in patients with multiple sclerosis (MS). The objective of this study was to give an insight on depression and LUTS in patients with MS in Croatia and to determine the possible association between LUTS and depression in patients with MS. SUBJECTS AND METHODS: This was a prospective cross-sectional study conducted in a tertiary healthcare center in Croatia. Hundred and one consecutive patients with MS (75 female, 26 male, mean age 42.09 (range 19-77) years, mean Expanded Disability Status Scale (EDSS) score 3.1 (range 0.0-7.0)) participated in this study. We evaluated LUTS and related quality of life (QoL) using three International Consultation on Incontinence Questionnaires (ICIQ) enquiring about overactive bladder (ICIQ-OAB), urinary incontinence short form (ICIQ-UI SF) and lower urinary tract symptoms related quality of life (ICIQLUTS-QoL). ICIQ-OAB and ICIQLUTS-QoL were for this purpose with permission successfully translated and validated into Croatian, while ICIQ-UI SF was already previously validated for the Croatian language. Information regarding treatment for depression was obtained during the medical interview. Data were analyzed and interpreted using IBM SPSS Statistics for Windows, version 23.0 (IBM Corp., Armonk, N.Y., USA). RESULTS: 89.10% (N=90) patients with MS reported urgency with urge urinary incontinence (UUI) present in 70.29% (N=71). 81.18% (N=82) patients reported nocturia, and 90.09% (N=91) reported feeling drowsy or sleepy during the day due to bladder symptoms. Neurological deficit measured by EDSS was found to positively correlate with LUTS on all three questionnaires: ICIQ-OAB (r=0.390, p<0.05), ICIQ-UI SF (r=0.477, p<0.01) and ICIQ-LUTSQoL (r=0.317, p<0.05). 25 patients were in treatment for depression. There were no significant differences between female and male patients regarding treatment for depression (χ2=0.018, df=1, p>0.05). Results on ICIQ-UI SF showed that depressive patients had more pronounced LUTS (t=2.067, df=99, p<0.05), which was also true for the ICIQ-LUTSQoL (t=-2.193, df=99, p<0.05). Positive correlations were found between depression and LUTS on ICIQ-UI SF (r=0.203, p<0.05) and ICIQ-LUTSQoL (r=0.215, p<0.05). CONCLUSION: This study gives insight into the presence of depression and LUTS in Croatian patients with MS for which purpose ICIQ-OAB and ICIQ-LUTSQoL were with permission successfully translated and validated into Croatian. The connection between depression and LUTS must be considered when managing patients with MS.

EIGHTY YEARS OF ELECTROCONVULSIVE THERAPY IN CROATIA AND IN SESTRE MILOSRDNICE UNIVERSITY HOSPITAL CENTRE.

In 1937, Ugo Cerletti and Lucio Bini performed electroconvulsive treatment (ECT) in Rome for the first time. That was the time when different types of 'shock therapy' were performed; beside ECT, insulin therapies, cardiazol shock therapy, etc. were also performed. In 1938, Cerletti and Bini reported the results of ECT. Since then, this method has spread rapidly to a large number of countries. As early as 1940, just two years after the results of the ECT had been published, it was also introduced in Croatia, at Sestre milosrdnice Hospital, for the first time in our hospital and in the then state of Yugoslavia. Since 1960, again the first in Croatia and the state, we performed ECT in general anesthesia and continued it down to the present, with a single time brake.

Sexual Dysfunction and Depression in Patients with Multiple Sclerosis in Croatia.

