Evaluation of 16S rRNA primer sets for characterisation of microbiota in paediatric patients with autism spectrum disorder.

intestinal microbiota is becoming a significant marker that reflects differences between health and disease status also in terms of gut-brain axis communication. Studies show that children with autism spectrum disorder (ASD) often have a mix of gut microbes that is distinct from the neurotypical children. Various assays are being used for microbiota investigation and were considered to be universal. However, newer studies showed that protocol for preparing DNA sequencing libraries is a key factor influencing results of microbiota investigation. The choice of DNA amplification primers seems to be the crucial for the outcome of analysis. In our study, we have tested 3 primer sets to investigate differences in outcome of sequencing analysis of microbiota in children with ASD. We found out that primers detected different portion of bacteria in samples especially at phylum level; significantly higher abundance of Bacteroides and lower Firmicutes were detected using 515f/806r compared to 27f/1492r and 27f*/1495f primers. So, the question is whether a gold standard of Firmicutes/Bacteroidetes ratio is a valuable and reliable universal marker, since two primer sets towards 16S rRNA can provide opposite information. Moreover, significantly higher relative abundance of Proteobacteria was detected using 27f/1492r. The beta diversity of sample groups differed remarkably and so the number of observed bacterial genera.

Specificity of gut microbiota in children with autism spectrum disorder in Slovakia and its correlation with astrocytes activity marker and specific behavioural patterns.

Recent research suggests the involvement of bidirectional gut-brain axis in autism spectrum disorder (ASD). The aim of this study was to establish the role of changed gut microbiota in behavioural and gastrointestinal manifestations, but also in astrocyte activation in children with ASD. Distinct faecal microbiota in children with ASD was found to be more heterogeneous compared to that in neurotypical children. Different bacterial abundance and correlation with behavioural and GI manifestations revealed several bacterial genera possibly important for ASD. Microbial-neuronal cross talk could be accomplished through S100B, released by glial cells, activated by low grade inflammation. More complex studies are required to understand ASD pathogenesis.

Plasma levels of glial cell marker S100B in children with autism.

Autism spectrum disorder (ASD) is a neurodevelopmental condition with increasing incidence. Recent evidences suggest glial cells involvement in autism pathophysiology. S100B is a calcium binding protein, mainly found in astrocytes and therefore used as a marker of their activity. In our study, children with autism had higher plasma concentrations of S100B compared to non-autistic controls. No association of S100B plasma levels with behavioral symptoms (ADI-R and ADOS-2 scales) was found. Plasma S100B concentration significantly correlated with urine serotonin, suggesting their interconnection. Correlation of plasma S100B levels with stool calprotectin concentrations was found, suggesting not only brain astrocytes, but also enteric glial cells may take part in autism pathogenesis. Based on our findings, S100B seems to have a potential to be used as a biomarker of human neurodevelopmental disorders, but more investigations are needed to clarify its exact role in pathomechanism of autism.

Inherited Thrombophilias Could Influence the Reproductive Outcome in Women with Systemic Lupus Erythematosus.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease associated with different reproductive complications in the affected women. Inherited thrombophilias are genetic factors increasing the risk for thromboembolism and recurrent pregnancy loss, but their influence on other reproductive disturbances in SLE patients has not been completely clarified. Two hundred and twenty-three Caucasian women (112 with SLE and 111 controls) were included in the study. Complete reproductive history of all SLE patients was carefully obtained. Genotyping for the FVLeiden, FIIG20210A, and MTHFRC677T polymorphisms was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. No significant differences in the prevalence of the FVLeiden, FIIG20210A, and MTHFRC677T polymorphisms between patients and controls were established. Patients with FVLeiden had fewer pregnancies (0.57 ± 0.98 vs. 2.18 ± 1.58; p = 0.007) than the others, while no significant differences in the reproductive history of FIIG20210A carriers and non-carriers were observed (p >0.05). In the SLE group, 41.67% of women with the MTHFRC677T TT genotype had at least one miscarriage in comparison to only 14.00% of the other female patients (p = 0.030). While the prevalence of the investigated thrombophilias was similar in patients with SLE and healthy women, a substantial influence of the inherited prothrombotic factors on the reproductive history of patients was revealed. The investigations of the FVLeiden and MTHFRC677T polymorphisms in SLE patients could help to identify women at highest risk for reproductive failure and thus, further studies in other ethnic groups would be of strong clinical importance.

