Adverse drug reactions caused by drug-drug interactions reported to Croatian Agency for Medicinal Products and Medical Devices: a retrospective observational study

Aim

To analyze potential and actual drug-drug interactions reported to the Spontaneous Reporting Database of the Croatian Agency for Medicinal Products and Medical Devices (HALMED) and determine their incidence.

Methods

In this retrospective observational study performed from March 2005 to December 2008, we detected potential and actual drug-drug interactions using interaction programs and analyzed them.

Results

HALMED received 1209 reports involving at least two drugs. There were 468 (38.7%) reports on potential drug-drug interactions, 94 of which (7.8% of total reports) were actual drug-drug interactions. Among actual drug-drug interaction reports, the proportion of serious adverse drug reactions (53 out of 94) and the number of drugs (n = 4) was significantly higher (P < 0.001) than among the remaining reports (580 out of 1982; n = 2, respectively). Actual drug-drug interactions most frequently involved nervous system agents (34.0%), and interactions caused by antiplatelet, anticoagulant, and non-steroidal anti-inflammatory drugs were in most cases serious. In only 12 out of 94 reports, actual drug-drug interactions were recognized by the reporter.

Conclusion

The study confirmed that the Spontaneous Reporting Database was a valuable resource for detecting actual drug-drug interactions. Also, it identified drugs leading to serious adverse drug reactions and deaths, thus indicating the areas which should be in the focus of health care education.Adverse drug reactions (ADR) are among the leading causes of mortality and morbidity responsible for causing additional complications (1,2) and longer hospital stays. Magnitude of ADRs and the burden they place on health care system are considerable (3-6) yet preventable public health problems (7) if we take into consideration that an important cause of ADRs are drug-drug interactions (8,9). Although there is a substantial body of literature on ADRs caused by drug-drug interactions, it is difficult to accurately estimate their incidence, mainly because of different study designs, populations, frequency measures, and classification systems (10-15).Many studies including different groups of patients found the percentage of potential drug-drug interactions resulting in ADRs to be from 0%-60% (10,11,16-25). System analysis of ADRs showed that drug-drug interactions represented 3%-5% of all in-hospital medication errors (3). The most endangered groups were elderly and polimedicated patients (22,26-28), and emergency department visits were a frequent result (29). Although the overall incidence of ADRs caused by drug-drug interactions is modest (11-13,15,29,30), they are severe and in most cases lead to hospitalization (31,32).Potential drug-drug interactions are defined on the basis of on retrospective chart reviews and actual drug-drug interactions are defined on the basis of clinical evidence, ie, they are confirmed by laboratory tests or symptoms (33). The frequency of potential interactions is higher than that of actual interactions, resulting in large discrepancies among study findings (24).A valuable resource for detecting drug-drug interactions is a spontaneous reporting database (15,34). It currently uses several methods to detect possible drug-drug interactions (15,29,35,36). However, drug-drug interactions in general are rarely reported and information about the ADRs due to drug-drug interactions is usually lacking.The aim of this study was to estimate the incidence of actual and potential drug-drug interactions in the national Spontaneous Reporting Database of ADRs in Croatia. Additionally, we assessed the clinical significance and seriousness of drug-drug interactions and their probable mechanism of action.     

Zagreb Amblyopia Preschool Screening Study: near and distance visual acuity testing increase the diagnostic accuracy of screening for amblyopia

AimTo present and evaluate a new screening protocol for amblyopia in preschool children.MethodsZagreb Amblyopia Preschool Screening (ZAPS) study protocol performed screening for amblyopia by near and distance visual acuity (VA) testing of 15 648 children aged 48-54 months attending kindergartens in the City of Zagreb County between September 2011 and June 2014 using Lea Symbols in lines test. If VA in either eye was >0.1 logMAR, the child was re-tested, if failed at re-test, the child was referred to comprehensive eye examination at the Eye Clinic.Results78.04% of children passed the screening test. Estimated prevalence of amblyopia was 8.08%. Testability, sensitivity, and specificity of the ZAPS study protocol were 99.19%, 100.00%, and 96.68% respectively.ConclusionThe ZAPS study used the most discriminative VA test with optotypes in lines as they do not underestimate amblyopia. The estimated prevalence of amblyopia was considerably higher than reported elsewhere. To the best of our knowledge, the ZAPS study protocol reached the highest sensitivity and specificity when evaluating diagnostic accuracy of VA tests for screening. The pass level defined at ≤0.1 logMAR for 4-year-old children, using Lea Symbols in lines missed no amblyopia cases, advocating that both near and distance VA testing should be performed when screening for amblyopia.Vision disorders in children represent important public health concern as they are acknowledged to be the leading cause of handicapping conditions in childhood (1). Amblyopia, a loss of visual acuity (VA) in one or both eyes (2) not immediately restored by refractive correction (3), is the most prevalent vision disorder in preschool population (4). The estimated prevalence of amblyopia among preschool children varies from 0.3% (4) to 5% (5). In addition, consequences of amblyopia include reduced contrast sensitivity and/or positional disorder (6). It develops due to abnormal binocular interaction and foveal pattern vision deprivation or a combination of both factors during a sensitive period of visual cortex development (7). Traversing through adulthood, it stands for the leading cause of monocular blindness in the 20-70 year age group (8). The main characteristic of amblyopia is crowding or spatial interference, referring to better VA when single optotypes are used compared to a line of optotypes, where objects surrounding the target object deliver a jumbled percept (9-12). Acuity is limited by letter size, crowding is limited by spacing, not size (12).Since amblyopia is predominantly defined as subnormal VA, a reliable instrument for detecting amblyopia is VA testing (13-15). Moreover, VA testing detects 97% of all ocular anomalies (13). The gold standard for diagnosing amblyopia is complete ophthalmological examination (4). There is a large body of evidence supporting the rationale for screening, as early treatment of amblyopia during the child’s first 5-7 years of life (8) is highly effective in habilitation of VA, while the treatment itself is among the most cost-effective interventions in ophthalmology (16). Preschool vision screening meets all the World Health Organization’s criteria for evaluation of screening programs (17). Literature search identified no studies reporting unhealthy and damaging effects of screening. The gold standard for screening for amblyopia has not been established (4). There is a large variety of screening methodologies and inconsistent protocols for referral of positives to complete ophthalmological examination. Lack of information on the validity (18,19) and accuracy (4) of such protocols probably intensifies the debate on determining the most effective method of vision screening (8,20-29). The unique definition of amblyopia accepted for research has not reached a consensus (4,5,30,31), further challenging the standardization of the screening protocols.Overall, two groups of screening methods exist: the traditional approach determines VA using VA tests, while the alternative approach identifies amblyogenic factors (27) based on photoscreening or automated refraction. The major difference between the two is that VA-based testing detects amblyopia directly, providing an explicit measure of visual function, while the latter, seeking for and determining only the level of refractive status does not evaluate visual function. In addition, the diagnosis and treatment of amblyopia is governed by the level of VA. On the other hand, amblyogenic factors represent risk factors for amblyopia to evolve. There are two major pitfalls in screening for amblyogenic factors. First, there is a lack of uniform cut-off values for referral and second, not all amblyogenic factors progress to amblyopia (19).Besides the issue of what should be detected, amblyopia or amblyogenic factors, a question is raised about who should be screened. Among literate children, both 3- and 4- year-old children can be reliably examined. However, 3-year-old children achieved testability rate of about 80% and positive predictive rate of 58% compared to >90% and 75%, respectively in the 4-year-old group (32). In addition, over-referrals are more common among 3-year-old children (32). These data determine the age of 4 years as the optimum age to screen for amblyopia. Hence, testability is a relevant contributor in designating the optimal screening test.If VA is to be tested in children, accepted standard tests should be used, with well-defined age-specific VA threshold determining normal monocular VA. For VA testing of preschool children Lea Symbols (33) and HOTV charts (22,32) are acknowledged as the best practice (34), while tumbling E (28,35,36) and Landolt C (28,37-39) are not appropriate as discernment of right-left laterality is still not a fully established skill (34,40). The Allen picture test is not standardized (34,41). Both Lea Symbols and HOTV optotypes can be presented as single optotypes, single optotypes surrounded with four flanking bars, single line of optotypes surrounded with rectangular crowding bars, or in lines of optotypes (22,33,34,41-53). The more the noise, the bigger the “crowding” effect. Isolated single optotypes without crowding overestimate VA (24), hence they are not used in clinical practice in Sweden (32). If presented in lines, which is recognized as the best composition to detect crowding, test charts can be assembled on Snellen or gold standard logMAR principle (34,42,51,54). Age-specific thresholds defining abnormal VA in preschool screening for amblyopia changed over time from <0.8 to <0.65 for four-year-old children due to overload of false positives (20).The outline of an effective screening test is conclusively demonstrated by both high sensitivity and high specificity. Vision screening tests predominately demonstrated higher specificity (4). Moreover, sensitivity evidently increased with age, whereas specificity remained evenly high (4). The criteria where to set the cut-off point if the confirmatory, diagnostic test is expensive or invasive, advocate to minimize false positives or use a cut-off point with high specificity.On the contrary, if the penalty for missing a case is high and treatment exists, the test should maximize true positives and use a cut-off point with high sensitivity (55). A screening test for amblyopia should target high sensitivity to identify children with visual impairment, while the specificity should be high enough not to put immense load on pediatric ophthalmologists (14). Complete ophthalmological examination as the diagnostic confirmatory gold standard test for amblyopia is neither invasive nor elaborate technology is needed, while the penalty for missing a case is a lifetime disability.In devising the Zagreb Amblyopia Preschool Screening (ZAPS) study protocol, we decided to use Lea Symbols in lines test and to screen preschool children aged 48-54 months to address the problems declared. Near VA testing was introduced in addition to commonly accepted distance VA testing (14,22,24,32,45,56-69) due to several reasons: first, hypermetropia is the most common refractive error in preschool children (70), hence near VA should more reliably detect the presence of hypermetropia; second, the larger the distance, the shorter the attention span is; and third, to increase the accuracy of the test.The pass cut-off level of ≤0.1 logMAR was defined because of particular arguments. Prior to 1992 Sweden used the pass cut-off level for screening of 0.8 (20). A change in the referral criteria to <0.65 for four-year-old children ensued, as many children referred did not require treatment (20). In addition, amblyopia treatment outcome of achieved VA>0.7 is considered as habilitation of normal vision (3,14). At last, the pass cut-off value ≤0.1 logMAR at four years can hardly mask serious visual problems, and even if they are present, we presume they are mild and can be successfully treated at six years when school-entry vision screening is performed. The aim of the ZAPS study is to present and evaluate new screening protocol for preschool children aged 48-54 months, established for testing near and distance VA using Lea Symbols in lines test. Furthermore, we aimed to determine the threshold of age-specific and chart-specific VA normative, testability of the ZAPS study protocol, and the prevalence of amblyopia in the City of Zagreb County. By delivering new evidence on amblyopia screening, guideline criteria defining optimal screening test for amblyopia in preschool children can be revised in favor of better visual impairment clarification.     