BACKGROUND: Both depression and sexual dysfunction (SD) may be present in patients with multiple sclerosis (MS). OBJECTIVE: The aim of this study was to evaluate a possible association between SD and depression in patients with MS in Croatia. SUBJECTS AND METHODS: This was a prospective cross-sectional study carried out in tertiary healthcare centre over 10 months, which included 101 consecutive pwMS (mean age 42.09 (range 19-77) years, 75 female, 26 male, EDSS score 3.1 (range 0.0-7.0)). SD was assessed using Multiple Sclerosis Intimacy and Sexuality Questionnaire (MSISQ), which was for this purpose successfully translated and validated into Croatian. Information on treatment for depression was obtained during the medical interview. Data were analysed and interpreted using parametric statistics (IBM Corp. Released 2015. IBM SPSS Statistics for Windows, Version 23.0. Armonk, NY: IBM Corp.). RESULTS: 89 patients completed MSISQ. 25 patients were in treatment for depression, while 75 did not have depressive symptoms. On MSISQ 57 (43 women, 14 men) patients had responded with 'almost always/ always' suggestive of SD. Majority of patients reported primary SD, followed by secondary and tertiary SD. Most difficulties were found regarding difficulty in getting or keeping a satisfactory erection (34.6% (N=9) men), followed by 32.9% (N=27) reporting that it takes too long to orgasm or climax, followed with bladder or urinary symptoms in 32.6% (N=29). There were no significant differences between female and male patients regarding treatment for depression (χ2=0.018, df=1, p>0.05). Results in all subcategories on t-test found that depressive patients had higher impact on SD when compared to non-depressive: overall (t=-2.691, df=87, p<0.01) and in regards to primary (t=-2.086, df=87, p<0.05), secondary (t=-2.608, df=87, p<0.05) and tertiary (t=-2.460, df=86, p<0.05) SD. Depressive patients on 7 questions showed significantly (p<0.05) higher SD symptoms: Muscle tightness or spasms in my arms, legs, or body; Tremors or shaking in hands or body; Pain, burning, or discomfort in their body; Feeling less attractive; Fear of being rejected sexually because of MS; Lack of sexual interest or desire; Less intense or pleasurable orgasms or climaxes. CONCLUSIONS: This study gives insight into the presence of depression and SD in Croatian patients with MS for which purpose valid questionnaire for the assessment of SD in MS patients MSISQ was with permission successfully translated and validated into Croatian. The connection between depression and SD must be considered when managing patients with MS.

Clinical trials in developing countries - ethical considerations.

When designing clinical trial or considering decision to take part in particular clinical trial as investigators, even before submission to responsible Central Ethic Committee, we always make certain private assessment about ethical justification of this clinical trial. When making assessment if any clinical trial is ethically justifiable, there should make no difference in which country this clinical trial will be executed. Physicians coming from developing countries must ensure that patient population of developing countries is not misused in any ethically questionable clinical trial. There must be careful assessment of clinical protocols by various independent local advisory committees (e.g. hospital review boards, hospital drug committees, hospital administration and whatever is applicable) to exclude the possibility that only one person or one group of people has concentrated power to make decisions for entire country. Many times physicians/clinical researchers from developing countries are faced with the criticisms that they are not of the same quality as physicians from developed countries and that they can be easily bribed by sponsors, which are based on the prejudice that any clinical trial can be executed in developing countries, no matter of quality or risks for patients. Physicians coming from developing countries must ensure that patient population of developing countries is not misused in any ethically questionable clinical trial.

Increased calcium-independent lipoprotein phospholipase A2 but not protein S100 in patients with schizophrenia.

BACKGROUND: The aim of this paper was to investigate serum concentrations of calcium-independent lipoprotein phospholipase A2 (PLA2) and protein S100 in schizophrenia patients in comparison to healthy controls and correlate them with the clinical severity, duration, and number of schizophrenia relapses. SUBJECTS AND METHODS: This study included 65 schizophrenia patients and 70 controls. Schizophrenia was diagnosed according to DSM-IV-TR criteria. Clinical severity was determined by PANSS. PLA2 and protein S100 concentration were assessed by the enzyme-linked immunosorbent assay (ELISA). RESULTS: PLA2 concentrations were higher in patients with schizophrenia, whereas protein S100 concentrations were not. Higher concentrations of PLA2 were positively correlated with the duration of illness and number of episodes, as determined by multivariate analysis. CONCLUSION: PLA2 might be considered a possible biochemical trait marker for schizophrenia. Further research with larger and more homogeneous clinical samples is required.

Association between PLA2 gene polymorphisms and treatment response to antipsychotic medications: A study of antipsychotic-naïve first-episode psychosis patients and nonadherent chronic psychosis patients.