Fecal calprotectin levels correlate with main domains of the autism diagnostic interview-revised (ADI-R) in a sample of individuals with autism spectrum disorders from Slovakia.

Autism spectrum disorders (ASD) are neurodevelopmental disorders characterized by impaired social interaction and communication, as well as repetitive behavior and restricted interests. There is convincing evidence that the intestinal inflammation is involved in etiology of ASD. Increased levels of inflammatory markers were shown to be associated with more aberrant behaviors and communication of subjects with ASD. Calprotectin in the feces is produced by activated neutrophils and epithelial cells of the gut mucosa, and its levels reflect local inflammation of the gastrointestinal tract. Concentration of fecal calprotectin was determined by ELISA method in 87 individuals with ASD and 51 controls, of that 29 siblings of children with ASD and 22 non-related controls. In non-relatives significantly lower values of fecal calprotectin were observed than in both subjects with ASD and their siblings. In the group with ASD significant correlations of fecal calprotectin with all domains of the ADI-R diagnostic tool were found: qualitative abnormalities in reciprocal social interaction and communication, restrictive and repetitive patterns of behavior. Results suggest that low grade intestinal inflammation may be one of factors implicated in the pathophysiology of ASD.

Melatonin receptor 1b polymorphisms in women with Systemic Lupus Erythematosus.

AIM: The pineal hormone melatonin could exert an important influence on the immune system and autoimmunity. Its effect on the immunocompetent cells might be mediated at least partially through specific melatonin receptors. However, the role of melatonin - melatonin receptor 1B (MTNR1B) interrelations in human autoimmune diseases is still unknown. Therefore, the present study aimed to investigate the possible influence of the MTNR1B gene polymorphisms for the development and clinical expression of systemic lupus erythematosus (SLE). METHODS: 109 female SLE patients and 101 healthy women were genotyped for the MTNR1B rs1562444, rs10830962 and rs10830963 polymorphisms. RESULTS: No genotype distribution differences were found between patients and controls. The presence of MTNR1B rs10830963 C/C genotype was related to increased prevalence of leucopenia compared to genotypes C/G and G/G after Bonferroni correction for multiple comparisons [36.5% vs. 14.5%, p=0.014]. Moreover, the rs10830963 G/G carriers had lower number of lupus criteria in comparison to patients with C/C genotype. CONCLUSIONS: The present data suggested that MTNR1B polymorphisms could influence the clinical features in lupus patients, and especially the susceptibility to leucopenia.

[CLINICAL EFFICACY OF THE RECEPTOR BETA AND ALPHA BLOCKERS - CARVEDILOL AND ORNITHINE-ASPARTATE IN PATIENTS WITH CHD AND CHF COMBINED WITH ALCOHOLIC LIVER DISEASE].

Aim: The aim of the paper is to study the clinical efficacy of carvedilol and ornithine-aspartate in the complex therapy (ACE inhibitors, diuretics, cardiac glycosides, nitrates indication), to assess their impact on quality of life, intracardiac hemodynamics, remodeling of the left (LV) and right ventricular (RV), indicators of the inflammatory enzyme activity in blood serum of patients with coronary heart disease with CHF II-III FC and alcoholic liver disease (ALD). Materials and Methods: 95 patients were studied 45-75 years (mean age - 58,2 ± 1,2) with CHF II-III FC and postinfarction cardiosclerosis, LVEF less than 45%. ALD was diagnosed in 58 patients. In 23 (39%) patients among them had steatosis, 18 (30.5%) - chronic hepatitis (CH), 17 (30.5%) - liver cirrhosis (LC). Patients were divided into 3 groups. Patients in the first group (37 people) with coronary artery disease and heart failure without a UPS received an average dose of carvedilol in - 32,8 ± 4,7 mg / day. Patients in the 2nd group (32 persons) suffering from coronary artery disease, heart failure, and UPS received carvedilol in an average dose of 25,4 ± 1,6- mg / day and L-ornithine-L-aspartate in a dose of 10 g granulate per day for 2 weeks, and then by 5g a day for 4 months. Patients in the third control group (26 people) with CHD and CHF and CHF ABP received basic therapy (without ß-blocker) and ademetionine at a dose of 800 mg / day for 2 weeks, followed by 400 mg / day for 4 months. Results: After 4 months of observation, it was noted that CHF patients with IHD in combination with BPO flows less favorably. In all groups, the clinical status of patients was improved on the background of the therapy, however, the clinical status was more pronounced while using carvedilol and ornithine-aspartate (Group 2): summary measure of quality of life has improved by 38 points, the speed of the test increased with a digital sequence up to 54.4, decreased shortness of breath, edema, ascites, portal hypertension effects, hepatocellular insufficiency and hepatic encephalopathy. In general, was shown the normalization of sleep rhythm, reducing sleepiness, improved memory, attention, reduced asterixis and sweeping hand tremor, asthenia. Conclusions: The use of carvedilol and ornithine-aspartate in the treatment of patients with CHF FC II-III with CHD and BPO improves the clinical condition of patients, quality of life, hemodynamics, reduces the severity of pulmonary hypertension and normalizes serum biochemical parameters.