Pharmacotherapy of treatment-resistant combat-related posttraumatic stress disorder with psychotic features

Aim

To assess retrospectively the clinical effects of typical (fluphenazine) or atypical (olanzapine, risperidone, quetiapine) antipsychotics in three open clinical trials in male Croatian war veterans with chronic combat-related posttraumatic stress disorder (PTSD) with psychotic features, resistant to previous antidepressant treatment.

Methods

Inpatients with combat-related PTSD were treated for 6 weeks with fluphenazine (n = 27), olanzapine (n = 28) risperidone (n = 26), or quetiapine (n = 53), as a monotherapy. Treatment response was assessed by the reduction in total and subscales scores in the clinical scales measuring PTSD (PTSD interview and Clinician-administered PTSD Scale) and psychotic symptoms (Positive and Negative Syndrome Scale).

Results

After 6 weeks of treatment, monotherapy with fluphenazine, olanzapine, risperidone, or quetiapine in patients with PTSD significantly decreased the scores listed in trauma reexperiencing, avoidance, and hyperarousal subscales in the clinical scales measuring PTSD, and total and subscales scores listed in positive, negative, general psychopathology, and supplementary items of the Positive and negative syndrome scale subscales, respectively (P<0.001).

Conclusion

PTSD and psychotic symptoms were significantly reduced after monotherapy with typical or atypical antipsychotics. As psychotic symptoms commonly occur in combat-related PTSD, the use of antipsychotic medication seems to offer another approach to treat a psychotic subtype of combat-related PTSD resistant to previous antidepressant treatment.In a world in which terrorism and conflicts are constant threats, and these threats are becoming global, posttraumatic stress disorder (PTSD) is a serious and global illness. According to the criteria from the 4th edition of Diagnostic and Statistical Manual of Mental Disorders (DSM-IV) (1), exposure to a life-threatening or horrifying event, such as combat trauma, rape, sexual molestation, abuse, child maltreatment, natural disasters, motor vehicle accidents, violent crimes, hostage situations, or terrorism, can lead to the development of PTSD (1,2). The disorder may also be precipitated if a person experienced, saw, or learned of an event or events that involved actual or threatened death, serious injury, or violation of the body of self or others (3,4). In such an event, a person’s response can involve intense fear, helplessness, or horror (3,4). However, not all persons who are exposed to a traumatic event will develop PTSD. Although the stress reaction is a normal response to an abnormal situation, some extremely stressful situations will in some individuals overwhelm their ability to cope with stress (5).PTSD is a chronic psychiatric illness. The essential features of PTSD are the development of three characteristic symptom clusters in the aftermath of a traumatic event: re-experiencing the trauma, avoidance and numbing, and hyperarousal (1,6). The core PTSD symptoms in the re-experiencing cluster are intrusive memories, images, or perceptions; recurring nightmares; intrusive daydreams or flashbacks; exaggerated emotional and physical reactions; and dissociative experiences (1,6,7). These symptoms intensify or re-occur upon exposure to reminders of the trauma, and various visual, auditory, or olfactory cues might trigger traumatic memories (3,4). The avoidance and numbing cluster of symptoms includes efforts to avoid thoughts, feelings, activities, or situations associated with the trauma; feelings of detachment or alienation; inability to have loving feelings; restricted range of affect; loss of interest; and avoidance of activity. The hyperarousal cluster includes exaggerated startle response, hyper-vigilance, insomnia and other sleep disturbances, difficulties in concentrating, and irritability or outbursts of anger. PTSD criteria include functional impairment, which can be seen in occupational instability, marital problems, discord with family and friends, and difficulties in parenting (3,4,8). In addition to this social and occupational dysfunction, PTSD is often accompanied by substance abuse (9) and by various comorbid diagnoses, such as major depression (10), other anxiety disorders, somatization, personality disorders, dissociative disorders (7,11), and frequently with suicidal behavior (12). Combat exposure can precipitate a more severe clinical picture of PTSD, which may be complicated with psychotic features and resistance to treatment. War veterans with PTSD have a high risk of suicide, and military experience, guilt about combat actions, survivor guilt, depression, anxiety, and severe PTSD are significantly associated with suicide attempts (12).The pharmacotherapy treatment of PTSD includes the use of antidepressants, such as selective serotonin reuptake inhibitors (fluvoxamine, fluoxetine, sertraline, or paroxetine) as a first choice of treatment, tricyclic antidepressants (desipramine, amitriptyline, imipramine), monoamine oxidase inhibitors (phenelzine, brofaromine), buspirone, and other antianxiety agents, benzodiazepines (alprazolam), and mood stabilizers (lithium) (13-16). Although the pharmacotherapy of PTSD starts with antidepressants, in treatment-refractory patients a new pharmacological approach is required to obtain a response. In treatment-resistant patients, pharmacotherapy strategies reported to be effective include anticonvulsants, such as carbamazepine, gabapentine, topiramate, tiagabine, divalproex, lamotrigine (14,17); anti-adrenergic agents, such as clonidine (although presynaptic α2-adrenoceptor agonist, clonidine blocks central noradrenergic outflow from the locus ceruleus), propranolol, and prazosin (13,14), opiate antagonists (13), and neuroleptics and antipsychotics (14,17,18).Combat exposure frequently induces PTSD, and combat-related PTSD might progress to a severe form of PTSD, which is often refractory to treatment (19-21). Combat-related PTSD is frequently associated with comorbid psychotic features (11,14,17,19-21), while psychotic features add to the severity of symptoms in combat-related PTSD patients (19,22-24). These cases of a more severe subtype of PTSD, complicated with psychotic symptoms, require the use of neuroleptics or atypical antipsychotic drugs (14,17,25-27).After the war in Croatia (1991-1995), an estimated million people were exposed to war trauma and about 10 000 of the Homeland War veterans (15% prevalence) have developed PTSD, with an alarmingly high suicide rate (28). The war in Croatia brought tremendous suffering, not only to combat-exposed veterans and prisoners of war (29), but also to different groups of traumatized civilians in the combat zones, displaced persons and refugees, victims of terrorist attacks, civilian relatives of traumatized war veterans and terrorist attacks victims, and traumatized children and adolescents (30). Among Croatian war veterans with combat-related PTSD, 57-62% of combat soldiers with PTSD met criteria for comorbid diagnoses (8-11), such as alcohol abuse, major depressive disorder, anxiety disorders, panic disorder and phobia, psychosomatic disorder, psychotic disorders, drug abuse, and dementia. In addition to different comorbid psychiatric disorders, a great proportion of war veterans with combat-related PTSD developed psychotic features (8,11,25,26), which consisted of psychotic depressive and schizophrenia-like symptoms (suggesting prominent symptoms of thought disturbances and psychosis). Psychotic symptoms were accompanied by auditory or visual hallucinations and delusional thinking in over two-thirds of patients (25,26). Delusional paranoid symptoms occurred in 32% of patients (25,26). The hallucinations were not associated exclusively with the traumatic experience, while the delusions were generally paranoid or persecutory in nature (25,26). Although psychotic PTSD and schizophrenia share some similar symptoms, there are clear differences between these two entities, since PTSD patients still retain some insight into reality and usually do not have complete disturbances of affect (eg, constricted or inappropriate) or thought disorder (eg, loose associations or disorganized responses).This proportion of veterans with combat-related PTSD refractory to treatment (18-20) and with co-occurring psychotic symptoms requires additional pharmacological strategies, such as the use of neuroleptics (25) or atypical antipsychotics (14,17,26). Studies evaluating the use of antipsychotics in combat-related PTSD with psychotic features are scarce, and antipsychotics were frequently added to existing medication in the treatment of PTSD.In this study, we compared retrospectively the clinical effects of four antipsychotic drugs – a neuroleptic drug (fluphenazine) and three atypical antipsychotics (olanzapine, risperidone and quetiapine) – in treatment-resistant male war veterans with combat-related PTSD with psychotic features.     