Here we investigated whether antipsychotic treatment was influenced by three polymorphisms: rs10798059 (BanI) in the phospholipase A2 (PLA2)G4A gene, rs4375 in PLA2G6, and rs1549637 in PLA2G4C. A total of 186 antipsychotic-naïve first-episode psychosis patients or nonadherent chronic psychosis individuals (99 males and 87 females) were genotyped by polymerase chain reaction analysis/restriction fragment length polymorphism. At baseline, and after 8 weeks of treatment with various antipsychotic medications, we assessed patients' Positive and Negative Syndrome Scale (PANSS) scores, PANSS factors, and metabolic syndrome-related parameters (fasting plasma lipid and glucose levels, and body mass index). We found that PLA2G4A polymorphism influenced changes in PANSS psychopathology, and PLA2G6 polymorphism influenced changes in PANSS psychopathology and metabolic parameters. PLA2G4C polymorphism did not show any impact on PANSS psychopathology or metabolic parameters. The polymorphisms' effect sizes were estimated as moderate to strong, with contributions ranging from around 6.2-15.7%. Furthermore, the polymorphisms' effects manifested in a gender-specific manner.

The Impact of Long-acting Paliperidone in Reducing Hospitalizations and Clinical Severity in Recent Onset Schizophrenia: A Mirror-image Study in Real-world Clinical Setting.

OBJECTIVE: Schizophrenia is a debilitating disease that disrupts the lives of many affected individuals and exerts a toll on the health system. Only few studies assessed once-monthly injectable formulation of paliperidone palmitate (PP-1M) and other long-acting antipsychotics in recent onset schizophrenia (ROS). To evaluate whether PP-1M is efficacious in reducing frequency and length of hospitalizations and psychosis symptom severity in patients with ROS. METHODS: This mirror-image study included 112 patients, suffering from ROS admitted in a psychiatric ward and successively treated with PP-1M for 1-year. Other psychotic disorders were excluded. We collected socio-demographic data of all subjects included, number and days of hospitalization, as well as Clinical Global Impression-Severity scale (CGI-S) and Clinician-Rated Dimensions of Psychosis Symptom Severity (CRDPSS) scores at the initiation and after 1-year of PP treatment. RESULTS: After 1-year PP-1M treatment, mean scores of both CGI and CRDPSS significantly decreased (p < 0.001), as well as the mean number of hospitalizations (p = 0.002) and total hospitalization days (p < 0.001) in comparison with those of the previous year. CONCLUSION: Our results suggest that PP-1M can be considered as an important therapeutic option in patients with ROS. Its use led to a meaningful reduction in the patient's use of hospital services, as well as a significant clinical improvement of psychotic symptoms in our sample.

Impairment of proprioception after whiplash injury.

Whiplash injury usually occurs in traffic accidents. Persons experienced this injury might have an impairment of proprioception clinically expressed as inability to determine the exact position of their heads. The aim of this study was to examine the loss of proprioception in people who had a whiplash injury. The study included 60 subjects with cervical spine injury, aged 20 to 50 years and 60 healthy volunteers matched by sex and age. The instrument used for cervical spine mobility assessment was the Cervical Measurement System (CMS), which determines the ability of subjects to return their head in the exact position as it was before they turned it 30 degrees left or right. Patients with cervical spine injury showed significant impairment of proprioception in comparison with healthy subjects (P < 0.001). The results support the hypothesis that subject with recent cervical spine injury have incorrect perception of their head position. Therefore, their rehabilitation should include the correction of proprioception and head coordination.

NEUROBIOLOGICAL BASES OF ALCOHOL ADDICTION.