[Intestinal microbiota and the brain: multilevel interactions in health and disease]. / Os crevo-mozog: význam interakcií medzi crevnou mikrobiotou a mozgom za fyziologických a patologických situácii.

Commensal bacteria of the digestive tract are separated from the brain by multiple barriers. Despite that bacteria residing in the intestine and the neurons of the brain interact by neural and humoral pathways. Mental processes, such as the stress response, may affect the composition and function of intestinal bacteria via the brain-gut axis. On the other hand, intestinal bacteria can influence the processes in the brain through the gut-brain axis. Disruption of these interactions may be involved in various alterations both in the function of the gastrointestinal tract and the brain function.

[PAL-1 5G/4G polymorphism in patients with systemic lupus erythematosus].

Systemic lupus erythematosus (SLE) is a connective tissue disease affecting predominantly women that has been widely associated with obstetric complications. Inherited thrombophilias are significant risk factors for pregnancy loss, but their role in patients with SLE, and especially in those without concomitant secondary antiphospholipid syndrome (APS) has not been clarified. The aim of the present study was to study PAI-1 5G/4G polymorphism in women with lupus. A total of 103 SLE patients as well as 69 healthy volunteers were genotyped for PAI-1 5G/4G (rs1799889). No significant differences in the PAI-1 5G/4G genotype prevalence between patients and controls were found. After exclusion of the women with secondary APS, the frequency of pregnancies and spontaneous abortions, as well as the number of live births were similar in the studied patients with different PAI-1 genotype (p> 0.05). PAI-1 5G/4G polymorphism was not significantly related to any of the lupus ACR criteria or disease activity (p > 0.05), but it could influence the platelet number in the studied patients (263.52 ± 91.10 [5G/5G genotype] versus 210.12 ± 71.79 [4G/4G genotype], p = 0.023). In conclusion, our results showed that PAI-1 4G/5G polymorphism did not worsen the reproductive outcome in SLE women without secondary APS.

[Nonspecific immunomodulation in obstetrics and gynecology with Ecomer preliminary report].

UNLABELLED: The article gives a brief description of "Ecomer", preparation containing shark liver oil. Main immunoactive ingredient of the shark liver oil are the alkylglycerols. The main characteristics of alkylglycerols are noted together with their mechanism of action. There is also a list of indications for Ecomer administration. There is a summary of the authors' experience with Ecomer, its efficacy in ObGyn being the main aim of this study. METHODS AND MATERIALS: For the purpose of the study Ecomer is given to two main groups of patients: I-pregnant women between 27 and 36 weeks of gestation having two subgroups: 1--with 8 women with naso-pharyngeal complaints and 2--with 13 patients with urinary tract complaints, suggestive of cysto-pyelitic disorders and II main group of patients operated for cervical cancer in different stages with two subgroups--1 with 17 patients with Ecomer intake started at the beginning of the radiation therapy, and 2 with 6 patients who began taking Ecomer before the beginning of the radiation therapy, after having a histologically proved diagnose. The I group took two capsules of Ecomer three times a day for 15 days or less if asymptomatic earlier; in the II group Ecomer was taken two capsules three times a day for 2 months, then one capsule three times a day for a month, followed by a repetition of the scheme. RESULTS: In the pregnant women group, improvement was noticed in 75% in the first subgroup and in 76% in the second subgroup. In the second main group the interpretation of results is hindered by the insufficient time interval. Nevertheless, fewer side effects of the radiation therapy was noticed in both groups. Improvement in the survival rate is yet to be followed up. CONCLUSIONS: Administration of Ecomer in pregnant women with naso-pharyngeal problems led to improvement in their general condition and less frequent need to prescribe antibiotics. In pregnant women with urinary tract problems, Ecomer resulted in easing the pain faster and lowered the recurrence risk. In women with cervical cancer, the treatment is quite aggressive and the opportunity to diminish the side effects by administration of a natural and harmless preparation should not be omitted.