Up-regulation of Synaptotagmin IV within amyloid plaque-associated dystrophic neurons in Tg2576 mouse model of Alzheimer’s disease

Aim

To investigate the involvement of the vesicular membrane trafficking regulator Synaptotagmin IV (Syt IV) in Alzheimer’s disease pathogenesis and to define the cell types containing increased levels of Syt IV in the β-amyloid plaque vicinity.

Methods

Syt IV protein levels in wild type (WT) and Tg2576 mice cortex were determined by Western blot analysis and immunohistochemistry. Co-localization studies using double immunofluorescence staining for Syt IV and markers for astrocytes (glial fibrillary acidic protein), microglia (major histocompatibility complex class II), neurons (neuronal specific nuclear protein), and neurites (neurofilaments) were performed in WT and Tg2576 mouse cerebral cortex.

Results

Western blot analysis showed higher Syt IV levels in Tg2576 mice cortex than in WT cortex. Syt IV was found only in neurons. In plaque vicinity, Syt IV was up-regulated in dystrophic neurons. The Syt IV signal was not up-regulated in the neurons of Tg2576 mice cortex without plaques (resembling the pre-symptomatic conditions).

Conclusions

Syt IV up-regulation within dystrophic neurons probably reflects disrupted vesicular transport or/and impaired protein degradation occurring in Alzheimer’s disease and is probably a consequence but not the cause of neuronal degeneration. Hence, Syt IV up-regulation and/or its accumulation in dystrophic neurons may have adverse effects on the survival of the affected neuron.The main pathological hallmarks of Alzheimer’s disease (AD) are the formation of amyloid plaques, neurofibrillary tangles, dystrophic neurites, and sometimes activation of glial cells in the brain (1,2). In the vicinity of amyloid plaques, neurons undergo dramatic neuropathological changes including metabolic disturbances such as altered energy metabolism, dysfunction of vesicular trafficking, neurite breakage, and disruption of neuronal connections (3-8).Synaptotagmin IV (Syt IV) is a protein involved in the regulation of membrane trafficking in neurons and astrocytes (9,10). In hippocampal neurons, it regulates brain-derived neurotrophic factor release (11) and is involved in hippocampus-dependent memory and learning (12,13). In astrocytes, it is implicated in glutamate release (10). Recent data show that Syt IV plays an important role in neurodegenerative processes (14). Syt IV expression could be induced by seizures, drugs, and brain injury. Its changes have been shown in several animal models of neurodegeneration (Parkinson’s disease, brain ischemia, AD) (14-25). However, the exact role of Syt IV in neurodegeneration is unknown.Our previous study showed that the expression of Syt IV mRNA and its protein in the hippocampus and cortex of Tg2576 mouse model for AD was increased in the tissue surrounding β-amyloid plaques (14). It is not clear whether Syt IV is expressed in astrocytes (10,26,27) or/and in neurons (28,29), ie, whether it regulates the release of pro- or anti-inflammatory cytokines from β-amyloid associated astrocytes or is involved in neuronal vesicular pathogenesis (5,30). Therefore, the present study aimed to determine the type of cells in which Syt IV up-regulation occurs.     

Uric Acid and Antioxidant Effects of Wine

The aim of this article is to review the role of uric acid in the context of antioxidant effects of wine and its potential implication to human health. We described and discussed the mechanisms of increase in plasma antioxidant capacity after consumption of moderate amounts of wine. Because this effect is largely contributed by acute elevation in plasma uric acid, we paid special attention to wine constituents and metabolic processes that are likely to be involved in uric acid elevation.The association between light-to-moderate wine consumption and risk reduction of cardiovascular diseases and/or reduction of all-cause mortality was confirmed by numerous epidemiological studies (1-7). Among other beneficial biological effects, it has been shown that wine can increase antioxidant capacity in humans (8-12) and reduce susceptibility of human plasma to lipid peroxidation (11,13). These effects of wine attracted significant research interest, as oxidative stress is implicated in the pathogenesis of various diseases such as cancer and cardiovascular and neurodegenerative diseases (14-19).     

The involvement of noradrenergic mechanisms in the suppressive effects of diazepam on the hypothalamic-pituitary-adrenal axis activity in female rats

Aim

To elucidate the involvement of noradrenergic system in the mechanism by which diazepam suppresses basal hypothalamic-pituitary-adrenal (HPA) axis activity.

Methods

Plasma corticosterone and adrenocorticotropic hormone (ACTH) levels were determined in female rats treated with diazepam alone, as well as with diazepam in combination with clonidine (α₂-adrenoreceptor agonist), yohimbine (α₂-adrenoreceptor antagonist), alpha-methyl-p-tyrosine (α-MPT, an inhibitor of catecholamine synthesis), or reserpine (a catecholamine depleting drug) and yohimbine.

Results

Diazepam administered in a dose of 2.0 mg/kg suppressed basal HPA axis activity, ie, decreased plasma corticosterone and ACTH levels. Pretreatment with clonidine or yohimbine failed to affect basal plasma corticosterone and ACTH concentrations, but abolished diazepam-induced inhibition of the HPA axis activity. Pretreatment with α-MPT, or with a combination of reserpine and yohimbine, increased plasma corticosterone and ACTH levels and prevented diazepam-induced inhibition of the HPA axis activity.

Conclusion

The results suggest that α₂-adrenoreceptors activity, as well as intact presynaptic noradrenergic function, are required for the suppressive effect of diazepam on the HPA axis activity.Benzodiazepines are used for their anxiolytic, sedative-hypnotic, muscle relaxant, and anticonvulsant properties in the treatment of a variety of neuropsychiatric disorders (1,2), including anxiety and depression, which are often related to disturbances in the activity of hypothalamic-pituitary-adrenal (HPA) axis (3,4). Although these drugs exert most of their pharmacological effects via γ-aminobutyric acid_A (GABA_A) receptors (5,6), benzodiazepine administration has been associated with alterations in neuroendocrine function both in experimental animals and humans (7-9). However, even after years of extensive studies, the complex mechanisms by which these widely used drugs produce their effects on the HPA axis are still not known.Although most of the previous studies have demonstrated that classical benzodiazepines such as diazepam decrease the HPA axis activity in stressful contexts (10-14), under basal conditions they have been shown to stimulate (9,11,15-18), inhibit (15,19-22), and not affect (17,23-25) the HPA axis activity. Such diverse results might be related to several factors such as the dose and gender (15,16,20,21,26-28), or may also be a consequence of the net effect of non-selective benzodiazepines on the various GABA_A receptor isoforms (9).Our previous studies demonstrated that while diazepam (1 mg/kg) produced no change in plasma corticosterone levels in male rats (15,20), it decreased basal levels of corticosterone in female rats (15,26). However, although diazepam inhibited the HPA axis activity of female rats following administration of lower doses (1 or 2 mg/kg) (15,20,21,26), it stimulated the HPA axis activity following administration of high doses (10 mg/kg) (15,16,26). Moreover, whereas the suppressive effect of the lower doses of diazepam (2.0 mg/kg) on the HPA axis activity in female rats involves the GABA_A receptor complex (21), increases in corticosterone levels by a higher dose of diazepam (10 mg/kg) do not involve the stimulation of GABA_A receptors (16). In addition, stimulatory effect of 10 mg/kg diazepam on the HPA axis activity in rats seems not to be mediated by the benzodiazepine/GABA/channel chloride complex or by peripheral benzodiazepine receptors, but rather by a cyclic adenosine monophosphate (AMP)-dependent mechanism (18).Since our previous results suggested that the effect of a high dose of diazepam on the activity of the HPA axis in female rats might be due to a blockade of α₂-adrenergic receptors (16), the aim of this study was to elucidate whether noradrenergic system also has a modulatory role in the inhibitory effect of 2.0 mg/kg diazepam on basal plasma adrenocorticotropic hormone (ACTH) and corticosterone levels in female rats.     