Alcohol addiction is a heterogeneous psychiatric disorder according to both phenotype and etiology. Difference in phenotype characteristics manifests in the manner the addiction arises, history of the alcoholic and history of drinking, comorbid disorders, and the phenomenon of abstinence difficulties. Concerning the etiology of alcoholism, the disease itself is considered to be a consequence of an interactive influence of the environment and genetic factors. Numerous researches conducted in the last decades discovered many aspects of the biochemical, cell and molecular bases of alcohol addiction, leading to a conclusion that alcoholism is, like many other addictions, a brain disease. By recognizing alcoholism as a disease which basically implies changes of the neurobiological mechanisms, as well as a clear genetic basis, it was supposed that the disease, having its basis solely in the symptomatology, is essentially heterogeneous. By trying to solve the problem of a clinically heterogeneous nature of the disease during the last fifty years, various sub-classifications of such patients have been suggested. According to Cloninger, subtypes of alcoholism differ also according to changes in the brain neurotransmission systems, i.e. it is supposed that patients suffering from alcoholism type 1 have a more pronounced dopaminergic transmission deficit, while dopaminergic transmission is not disturbed significantly in patients diagnosed with alcoholism type 2, who, however, have a significant lack of serotonergic transmission. In such a way, Cloninger actually presented the basis of the so-called neurobiological alcoholism model. Since he has connected differences in neurotransmission with differences in personality characteristics, this model is also known as the psychobiological model of alcoholism. The characteristic of alcoholism type 1 is avoiding damage (Harm Avoidance, HA) decreased dopamine transmission and increased serotonin transmission, while the significant characteristic of alcoholism type 2 is seeking for excitement (Novelty Seeking, NS), unchanged dopamine transmission and decreased serotonin transmission. These neurochemical differences among alcoholism subtypes represent the basis for a different therapy approach. Intake of alcohol changes different gene expression in the human brain. The inheritance model of alcoholism is not fully explained, however, it is considered that the disease is connected to a larger gene number included in neurotransmission, cell mechanisms and general metabolic function, with a simultaneous influence of the environment. The contribution of genetic factors is stronger in certain types of alcoholism and thus we have been confronted in the last years of alcoholism research with studies researching the connections of some alcoholism subtypes with the polymorphism phenomenon in the genes coding the synaptic proteins included in the alcoholism etiology. The primary role of monoamine oxidase (MAO) in the brain is catalysis of deamination of the oxidative neurotransmitter amines, i.e. serotonin, adrenaline, noradrenaline and dopamine. Thus, this enzyme is the key factor for maintaining cytoplasmic concentration of various neurotransmitters and for regulation of the neurotransmitting synaptic activity. Taken this MAO function into consideration, MAO is the enzyme included in the etiology and pathogenesis of various neuropsychiatric and neurological disorders. The finding of the decreased platelet MAO activity in various psychiatric disorders has brought us to the assumption that this enzyme may be a constitutional/genetic indicator (trait marker) or an indicator of disease condition (state marker) in biologic psychiatry. There are only a few studies of alcohol addiction researching the connections of the MAO coding gene polymorphism and alcoholism; however, these studies are primarily related to the variable number of tandem repeats (VTNR) polymorphism in the regulatory gene region for MAO-A, considered to influence the transcription activity/functionality of the enzyme.

Platelet serotonin concentration and depressive symptoms in patients with schizophrenia.

Depressive symptoms seem to be frequent in schizophrenia, but so far they have received less attention than other symptom domains. Impaired serotonergic neurotransmission has been implicated in the pathogenesis of depression and schizophrenia. The objectives of this study were to investigate platelet serotonin concentrations in schizophrenic patients with and without depressive symptoms, and to investigate the association between platelet serotonin concentrations and symptoms of schizophrenia, mostly depressive symptoms. A total of 364 patients were included in the study, 237 of which had significant depressive symptoms. Significant depressive symptoms were defined by the cut-off score of 7 or more on Calgary Depression Rating Scale (CDSS). Platelet serotonin concentrations were assessed by the enzyme-linked immunosorbent assay (ELISA). Prevalence of depression in patients with schizophrenia was 65.1%. Schizophrenic patients with depressive symptoms showed lower platelet serotonin concentrations (mean±SD; 490.6±401.2) compared to schizophrenic patients without depressive symptoms (mean±SD; 660.9±471.5). An inverse correlation was established between platelet serotonin concentration and depressive symptoms, with more severe symptoms being associated with lower platelet serotonin concentrations. Depressive symptoms in schizophrenic patients may be associated with reduced concentrations of platelet serotonin.