Anti-Müllerian hormone and inhibin B levels reflect altered Sertoli cell function in men with metabolic syndrome.

The aim of the present study was to investigate the Sertoli cell markers inhibin B and anti-Müllerian hormone (AMH) in men with metabolic syndrome (MS). Twenty patients with MS according to the criteria of the International Diabetes Federation and 20 non obese age-matched men were investigated. The levels of testosterone, sex hormone binding globulin (SHBG), gonadotropins, inhibin B and AMH were measured in all of them. In obese patients with MS total testosterone (15.74 ± 6.95 versus 27.84 ± 12.80 nmol l(-1), P = 0.001), SHBG (21.71 ± 11.08 versus 38.80 ± 17.51 nmol l(-1), P = 0.001) and free testosterone (430.35 ± 237.40 versus 613.85 ± 303.65 pmol l(-1), P = 0.040) were significantly lower than in the controls. Interestingly, the inhibin B (103.64 ± 56.77 versus 149.88 ± 68.31 pg ml(-1), P = 0.025) and AMH levels (30.84 ± 13.14 versus 43.14 ± 9.66 pmol l(-1), P = 0.002) were also significantly lower in MS group in comparison to the other participants. The lowest levels of AMH were found in patients with MS and carbohydrate disturbances. The decreased concentrations of testosterone, inhibin B and AMH in patients with MS could reflect an impaired Leydig and Sertoli cell function. Further studies in men with obesity, insulin resistance and diabetes type 2 could reveal more information about the interrelations between the metabolic disturbances and reproductive function in men.

[Optimization of treatment of patients with chronic cardiac insufficiency of the ischemic genesis and hobnail liver].

Clinical efficacy of combined therapy including the use of rifaximin and L-ornithin-l-aspartate, as well as the dynamics of the biochemical indices, the manifestation levels of portal-systemic-encephalopathy and intestinal microbiocynosis were investigated in patients with chronic cardiac insufficiency of ischemic genesis and hobnail liver. The combined therapy resulted in improvement of the patients clinical state, lower levels of the portal-systemic encephalopathy manifectation by decreasing hyperammonium, normalization of the large intestine microflora, and blood serum biochemical parameters.

Anti-Müllerian hormone in women with polycystic ovary syndrome before and after therapy with metformin.

Anti-Müllerian hormone (AMH) is largely expressed throughout folliculogenesis and its levels may represent both the quantity and quality of ovarian follicle pool. We conducted this study to evaluate the levels of AMH in women with polycystic ovarian syndrome (PCOS) before and after metformin therapy. 22 consecutive patients with PCOS and 20 healthy age-matched controls were investigated. The patients received 2 550 mg/day metformin for 6 months. Serum levels of AMH, sex hormones, insulin, blood glucose, and lipids were measured before and after metformin therapy. The basal AMH levels in patients with PCOS (42.34±6.42 pmol/l) were significantly elevated in comparison with the controls (21.58±3.41 pmol/l), p=0.008. 17 patients completed 6 months therapy with metformin. Of them, 13 responded clinically by restoration of regular menstrual cycles. The AMH levels of these 13 women decreased from 45.67±9.30 pmol/l to 38.25±6.89 pmol/l (16.27%). In the other 4 patients who did not show satisfactory clinical response to metformin, AMH levels increased from 31.30±16.52 to 80.77±12.73 (p=0.021). The patients who responded to metformin were significantly overweight, had higher BMI, waist circumference, body fat, and blood pressure as compared to nonresponders. AMH levels are significantly elevated in women with PCOS and they may serve as a marker for evaluation of treatment efficacy with metformin. Furthermore, obese PCOS patients are more likely to respond to metformin therapy with maximal doses as compared to the ones with low body mass index.