Use of concentrated bone marrow aspirate and platelet rich plasma during minimally invasive decompression of the femoral head in the treatment of osteonecrosis

The aim of this paper is to describe our surgical procedure for the treatment of osteonecrosis of the femoral head using a minimally invasive technique. We have limited the use of this procedure for patients with pre-collapse osteonecrosis of the femoral head (Ficat Stage I or II). To treat osteonecrosis of the femoral head at our institution we currently use a combination of outpatient, minimally invasive iliac crest bone marrow aspirations and blood draw combined with decompressions of the femoral head. Following the decompression of the femoral head, adult mesenchymal stem cells obtained from the iliac crest and platelet rich plasma are injected into the area of osteonecrosis. Patients are then discharged from the hospital using crutches to assist with ambulation. This novel technique was utilized on 77 hips. Sixteen hips (21%) progressed to further stages of osteonecrosis, ultimately requiring total hip replacement. Significant pain relief was reported in 86% of patients (n = 60), while the rest of patients reported little or no pain relief. There were no significant complications in any patient. We found that the use of a minimally invasive decompression augmented with concentrated bone marrow and platelet rich plasma resulted in significant pain relief and halted the progression of disease in a majority of patients.Osteonecrosis of the femoral head (ONFH) occurs when the cells of the trabecular bone and marrow in the femoral head spontaneously die, leading to fracture and collapse of the articular surface (1,2). In the US, every year ONFH occurs in 10 000-20 000 adults between the ages of 20 and 60 (1,3,4). Once collapse occurs, severe pain ensues, and the disease course rarely regresses (5-8). In order to halt disease progression and provide pain relief, 80% of patients suffering from ONFH will require a total hip arthroplasty (THA); typically at a younger age than patients undergoing a THA for osteoarthritis (9-11).Although ONFH is a common indication for THA, the etiology of the disease is still unknown (12,13). ONFH is thought to be a multifactorial disease, with patients reporting a history of exposure to one or more risk factors, including trauma to the hip, alcohol abuse, corticosteroid use, hemoglobinopathies, pregnancy, coagulopathies, organ transplant, chemotherapy, Caisson disease, HIV, and autoimmune conditions; however in some patients the risk factor remains unknown, and the disease is termed “idiopathic” ONFH (12-16). Recent studies looking at the gentics risks of ONFH have resulted in identifying an autosomal dominant mutation in collagen type II gene (COL2 A1 gene) (17); which has been associated with genetic polymorphisms in alcohol metabolizing enzymes and the drug transport proteins (18,19).If the disease course is recognized before collapse of the subchondral bone and cartilage, patients can be treated with core decompression of the femoral head including Ficat Stage I or II (12,20,21). This technique has been used for over four decades, however randomized control trials have failed to show that this procedure alone halts disease progression and collapse (4). Recently, concentrated bone marrow autograft has been used to augment the decompression site to attempt to repopulate the femoral head with human mesenchymal stem cells (hMSC) (13,22,23). This aim of this paper is to describe our surgical technique and early clinical results using autologous bone marrow concentrate with platelet rich plasma and a minimally invasive decompression for the treatment of ONFH.     

Attitudes of Slovenian family practice patients toward changing unhealthy lifestyle and the role of family physicians: cross-sectional study

Aim

To assess patients’ attitudes toward changing unhealthy lifestyle, confidence in the success, and desired involvement of their family physicians in facilitating this change.

Methods

We conducted a cross-sectional study in 15 family physicians’ practices on a consecutive sample of 472 patients (44.9% men, mean age  [± standard deviation] 49.3 ± 10.9 years) from October 2007 to May 2008. Patients were given a self-administered questionnaire on attitudes toward changing unhealthy diet, increasing physical activity, and reducing body weight. It also included questions on confidence in the success, planning lifestyle changes, and advice from family physicians.

Results

Nearly 20% of patients planned to change their eating habits, increase physical activity, and reach normal body weight. Approximately 30% of patients (more men than women) said that they wanted to receive advice on this issue from their family physicians. Younger patients and patients with higher education were more confident that they could improve their lifestyle. Patients who planned to change their lifestyle and were more confident in the success wanted to receive advice from their family physicians.

Conclusion

Family physicians should regularly ask the patients about the intention of changing their lifestyle and offer them help in carrying out this intention.Unhealthy lifestyle, including unhealthy diet and physical inactivity, is still a considerable health problem all over the world. Despite publicly available evidence about the health risks of unhealthy lifestyle, people still find it hard to improve their unhealthy diet and increase physical activity. Previous studies have shown that attitudes toward lifestyle change depended on previous health behavior, awareness of unhealthy lifestyle, demographic characteristics, personality traits, social support, family functioning, ongoing contact with health care providers, and an individual’s social ecology or network (1-4).As community-based health education approaches have had a limited effect on health risk factors reduction (3,5), the readiness-to-change approach, based on two-way communication, has become increasingly used with patients who lead an unhealthy lifestyle (3,6,7). Family physicians are in a unique position to adopt this approach, since almost every patient visits his/hers family physician at least once in five years (8). Previous studies showed that patients highly appreciated their family physicians’ advice on lifestyle changes (9,10). Moreover, patients who received such advice were also more willing to change their unhealthy habits (3,7,11). The reason for this is probably that behavioral changes are made according to the patient’s stage of the motivational circle at the moment of consultation (12), which can be determined only by individual approach.Although family physicians are convinced that it is their task to give advice on health promotion and disease prevention, in practice they are less likely to do so (13). The factors that prevent them from giving advice are time (14,15), cost, availability, practice capacity (14), lack of knowledge and guidelines, poor counseling skills (16), and personal attitudes (17). It also seems that physicians’ assessment varies considerably according to the risk factor in question. For example, information on diet and physical activity are often inferred from patients’ appearance rather than from clinical measurements (14). Also, health care professionals seldom give advice on recommended aspects of intervention that could facilitate behavioral change (18). As a large proportion of primary care patients are ready to lose weight, improve diet, and increase exercise (19), it is even more important that their family physicians provide timely advice.So far, several studies have addressed patients’ willingness to make lifestyle change (2-5,20) and the provision of professional advice (3,5,7,10,11). However, none of these studies have investigated the relation between these factors. So, the aim of our study was to assess the relation between patients’ attitudes toward changing unhealthy lifestyle, confidence in success, and the desired involvement of their family physicians in facilitating the change.     

Psychiatric heredity and posttraumatic stress disorder: survey study of war veterans

Aim

To explore the prevalence of psychiatric heredity (family history of psychiatric illness, alcohol dependence disorder, and suicidality) and its association with the diagnosis of stress-related disorders in Croatian war veterans established during psychiatric examination.

Methods

The study included 415 war veterans who were psychiatrically assessed and diagnosed by the same psychiatrist during an expert examination conducted for the purposes of compensation seeking. Data were collected by a structured diagnostic procedure.

Results

There was no significant correlation between psychiatric heredity of psychiatric illness, alcohol dependence, or suicidality and diagnosis of posttraumatic stress disorder (PTSD) or PTSD with psychiatric comorbidity. Diagnoses of psychosis or psychosis with comorbidity significantly correlated with psychiatric heredity (φ = 0.111; P = 0.023). There was a statistically significant correlation between maternal psychiatric illness and the patients’ diagnoses of partial PTSD or partial PTSD with comorbidity (φ = 0.104; P = 0.035) and psychosis or psychosis with comorbidity (φ = 0.113; P = 0.022); paternal psychiatric illness and the patients’ diagnoses of psychosis or psychosis with comorbidity (φ = 0.130; P = 0.008), alcohol dependence or alcohol dependence with comorbidity (φ = 0.166; P = 0.001); psychiatric illness in the primary family with the patients’ psychosis or psychosis with comorbidity (φ = 0.115; P = 0.019); alcohol dependence in the primary family with the patients’ personality disorder or personality disorder with comorbidity (φ = 0.099; P = 0.044); and suicidality in the primary family and a diagnosis of personality disorder or personality disorder with comorbidity (φ = 0.128; P = 0.009).