The dissections of craniocervical arteries.

Dissection of craniocervical arteries internal carotid artery (ICA), or vertebral artery (VA) is an increasingly recognized entity and infrequent cause of stroke. We investigated 8 patients (4 women and 4 men) with dissections of the craniocervical arteries. Diagnostic procedures for detection of craniocervical dissection included: extracranial ultrasound-color Doppler flow imaging (CDFI) of carotid and vertebral arteries, transcranial Doppler sonography (TCD) and radiological computed tomography (CT) and digital subtractive angiography (DSA) examinations. Ultrasound findings (CDFI of carotid and vertebral arteries) were positive for vessel dissection in seven patients (or 87.5 per cent) and negative in one patient. DSA was consistent with dissection in five patients (or 62.5 per cent), negative in one, while in two patients the examination was not performed due to known allergy to contrast media. Five patients (62.5 per cent) were treated with anticoagulants, one with suppressors of platelet aggregation, and two patients were operated. Six patients (75 per cent) after the treatment showed partial recovery of neurological defects, and an improvement of ultrasound finding of dissected arteries. In one patient, following operation, stroke developed with deterioration of motor deficit, and one patient was readmitted three months later due to a newly developed stroke and soon died. The diagnosis should be suspected in any young or middle-age patient with new onset of otherwise unexplained unremitting headache or neck ache, especially in association with transient or permanent focal neurological deficits.

Arachnoid cyst as the cause of bipolar affective disorder: case report.

This report presents the course of diagnostic examinations and treatment of a 20-year-old man with bipolar affective disorder for which an organic basis was demonstrated. Computed tomography of the brain revealed an arachnoid cyst that was surgically treated. The patient underwent both psychiatric and neurosurgical treatment. After two-year follow-up and medicamentous treatment prescribed, the patient was symptom-free requiring no psychopharmacotherapy for the next 5.5 years. His overall life functioning is normal, with no signs of disease.

A review of the psychoneuroimmunologic concepts on the etiology of depressive disorders.

The brain is no longer considered an immunoprivileged organ which is completely separated from the circulating immune cells by the blood-brain barrier and which shows a lowered or changed immunoreactivity. It has become clear that there are numerous interactions between the neurological, immune and neuroendocrinologic systems. The psychiatric disorder which is supposed to be connected to changes in the functioning of the immune system is depression. One of the hypotheses suggesting the pathophysiology of depression is the cytokine hypothesis of depression. According to it, the behavior changes in depressed patients are a consequence of changes in cytokines. Physiological and psychological effects of the immune activation during an infection, primarily mediated by central activity of peripherally excreted proinflammatory cytokines, are called "sickness behavior". Depression is connected with the activation of the inflammatory response system. When it comes to the immune characteristics of depressive disorders, it should be stressed that depression is a heterogeneous disorder, so different types of depression can differ not only psychopathologically but also at the immune level. Depression is characterized by disorders in noradrenergic and serotonergic neurotransmission. Proinflammatory cytokines are included in the noradrenergic and serotonergic neurotransmission in the brain areas that are thought to be involved in the pathogenesis of depression. According to this model, depression can be considered a psychoneuroimmune disease in which the peripheral immune activation is responsible (by excreting the inflammatory mediator) for various behavioral, neuroendocrinologic and neurochemical changes connected to the psychiatric condition.

Treatment of multiple sclerosis.

Multiple sclerosis is an autoimmune inflammatory demyelinating disease of the central nervous system, characterized by multifocal inflammatory destruction of myelin, axonal damage and loss of oligodendrocytes. The disease is carried through two stages: inflammatory and degenerative. The most common form of disease in approximately 85% of the cases is RRMS (relapsing-remitting form). The treatment of MS is devided into: treatment of the acute phase of illness, prevention of new relapses and disease progression, and symptomatic treatment. Most of the changes in treatment of multiple sclerosis and most of the news in recent years concerning new drugs are used in the treatment of progression of the disease and prevention of disease relapses. Some of these drugs are registrated in most Europian countries and USA, and others are in various stages of research.