Timing of pubertal maturation according to the age at first conscious ejaculation.

The first conscious ejaculation (ejacularche) is an important event in the somatic and psychic development of boys. The aim of this study was to reveal the age at first ejaculation in Bulgarian boys and to observe the secular trend of this marker for puberty. A total of 1582 (901 from urban and 681 from rural origin respectively) gave a positive answer admitting the age in years at the first ejaculation. The mean age ± standard deviation (SD) of ejacularche was 13.27 ± 1.08 years, and the median was 13.0. It was found a significant difference between the urban (13.34 ± 1.07) and rural (13.18 ± 1.08) inhabitants in relation to the age of their remembered first ejaculation (P = 0.003). A secular trend in appearance of ejacularche was revealed when our results were compared with those from the previous studies in Bulgaria. We can suggest that in the presence of testicular volume ≥6 ml asking about ejaculation is reasonable and not superfluous. Self-reported spontaneous ejaculation can be used as an index of male pubertal timing.

[Menarche in Bulgarian--secular trend in twenty century].

UNLABELLED: The aim of the study was to determine the time of menarche in Bulgarian girls and specify the changes in menarcheal age during 20 century. MATERIAL AND METHODS: We examined in a longitudinal study 93 girls from 3 schools in Sofia in the period from 1994-2000. RESULTS: Mean age of menarche in girls was 11.96 + 0.75 years, (x + SD), median 12, 00 years. At the age of eleven 41.9% of the investigated girls have already had menarche and at the age of 10--4.3%. By the completion of the 12 years 90.3% were with menarche and at 14 years of age--100%. In a study in Bulgaria, done by the beginning of 20 century (1904-1906), the mean age of menarche was 15.0 + 3.32 years, 3 years later than it was found by us at the end of the century. CONCLUSION: We observed a secular trend of earlier time of menarche in Bulgarian girls during 20th century.

[Sperm disorder in males with obesity and metabolic syndrome--pilot study].

UNLABELLED: The aim of the study was to compare the semen quality in men with metabolic syndrome /MS/ and controls. MATERIALS AND METHODS: Semen samples were collected from 42 males (mean age--27.69 +/- 7.98 years). 21 of them had the features of the metabolic syndrome according to the IDF definition and 21 were healthy volunteers. The semen samples were analyzed according to the World Health Organization 1999 guidelines. RESULTS: The patients with MS had similar age, ejaculate volume, percentage of spermatozoa with normal morphology, sperm concentration (in million per milliliter), and total sperm count (in million) compared to controls. However, they had lower percentage of motile spermatozoa (p = 0.002). Men with obesity (BMI > 30) had significantly lower sperm concentration and total sperm count in comparison to normal- or overweight males (BMI < 30). CONCLUSION: reduced semen quality could be established in patients with obesity and MS. Further investigations are necessary to clarify the changes in the exocrine testicular function in males with MS and their consequences for the reproduction.

[Mechanism of interaction of tumor necrosis factor (TNF-alpha) of macroorganism with the Salmonella enterica cells (ser. Typhimurium)].

The goal of this work was to elucidate the mechanism of direct interaction of bacterial cells with tumor necrosis factor (TNF-alpha; cytokine). It was shown earlier that this interaction facilitated activation of bacterial growth and recultivation of non-cultivated forms in vitro and in vivo. It was shown in experiments with mice deficient in the genes encoding eucaryotic TNF-alpha receptors and infected with salmonella that addition of exogenous TNF-alpha to suspension of infection cells caused a one-day acceleration in the infection start (bacteria planting from spleen) in both knockouted and control mice relative to the same animals infected with the same bacteria without cytokine. Thus, bacteria are able to interact with cytokine even in the absence of eucaryotic receptors. Specificity of the bacterium-cytokine interaction and bacterial protein EF-Tu mediating direct interaction of bacteria with cytokine were identified using the method of immobilization of recombinant protein TNF-alpha-spacer-CSD on cellulose.

Melatonin-insulin interactions in patients with metabolic syndrome.