Conclusion

The study confirmed that parental and familial positive history of psychiatric disorders puts the individual at higher risk for developing psychiatric illness or alcohol or drug dependence disorder. Psychiatric heredity might not be necessary for the individual who was exposed to severe combat-related events to develop symptoms of PTSD.There are several risk factors associated with the development of posttraumatic stress disorder (PTSD), such as factors related to cognitive and biological systems and genetic and familial risk (1), environmental and demographic factors (2), and personality and psychiatric anamnesis (3).They are usually grouped into three categories: factors that preceded the exposure to trauma or pre-trauma factors; factors associated with trauma exposure itself; and post-trauma factors that are associated with the recovery environment (2,4).There are many studies which support the hypothesis that pre-trauma factors, such as ongoing life stress, psychiatric history, female sex (3), childhood abuse, low economic status, lack of education, low intelligence, lack of social support (5), belonging to racial and ethnic minority, previous traumatic events, psychiatric heredity, and a history of perceived life threat, influence the development of stress related disorders (6). Many findings suggest that ongoing life stress or prior trauma history sensitizes a person to a new stressor (2,7-9). The same is true for the lack of social support, particularly the loss of support from significant others (2,9-11), as well as from friends and community (12-14). If the community does not have an elaborated plan for providing socioeconomic support to the victims, then the low socioeconomic status can also be an important predictor of a psychological outcome such as PTSD (2,10,15). Unemployment was recognized as a risk factor for developing PTSD in a survey of 374 trauma survivors (16). It is known that PTSD commonly occurs in patients with a previous psychiatric history of mental disorders, such as affective disorders, other anxiety disorders, somatization, substance abuse, or dissociative disorders (17-21). Epidemiological studies showed that pre-existing psychiatric problems are one of the three factors that can predict the development of PTSD (2,22). Pre-existing anxiety disorders, somatoform disorders, and depressive disorders can significantly increase the risk of PTSD (23). Women have a higher vulnerability for PTSD than men if they experienced sexually motivated violence or had pre-existing anxiety disorders (23,24). A number of studies have examined the effects of gender differences on the predisposition for developing PTSD, with the explanation that women generally have higher rates of depression and anxiety disorders (3,25,26). War-zone stressors were described as more important for PTSD in men, whereas post-trauma resilience-recovery factors as more important for women (27).Lower levels of education and poorer cognitive abilities also appear to be risk factors (25). Golier et al (25) reported that low levels of education and low IQ were associated with poorer recall on words memorization tasks. In addition, this study found that the PTSD group with lower Wechsler Adult Intelligence Scale-Revised (WAIS-R) scores had fewer years of education (25). Nevertheless, some experts provided evidence for poorer cognitive ability in PTSD patients as a result or consequence rather than the cause of stress-related symptoms (28-31). Studies of war veterans showed that belonging to racial and ethnic minority could influence higher rates of developing PTSD even after the adjustment for combat exposure (32,33). Many findings suggest that early trauma in childhood, such as physical or sexual abuse or even neglect, can be associated with adult psychopathology and lead to the development of PTSD (2,5,26,34,35). Surveys on animal models confirm the findings of lifelong influences of early experience on stress hormone reactivity (36).Along with the reports on the effects of childhood adversity as a risk factor for the later development of PTSD, there is also evidence for the influence of previous exposure to trauma related events on PTSD (9,26,28). Breslau et al (36) reported that previous trauma experience substantially increased the risk for chronic PTSD.Perceived life threats and coping strategies carry a high risk for developing PTSD (9,26). For instance, Ozer et al (9) reported that dissociation during trauma exposure has high predictive value for later development of PTSD. Along with that, the way in which people process and interpret perceived threats has a great impact on the development or maintenance of PTSD (37,38).Brewin et al (2) reported that individual and family psychiatric history had more uniform predictive effects than other risk factors. Still, this kind of influence has not been examined yet.Keeping in mind the lack of investigation of parental psychiatric heredity on the development of stress-related disorders, the aim of our study was to explore the prevalence and correlation between the heredity of psychiatric illness, alcohol dependence, suicidality, and the established diagnosis of stress-related disorders in Croatian 1991-1995 war veterans.     

Breast and gynecological cancers in Croatia, 1988-2008

Aim

To analyze and interpret incidence and mortality trends of breast and ovarian cancers and incidence trends of cervical and endometrial cancers in Croatia for the period 1988-2008.

Methods

Incidence data were obtained from the Croatian National Cancer Registry. The mortality data were obtained from the World Health Organization (WHO) mortality database. Trends of incidence and mortality were analyzed by joinpoint regression analysis.

Results

Joinpoint analysis showed an increase in the incidence of breast cancer with estimated annual percent of change (EAPC) of 2.6% (95% confidence interval [CI], 1.9 to 3.4). The mortality rate was stable, with the EAPC of 0.3% (95% CI, -0.6 to 0.0). Endometrial cancer showed an increasing incidence trend, with EAPC of 0.8% (95% CI, 0.2 to 1.4), while cervical cancer showed a decreasing incidence trend, with EAPC of -1.0 (95% CI, -1.6 to -0.4). Ovarian cancer incidence showed three trends, but the average annual percent change (AAPC) for the overall period was not significant, with a stable trend of 0.1%. Ovarian cancer mortality was increasing since 1992, with EAPC of 1.2% (95% CI, 0.4 to 1.9), while the trend for overall period was stable with AAPC 0.1%.

Conclusion

Incidence trends of breast, endometrial, and ovarian cancers in Croatia 1988-2008 are similar to the trends observed in most of the European countries, while the modest decline in cervical cancer incidence and lack of decline in breast cancer mortality suggest suboptimal cancer prevention and control.Breast and gynecological cancers are among the seven most common female cancers in Croatia: in 2008 breast cancer was the most common cancer with the proportion of 26% of all cancer sites, endometrial cancer ranked fourth (6%), ovarian cancer (with fallopian tubes cancer) sixth (5%), and cervical cancer seventh (4%) (1).Breast, endometrial, and ovarian cancers share some similar risk factors like early menarche, late menopause, obesity, and low parity (2-5). Also, breast cancer in personal history increases the risk of endometrial and ovarian cancer (6). Delayed childbearing increases the risk of breast cancer but seems to have no impact on the development of ovarian and endometrial cancer (3-5). Diabetes mellitus increases the risk of endometrial and breast cancer (7,8). Use of tamoxifen or other selective estrogen receptor modulators increases the risk of endometrial and ovarian cancer, while the use of combined oral contraceptives is a protective factor (2,9,10). Also, tobacco smoking and alcohol intake reduce the risk of endometrial cancer (2,11,12). Alcohol intake and both oral contraceptives and hormonal replacement therapy are risk factors for breast cancer (2,13,14). Multiparty and physical activity are protective factors for all three cancers (2,4,15,16). Low socioeconomic status, sexually transmitted diseases, promiscuity, unprotected sexual behavior, earlier age of first intercourse, and smoking are risk factors for cervical cancer (2,17-23). Infection with human papillomavirus is considered as a necessary cause of cervical cancer (24).The aim of this study was to report the incidence and mortality of breast and ovarian cancers and incidence of endometrial and cervical cancers, analyze the trends in the period 1988-2008, and compare them to other European countries.     

Comparison of peritonsillar infiltration effects of ketamine and tramadol on post tonsillectomy pain: a double-blinded randomized placebo-controlled clinical trial

Aim

To assess the effect of peritonsillar infiltration of ketamine and tramadol on post tonsillectomy pain and compare the side effects.

Methods

The double-blind randomized clinical trial was performed on 126 patients aged 5-12 years who had been scheduled for elective tonsillectomy. The patients were randomly divided into 3 groups to receive either ketamine, tramadol, or placebo. They had American Society of Anesthesiologists physical status class I and II. All patients underwent the same method of anesthesia and surgical procedure. The three groups did not differ according to their age, sex, and duration of anesthesia and surgery. Post operative pain was evaluated using CHEOPS score. Other parameters such as the time to the first request for analgesic, hemodynamic elements, sedation score, nausea, vomiting, and hallucination were also assessed during 12 hours after surgery.

Results

Tramadol group had significantly lower pain scores (P = 0.005), significantly longer time to the first request for analgesic (P = 0.001), significantly shorter time to the beginning of liquid regimen (P = 0.001), and lower hemodynamic parameters such as blood pressure (P = 0.001) and heart rate (P = 0.001) than other two groups. Ketamine group had significantly greater presence of hallucinations and negative behavior than tramadol and placebo groups. The groups did not differ significantly in the presence of nausea and vomiting.

Conclusion

Preoperative peritonsillar infiltration of tramadol can decrease post-tonsillectomy pain, analgesic consumption, and the time to recovery without significant side effects.Registration No: IRCT201103255764N2Postoperative pain has not only a pathophysiologic impact but also affects the quality of patients’ lives. Improved pain management might therefore speed up recovery and rehabilitation and consequently decrease the time of hospitalization (1). Surgery causes tissue damage and subsequent release of biochemical agents such as prostaglandins and histamine. These agents can then stimulate nociceptors, which will send the pain message to the central nervous system to generate the sensation of pain (2-4). Neuroendocrine responses to pain can also cause hypercoagulation state and immune suppression, leading to hypoglycemia, which can delay wound healing (5).Tonsillectomy is a common surgery in children and post-tonsillectomy pain is an important concern. Duration and severity of pain depend on the surgical technique, antibiotic and corticosteroid use, preemptive and postoperative pain management, and patient’s perception of pain (6-9). There are many studies that investigated the control of post tonsillectomy pain using different drugs such as intravenous opioids, non-steroidal anti-inflammatory drugs, steroids, ketamine, as well as peritonsillar injection of local anesthetic, opioid, and ketamine (6,7,10-14).Ketamine is an intravenous anesthetic from phencyclidin family, which because of its antagonist effects on N methyl-D-aspartate receptors (that are involved in central pain sensitization) has regulatory influence on central sensitization and opium resistance. It can also band with mu receptors in the spinal cord and brain and cause analgesia. Ketamine can be utilized intravenously, intramuscularly, epidurally, rectally, and nasaly (15,16). Several studies have shown the effects of sub-analgesic doses of ketamine on postoperative pain and opioid consumption (7,13,15-17). Its side effects are hallucination, delirium, agitation, nausea, vomiting, airways hyper-secretion, and increased intra cerebral pressure and intra ocular pressure (10,11,15,16).Tramadol is an opium agonist that mostly effects mu receptors, and in smaller extent kappa and sigma receptors, and like anti depressant drugs can inhibit serotonin and norepinephrine reuptake and cause analgesia (6,12,18). Its potency is 5 times lower than morphine (6,12), but it has lower risk of dependency and respiratory depression, without any reported serious toxicity (6,12). However, it has some side effects such as nausea, vomiting, dizziness, sweating, anaphylactic reactions, and increased intra-cerebral pressure. It can also lower the seizure threshold (6,12,18,19).Several studies have confirmed the efficacy of tramadol and ketamine on post-tonsillectomy pain (6,10-12,20). In previous studies, effects of peritonsillar/ IV or IM infiltration of tramadol and ketamine were compared to each other and to placebo, and ketamine and tramadol were suggested as appropriate drugs for pain management (6,7,10-19,21). Therefore, in this study we directly compared the effect of peritonsillar infiltration of either tramadol or ketamine with each other and with placebo.     

Patients with Combat-related and War-related Posttraumatic Stress Disorder 10 Years After Diagnosis

Aim

To establish how many patients diagnosed with posttraumatic stress disorder (PTSD) in 1996 used psychiatric facilities and had psychiatric symptoms 10 years later, and assess their sociodemographic characteristics, comorbid disorders, and type of treatment.

Methods

Medical records of patients diagnosed with PTSD in 1996 were reviewed in the period 2007-2009 and the patients who contacted a psychiatrist in that period (n = 85) and those who did not (n = 158) were compared.

Results

There were 36.7% of men and 20% of women diagnosed with PTSD in 1996 who contacted a psychiatrist in the period 2007-2009. Patients who contacted a psychiatrist and those who did not did not differ in sex, age, the number of visits and hospitalizations in 1996, and employment status. The majority of patients still had PTSD and/or were enduring personality change in the period 2007-2009, and 54.8% had some comorbidity (mostly depression, alcohol-related disorders, and personality disorders). Patients were most often treated with anxiolytics and antidepressants.

Conclusion

Ten years after the traumatic experience, one third of patients with PTSD received psychiatric help, regardless of their sex, age, and employment status. Half of them had comorbid disorders and the majority of them were treated with anxiolytics and antidepressants.Posttraumatic stress disorder (PTSD) is a mental disorder that develops in 9-25% of war veterans (1-4), mainly in the first two years after the traumatic experience (5,6), but sometimes can develop years later (7,8). A similar prevalence was also found among Croatian war veterans (5-9). In the majority of cases (80-98%), PTSD is comorbid with other mental disorders: alcohol abuse, depression, anxiety disorders, and somatization (5,9-11).PTSD can also develop after a war-related trauma that is not necessarily combat-related, and the lifetime prevalence of PTSD in this population is 15-38% (12) and the prevalence of anxiety and depressive disorders is even higher (13,14).Many patients in Croatia had symptoms of PTSD and used health facilities for treatment years after the war (5-9). In the former Yugoslavia, 84% of untreated war-related PTSD patients still had PTSD symptoms years after the war (15). Resolution of PTSD is observed in 50-60% of cases (4,16).Combat-related PTSD causes more functional impairment and is less responsive to treatment than PTSD related to other traumas (17-19). It is unclear whether this happens because there is indeed a difference between the two types of PTSD or some of the patients aggravate their symptoms in order to get compensation (17,18,20). Some studies show that the use of health facilities decreases after obtaining war veteran status and compensation, but others show that the patients who had obtained the status and compensation used medical facilities more often than those who had not (20-23).The aim of this study was to establish how many patients diagnosed with PTSD in 1996 used psychiatric facilities and had psychiatric symptoms 10 years later and assess their comorbidities, sociodemographic characteristics, and type of treatment.     

Prediction of eye color in the Slovenian population using the IrisPlex SNPs

Aim

To evaluate the accuracy of eye color prediction based on six IrisPlex single nucleotide polymorphisms (SNP) in a Slovenian population sample.

Methods

Six IrisPlex predictor SNPs (HERC2 – rs12913832, OCA2 – rs1800407, SLC45A2 – rs16891982 and TYR – rs1393350, SLC24A4 – rs12896399, and IRF4 – rs12203592) of 105 individuals were analyzed using single base extension approach and SNaPshot chemistry. The IrisPlex multinomial regression prediction model was used to infer eye color probabilities. The accuracy of the IrisPlex was assessed through the calculation of sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and the area under the receiver characteristic operating curves (AUC).

Results

Blue eye color was observed in 44.7%, brown in 29.6%, and intermediate in 25.7% participants. Prediction accuracy expressed by the AUC was 0.966 for blue, 0.913 for brown, and 0.796 for intermediate eye color. Sensitivity was 93.6% for blue, 58.1% for brown, and 0% for intermediate eye color. Specificity was 93.1% for blue, 89.2% for brown, and 100% for intermediate eye color. PPV was 91.7% for blue and 69.2% for brown color. NPV was 94.7% for blue and 83.5% for brown eye color. These values indicate prediction accuracy comparable to that established in other studies.

Conclusion

Blue and brown eye color can be reliably predicted from DNA samples using only six polymorphisms, while intermediate eye color defies prediction, indicating that more research is needed to genetically predict the whole variation of eye color in humans.Prediction of human visible characteristics by genotyping informative polymorphisms in DNA opens up a new perspective in the forensic field. Multiple genes including HERC2, OCA2, MC1R, SLC24A5, SLC45A2, TYR, TYRP1, ASIP, SLC24A4, TPCN2, KITLG, and IRF4 have been associated with eye, hair, and skin color in European populations and they have been used in studies dealing with eye color prediction (1-14). Variation of iris color depends on the content of eumelanine, a brown light-absorbing biopolymer, which is present in higher concentrations in brown-eyed individuals (15,16). Although eye color is evidently a continuous variable, it has been often classified into three categories – blue, brown, and intermediate (4,14). Eye color variability is particularly striking in European populations, constituting a highly differentiating trait of potential use in forensic investigations (7,14,17). Recent studies have shown that a significant fraction of human iris color variation can be explained by polymorphisms within a single region in the human genome, comprising the evolutionary conserved HERC2 gene and the neighboring OCA2 gene located on the chromosome 15. It is assumed that the level of expression of the known pigmentation gene – OCA2 – is controlled by polymorphism rs12913832 on HERC2 locus (18,19). The remaining genes that have been shown to contribute to eye color variation are SLC24A4, SLC45A2, TYR, and IRF4 (4,20,21). However, their impact on eye color prediction is lower and it seems to vary between populations (8,14,22,23). Since such differences may potentially affect accuracy of prediction in various populations, we further addressed this issue and analyzed a population sample of individuals with defined eye color from Slovenia.Several prediction models have already been proposed to be useful in eye color prediction (4,8,9,17,23,24). Here we used six IrisPlex predictors, which were selected by Liu et al (4) from a larger set of polymorphisms potentially influencing pigmentation in humans and included into the IrisPlex prediction system (4,13,17). The IrisPlex prediction model is based on a multinomial logistic regression method and uses phenotype and genotype data from 3804 Dutch individuals. Based on these data the model gives three probabilities for blue, brown, and intermediate eye color (13). From the obtained probabilities, the most probable iris color is predicted based on recommendations given in Walsh et al (13).     

Development of a multiplex single base extension assay for mitochondrial DNA haplogroup typing

Aim

To provide a screening tool to reduce time and sample consumption when attempting mtDNA haplogroup typing.