Metabolic syndrome (MS) as a group of risk factors is strongly associated with diabetes type 2 and cardiovascular disease. Insulin resistance plays a key role in the pathogenesis of MS. Recent studies have shown that melatonin may influence insulin secretion and glucose homeostasis. Therefore, the present study analyzed the relationships between the melatonin and the insulin in patients with MS and controls. The melatonin rhythm, insulin and lipid levels were studied in 40 subjects (21 patients and 19 controls) in reproductive age. The night melatonin-insulin ratio was correlated negatively with low-density lipoprotein cholesterol (r = -0.370, p = 0.024) and total cholesterol (r = -0.348, p = 0.030), and positively with high-density lipoprotein cholesterol levels (r = +0.414, p = 0.010). Night-time melatonin levels were related to night-time insulin concentrations (r = +0.313, p = 0.049). The correlation was pronounced in patients with MS (r = +0.640, p = 0.002), but did not reach statistical significance in controls (P > 0.05). In the patients with MS unlike the controls the night-day melatonin difference (%) correlated negatively with the fasting glucose (r = -0.494, p = 0.023) and positively to daily insulin (r = +0.536, p = 0.012). Our results show that melatonin-insulin interactions may exist in patients with MS, as well as relationships between melatonin-insulin ratio and the lipid profile. Pineal disturbances could influence the pathogenesis and the phenotype variations of the MS. Larger studies are needed to confirm or reject this hypothesis and to clarify the role of the melatonin in the metabolic disturbances.

Influence of ageing and some lifestyle factors on male gonadal function: a study in Bulgaria.

There are few systematic studies on the relationship between blood testosterone concentrations and the symptoms of androgen deficiency in ageing males. To assess the changes in sex hormone levels with age in relation with some lifestyle factors, the serum levels of total testosterone (TT), sex-hormone binding globulin (SHBG), luteinising hormone (LH) and follicle stimulating hormone (FSH) were measured in 33 men, age range 40-89 years. In addition, free testosterone (FT) and the free androgen index (FAI) were calculated. Seventeen healthy men under 40 years were involved as controls. The men over 40 years revealed significantly decreased TT, FT and FAI, and in the subgroup of men over 60 years, FSH and SHBG were significantly increased. Pearson's analysis showed that TT levels were significantly correlated with body mass index (BMI) (r = -0.464, P < 0.01) and body weight (r = -0.413, P < 0.05). SHBG levels were significantly correlated not only with age (r = +0.407, P < 0.05), but also with LH (r = +0.605, P < 0.001) and alcohol consumption (r = +0.382, P < 0.05). In conclusion, the TT, FT and FAI decreased in males over 40 years, but the alterations in hormone levels with age are more pronounced in men over 60 years. The important determinants of sex hormones are age, BMI and some lifestyle factors.

Disturbances in gonadal axis in women with anorexia nervosa.

UNLABELLED: Anorexia nervosa negatively affects multiple body systems including the reproductive system. AIM: To assess the disturbances in the hypothalamic-pituitary-gonadal axis (HPG) and the relationship between the gonadotropins and body weight, duration of the disease and amenorrhea we studied 40 female anorexic patients (aged 14-31 years) with a body mass index (BMI) 15.14+/-1.80 kg/m(2) and a degree of weight loss 28.67+/-8.74%. Fifteen healthy, age-matched women with normal weight served as controls. METHODS: We investigated the disturbances in the gonadotropin levels before and after stimulation with gonadotropin-releasing hormone (GnRH) 100 microg i.v. One week later 100 mg of clomiphene citrate (CC) was administered orally for 5 days. RESULTS: Basal levels of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were significantly lower in the patients. The responses of LH to GnRH were diminished, but those of FSH were exaggerated. However, after clomiphene citrate administration, LH increased 5.4 times whereas FSH increased 1.7 times. The basal levels of LH were significantly correlated with body weight (r=+0.373, p<0.05), BMI (r=+0.385, p<0.01) and percentage of the weight loss (r=-0.356, p<0.05). FSH levels were positively correlated with the duration of the disease (r=+0.481, p<0.01) and amenorrhea (r=+0.540, p<0.01). CONCLUSIONS: Our study demonstrates dissociation in the secretion of gonadotropins after hypothalamic stimulation in anorexic patients. It also reveals the relationship between alterations in the hormones of the HPG axis, not only with the changes in body weight, but also with the duration of the disease.