Methods

A single base primer extension assay was developed to enable typing, in a single reaction, of twelve mtDNA haplogroup specific polymorphisms. For validation purposes a total of 147 samples were tested including 73 samples successfully haplogroup typed using mtDNA control region (CR) sequence data, 21 samples inconclusively haplogroup typed by CR data, 20 samples previously haplogroup typed using restriction fragment length polymorphism (RFLP) analysis, and 31 samples of known ancestral origin without previous haplogroup typing. Additionally, two highly degraded human bones embalmed and buried in the early 1950s were analyzed using the single nucleotide polymorphisms (SNP) multiplex.

Results

When the SNP multiplex was used to type the 96 previously CR sequenced specimens, an increase in haplogroup or macrohaplogroup assignment relative to conventional CR sequence analysis was observed. The single base extension assay was also successfully used to assign a haplogroup to decades-old, embalmed skeletal remains dating to World War II.

Conclusion

The SNP multiplex was successfully used to obtain haplogroup status of highly degraded human bones, and demonstrated the ability to eliminate possible contributors. The SNP multiplex provides a low-cost, high throughput method for typing of mtDNA haplogroups A, B, C, D, E, F, G, H, L1/L2, L3, M, and N that could be useful for screening purposes for human identification efforts and anthropological studies.Mitochondrial DNA (mtDNA) is a 16 569 bp circular molecule present, on average, in 500 copies per cell (1). MtDNA analysis is utilized in several areas of science including, but not limited to, anthropology, evolutionary studies, and forensic science (2-5). The high copy number, and possibly the cellular location and molecular features of mtDNA, allow for increased recovery, thus providing a distinct advantage over nuclear DNA when working with highly compromised samples (6). The maternal inheritance and high mutation rate are characteristics extremely useful for evolutionary studies (7,8); in fact, mtDNA has been used to resolve evolutionary questions related to extinct species and to human migrations throughout the continents (9-12). The field of forensic science also relies upon mtDNA to identify missing persons, locate maternal relatives, identify victims in mass disasters, and, in some situations, include an individual at a crime scene (13-19).Early studies of the mtDNA genome revealed patterns of variation that were linked to geographic regions. Individuals with the same sequence variations were clustered into haplogroups defined by mutations at particular nucleotide positions (20-27). A closer examination of the mtDNA genomes of various populations led to the following assumptions: 1) several of the mtDNA mutations are highly correlated with the ethnic and geographic origin of the individual, 2) all mutations originated from a single mtDNA tree, and 3) the greatest variation and deepest root of the mtDNA tree is present in the African population. Furthermore, a calculation of the variation between mtDNA haplogroups demonstrated that 35% of the mutations were continent-specific, and therefore useful indicators of geographic origin (24,25,28-31).Before the advent of modern sequencing methods, the primary approach to identifying polymorphic sites throughout the mtDNA coding region was restriction fragment length polymorphism (RFLP) analysis. While this methodology is still utilized in certain contexts, direct sequencing of the mtDNA molecule is rapidly gaining acceptance as the method of choice for haplogroup typing (20,23,30,32-35). Single base primer extension, also known as minisequencing, is an example of a direct sequencing technique that is currently utilized for mtDNA haplogroup typing (36-42). This methodology, described in detail by Fiorentino et al (43), offers several advantages to the investigator over RFLP and conventional sequence analysis methods including the use of small amplicons (<150 bp), increased sensitivity and robustness, and multiplexing capability. Multiplexing capability is particularly important, especially in regard to forensic DNA analysis, as it reduces sample consumption while increasing throughput of sample processing and data analysis. Increased sensitivity allows for improved amplification success with DNA samples that contain limited starting template. Additionally, the possibility for high throughput processing can aid in population screening studies in situations where numerous samples need to be typed (44). This is particularly true in mass disaster or other mass screening situations, where a simple and rapid population screening tool that consumes little extract could effectively direct subsequent identification testing. In these situations, coding region sequencing would be expensive and time-consuming, and the subsequent data analysis a lengthy, burdensome, and potentially error-prone process (42,45-48). Furthermore, the possibility of obtaining interpretable results from poor quality polymerase chain reaction (PCR) products while simultaneously typing several polymorphisms throughout the mtDNA genome make it a more feasible method than conventional PCR fragment sequence analysis, especially in forensic cases and anthropological studies involving highly degraded or otherwise compromised human remains (16,36-38,49,50).In this article, we describe the development of a multiplex assay designed to simultaneously type twelve polymorphic positions located throughout the coding region of the mtDNA genome for the identification of haplogroups A, B, C, D, E, F, G, H, I, L1/L2, L3, M, N, and X. The intended utility of this assay is haplogroup typing of highly degraded human remains for either forensic casework or as a low cost, high throughput alternative for screening anthropological specimens. Validation of this assay included the analysis of 20 samples previously haplogroup typed by RFLP, and 94 samples for which haplogroup had been inferred based on control region (CR) sequence data. Additionally, and to evaluate the potential application of the assay for population screening, 31 samples were tested for which only the population of origin, and not the mtDNA haplogroup, was known. Finally, to verify robustness and sensitivity of the assay, we also tested two highly degraded human bones embalmed and buried in the early 1950s.     

Sex-specific chronic stress response at the level of adrenal gland modifies sexual hormone and leptin receptors

Aim

To compare cardiometabolic risk-related biochemical markers and sexual hormone and leptin receptors in the adrenal gland of rat males, non-ovariectomized females (NON-OVX), and ovariectomized females (OVX) under chronic stress.

Methods

Forty six 16-week-old Sprague-Dawley rats were divided into male, NON-OVX, and OVX group and exposed to chronic stress or kept as controls. Weight, glucose tolerance test (GTT), serum concentration of glucose, and cholesterol were measured. Adrenal glands were collected at the age of 28 weeks and immunohistochemical staining against estrogen beta (ERβ), progesterone (PR), testosterone (AR), and leptin (Ob-R) receptors was performed.

Results

Body weight, GTT, serum cholesterol, and glucose changed in response to stress as expected and validated the applied stress protocol. Stressed males had significantly higher number of ERβ receptors in comparison to control group (P = 0.028). Stressed NON-OVX group had significantly decreased AR in comparison to control group (P = 0.007). The levels of PR did not change in any consistent pattern. The levels of Ob-R increased upon stress in all groups, but the significant difference was reached only in the case of stressed OVX group compared to control (P = 0.033).

Conclusion

Chronic stress response was sex specific. OVX females had similar biochemical parameters as males. Changes upon chronic stress in adrenal gland were related to a decrease in testosterone receptor in females and increase in estrogen receptor in males.Maintaining homeostasis is often challenged by different types of stressors (1). Homeostasis is regulated by a complex endocrine processes engaging the hypothalamic-pituitary-adrenal axis (HPA) and sympathetic autonomic system (2-4). Stress can occur either in acute or chronic form with different consequences – the acute stress mostly induces the ˝fight or flight˝ response, while chronic stress promotes long term changes, which can lead to a variety of diseases (5,6). If stress is of sufficient magnitude and duration, the action of HPA is unsuppressed and results in prolonged elevation of cortisol (7), induced production of energy, vasoconstriction, lipolysis, proteolysis, immunosuppression, and suppression of reproductive function to save energy and retain overall homeostasis (8). Women are generally less susceptible to chronic stress up to the period of menopause, when the loss of protective hormones, estrogen and progesterone, occurs and thus they become prone to development of depression, anxiety, or schizophrenia (9). In contrast, men are generally more susceptible and sensitive to chronic stress, showing changes in feeding habits and decreased body weight (10,11).Chronic stress can cause the development of cardiovascular disorder, obesity, and diabetes, which can be reflected in serum cholesterol, glucose, and decreased glucose tolerance (12-14). There is a strong correlation between stress and sexual hormones, but the mechanisms by which estrogen, testosterone, and progesterone exert their possible protective role under stress conditions are not fully explored. Sexual hormones affect stress outcome and stress hormones affect the levels of sexual hormones (15-17). Testosterone is activated during stress response in rats and humans (18,19) and tends to increase more in men than women (20). Estrogen lowers the stress-induced response in women and men (9,21). Estrogens and progesterone are produced even after ovariectomy by adrenal glands (22) but it is not known if such compensation can withstand additional challenge like stress. Another possible player in stress response is leptin (Ob), hormone responsible for maintaining body weight, which is synthesized and secreted by adipose tissue (23), exerting its effects through the leptin receptor (Ob-R) (24). Chronic stress models imply a direct link between stress response and leptin (25,26). Receptors for leptin are present in the adrenal gland (27). The aim of this study was to investigate cardiovascular risk parameters and changes in leptin and sexual hormone receptors in adrenal gland during chronic stress. There is a clinically relevant change in the onset of cardiometabolic risk between healthy women and women with premature ovarian failure (28) and because of that ovariectomized female rats were included in the study.     

Venous thromboembolism in Croatia – Croatian Cooperative Group for Hematologic Diseases (CROHEM) study

Aim

To analyze the incidence and characteristics of venous thromboembolism (VTE) in Croatia.

Methods

The Croatian Cooperative Group for Hematologic Diseases conducted an observational non-interventional study in 2011. Medical records of patients with newly diagnosed VTE hospitalized in general hospitals in 4 Croatian counties (Šibenik-Knin, Koprivnica-Križevci, Brod-Posavina, and Varaždin County) were reviewed. According to 2011 Census, the population of these counties comprises 13.1% of the Croatian population.

Results

There were 663 patients with VTE; 408 (61.54%) had deep vein thrombosis, 219 (33.03%) had pulmonary embolism, and 36 (5.43%) had both conditions. Median age was 71 years, 290 (43.7%) were men and 373 (56.3%) women. Secondary VTE was found in 57.3% of participants, idiopathic VTE in 42.7%, and recurrent VTE in 11.9%. There were no differences between patients with secondary VTE and patients with idiopathic VTE in disease recurrence and sex. The most frequent causes of secondary VTE were cancer (40.8%), and trauma, surgery, and immobilization (38.2%), while 42.9% patients with secondary VTE had ≥2 causes. There were 8.9% patients ≤45 years; 3.3% with idiopathic or recurrent VTE. Seventy patients (10.6%) died, more of whom had secondary (81.4%) than idiopathic (18.6%) VTE (P < 0.001), and in 50.0% VTE was the main cause of death. Estimated incidence of VTE in Croatia was 1.185 per 1000 people.

Conclusion

Characteristics of VTE in Croatia are similar to those reported in large international studies. Improved thromboprophylaxis during the presence of risk factors for secondary VTE might substantially lower the VTE burden.Venous thromboembolism (VTE), including deep venous thrombosis (DVT) and pulmonary embolism (PE), is a major health problem in the world, associated with significant morbidity and mortality (1-9). Incidence rates for VTE mostly vary from 1 to 2 in 1000 individuals per year (1-5,7,9). PE, the most serious manifestation of VTE, has a mortality rate of more than 15% in the first 3 months after diagnosis, with short-term survival of less than 60% (10,11). Cohen et al estimated that the number of VTE-related deaths across the European Union (EU) was 543 454 per year, which was more than double the number of combined deaths in EU due to AIDS, breast and prostate cancer, and traffic accidents (8).VTE in survivors is associated with several chronic consequences of the disease that can severely impair the patients’ quality of life, including post-thrombotic syndrome (PTS) and pulmonary hypertension (PH), as well as recurrent VTE. PTS affects at least one-third of patients after DVT (8,12-15) and PH affects 4%-5% of patients after PE (8,16,17). VTE has significant incidence of recurrence: 10.1% at 6 months, 12.9% after 1 year, and 30.4% after 10 years (18).Total VTE-related costs to health care system are enormous. For example, the total cost of VTE to the UK National Health Service in 1993 was £235-£257 million (€349-€382 million), and the combined direct and indirect costs in 2004/2005 were approximately £640 million (€950 million), and are even higher when PTS is taken into account (8,19,20).VTE is a multifactorial disease, resulting from a complex interaction of genetic and acquired factors. Although some studies estimated that genetics was related to up to 60% of the risk of VTE (including FV Leiden and prothrombin G20210A mutations, deficiencies of protein C, S and antithrombin, and elevations of some procoagulant factors) (21), there is also a large number of acquired risk factors for VTE such as immobilization, surgery, trauma, cancer, pregnancy and puerperium, oral contraceptives, autoimmune diseases, and other disorders (1-8,21).In spite of the importance of VTE, there is not enough data on its incidence and characteristics in transitional countries. Also, although several studies analyzed the epidemiology of VTE in different study settings (1-9), there is still not much information on conditions present at the diagnosis of thrombosis, comparing idiopathic and secondary (provoked) VTE. Therefore, the Croatian Cooperative Group for Hematologic Diseases (CROHEM) analyzed the incidence and characteristics of idiopathic and secondary newly diagnosed VTE in Croatia in 2011, the year of the most recent national population census.     

DNA analysis of early mediaeval individuals from the Zvonimirovo burial site in Northern Croatia: investigation of kinship relationships by using multiplex system amplification for short tandem repeat loci

Aim

To perform initial DNA analysis of four selected early mediaeval individuals from the Zvonimirovo burial site in Northern Croatia.

Methods

Investigation of genetic matching of individuals from a “double burial” and of individuals with shared cranial non-metric/metric traits from 2 single inhumations, located in another block of the cemetery complex, was carried out. DNA from four teeth samples was extracted, quantified, and amplified by polymerase chain reaction (PCR) for short tandem repeat loci, using AmpFlSTR Profiler™ PCR Amplification Kit.

Results

Autosomal short tandem repeat (STR) genotyping generated high parentage probability (PP) as to the matching of the 2 individuals from the “double burial” (PP 98.63%), and of 2 women with shared cranial non-metric/metric traits from neighboring single burials (PP 90.07%). Parentage probability calculations of a possible genetic matching of the subadult from a “double burial” with the adults from single burials 4 and 3 were significantly lower (PP 60.45% and 38.52%). DNA typing for amelogenin confirmed the sex of the 3 female individuals, estimated previously by morphology. The unknown sex of the subadult was also determined as female.

Conclusion

Increased parentage probability for autosomal STR loci matches and the presence of a rare allele shared among matched individuals support their possible kinship relationship, in accordance with bioarchaeological data. We assume an intentional double burial based on a close familial relationship, ie 2 single neighboring inhumations based on consanguinity, rather than a strong social relationship. The kinship lineages remain unknown at this point.DNA analysis has recently become one of the most advanced tools broadly employed in the investigation of relatedness within burial groups (1-5). Genetic relationships within and between burial sites are of help in understanding both the organization of inhumation places and the origin of unearthed individuals in reference to a single or limited number of family groups (5). Kinship analyses of double burials are particularly interesting in terms of the possibility of uncovering consanguinity or strong social relationships among such individuals (6-8).However, except for a few cases of ancient DNA studies (9-11), the full advantage of DNA analysis is still not being taken in Croatian bioarchaeology (12). The major problem appears to be the cost of chemicals used in the procedure, along with other priorities for DNA typing in Croatia, such as the identification of war victims from mass graves (13-17) and in forensic medicine (9,14,18,19). A further problem is DNA degradation, contamination, or the presence of inhibitors (11,19). Damaged DNA templates in very old bones/teeth with a minute amount of cells occasionally lead to the elimination of a single or, in the worst case, all alleles, providing only non-reproducible results (20). Water or soil can also damage genomic DNA and make personal identification extremely difficult (18).As in forensic DNA testing (18), genetic analysis of bioarchaeological samples requires the use of short sized loci, amplified by polymerase chain reactions (PCR), with short tandem repeat (STR) loci. Autosomal STRs have been used in the study of close parentage relationships due to excellent discrimination power (5,18) and are suitable markers for ancient DNA typing because of their small size and recognition of sample contamination by modern DNA (21). Because of the possibility of simultaneous amplification and reduction to an absolute minimum of the amount of sample material necessary for kinship analysis (5,9), we gave priority to STRs in performing an investigation of possible kinship relationships of mediaeval individuals from the Zvonimirovo site in Northern Croatia. To the best of our knowledge, this work represents the only attempt in Croatian bioarchaeology to perform an investigation of possible kinship relationships among individuals from a burial site.     

Why pulsatility still matters: a review of current knowledge

Continuous-flow left ventricular assist devices (LVAD) have become standard therapy option for patients with advanced heart failure. They offer several advantages over previously used pulsatile-flow LVADs, including improved durability, less surgical trauma, higher energy efficiency, and lower thrombogenicity. These benefits translate into better survival, lower frequency of adverse events, improved quality of life, and higher functional capacity of patients. However, mounting evidence shows unanticipated consequences of continuous-flow support, such as acquired aortic valve insufficiency and acquired von Willebrand syndrome. In this review article we discuss current evidence on differences between continuous and pulsatile mechanical circulatory support, with a focus on clinical implications and potential benefits of pulsatile flow.During the last few decades, mechanical circulatory support has evolved into a standard therapy for patients with advanced heart failure – as a bridge to cardiac transplantation (1-3), bridge to myocardial recovery (4-7), or as destination therapy (8-10). This success can in most part be attributed to the use of continuous-flow devices and their advantages over previously used pulsatile pumps: they offer improved durability, less surgical trauma due to their smaller size, higher energy efficiency, and lower thrombogenicity. These benefits translate into better survival, lower frequency of adverse events, improved quality of life, and higher functional capacity of patients (11-13).The debate on the importance of pulsatility began several decades ago with the research on the effects of nonpulsatile flow during the cardiopulmonary bypass (CPB) (14,15). It is still alive today, after nearly a decade of use of continuous-flow devices, especially after evidence has shown that this therapy is complicated by diminished pulsatility. In this review article we discuss current evidence on differences between continuous and pulsatile ventricular assist devices, with a focus on clinical implications and potential benefits of pulsatile flow.