Slobodan Apostolski

Serum and CSF immunological findings in ALS

Serum and CSF immunological findings were analysed in 37 patients with amyotrophic lateral sclerosis (ALS). ALS patients had significantly higher mean values of serum IgG and complement component C4 and significantly lower mean value of total haemolytic titre of complement (THC) compared with normal controls. Incidence of immune complexes (ICs) was significantly higher in sera of ALS patients than in normal controls. There was no significant difference regarding mean serum levels of IgM, IgA, and complement components C3 and Factor B between patients and controls. The blood‐brain barrier (BBB) damage was found in 46% of patients. Intrathecal IgG synthesis was detected in six patients (16%). These results support the hypothesis of immune system involvement in ALS.

Epidemiological and clinical characteristics of myasthenia gravis in Belgrade, Yugoslavia (1983–1992)

This is the first epidemiological study of myasthenia gravis (MG) in the area of Belgrade. During the survey period (1983–1992), 124 incidental cases of MG were observed, producing an average annual incidence rate of 7.1 per million population (women, 8.3; men, 5.8). Age and sex specific incidence rates for females demonstrated a bimodal pattern, with the first peak in the age group between 20 and 40, and the second peak in the age group 70–80. The age‐specific rates for males showed unimodal pattern, reaching a maximum in the age group between 60 and 80. There was a tendency of more frequent disease appearance in the urban as opposed to the suburban districts. On the prevalence day, December 31, 1992, the point prevalence rate was 121.5 per million (women, 142.5; men, 98.8). Only for incidental cases, the point prevalence rate was 77.1 (women, 83.2; men, 70.4). The average annual mortality rate was 0.47 per million (females, 0.52; males, 0.42), while cumulative lethality was 5.6 (women, 5.6; men, 5.7). Most frequently initial symptoms were ocular, occurring in 58% patients. Through the period of investigation ocular symptoms were generalized in 68%, most frequently in the first 2 years (62.5%). Thymoma was confirmed in 11.3% of patients. In this group there was equal presence of both sexes, older median age at onset, and more severe clinical course of MG. Associated autoimmune disease was found in 17 out of 124 incidental cases (13.7%). The most common were thyroid diseases (7.3%). Family history of MG was recorded in 2 cases belonging to 1 family (1.6%).

Cyclosporine in the treatment of myasthenia gravis

Cyclosporine A (CsA) treatment was evaluated in 52 patients with severe generalized myasthenia gravis (MG) whose illness was not controlled by anticholinesterase drugs, thymectomy, corticosteroids, and azathioprine. The efficacy of CsA treatment was expressed by mean disability score quotient (MDSQ), which was obtained by comparing mean disability score (MDS) at the beginning of the treatment with the MDS at the end of the follow‐up period. For the entire group of patients MDSQ was 53.3%, indicating moderate improvement. Analyzing individual cases, eight patients (15%) did not improve, 17 (33%) showed moderate improvement, 20 (38%) showed remarkable improvement, and seven patients (14%) achieved complete remission. The most common side effects were rise of serum creatinine (seven), hypertension (two), gingival hyperplasia (two), hypertrichosis (six), myalgia (10), and ‘flu‐like’ symptoms (10 patients). The results of this study suggest that CsA is efficacious and safe treatment in severe and resistant forms of MG.

Leflunomide prevents the development of experimentally induced myasthenia gravis

Myasthenia gravis is an autoimmune disease in which autoantibodies specific to the acetylcholine receptor (AChR) are formed, leading to a gradual destruction of the receptors in muscles that are responsible for picking up nerve impulses, and results in weakness and eventual loss of muscle function. The novel immunomodulating drug leflunomide (HWA 486) has been shown to be very effective in preventing and halting ongoing disease in an array of experimental autoimmune disorders and reactions leading to organ graft rejection. Further, recent data from phase II clinical trials indicate that this drug is efficacious and is safe in humans with rheumatoid arthritis. In the studies reported here, we found that rats immunized with AChR-protein and not receiving leflunomide developed experimental myasthenia gravis (EMG) between day 7 and 11 post-immunization, and about 79% of these animals expressed clinical signs of disease. Treatment of AChR-protein immunized rats with leflunomide, from the day of disease induction, totally suppressed the development of EMG. Thus, the results we have obtained using leflunomide in EMG indicate that this drug could be beneficial in combating myasthenia gravis in humans.

Impairment of cardiac autonomic control in patients with amyotrophic lateral sclerosis

Abstract The aim of this study was to investigate autonomic cardiac control in patients with amyotrophic lateral sclerosis (ALS). Fifty-five patients with sporadic ALS (28 female and 27 male; average age 56.00 ± 10.34 years) were compared to 30 healthy controls (17 female and 13 male; average age 42.87 ± 11.91 years). Patients with previous history of cardiac disease, diabetes mellitus, and impaired respiratory function were excluded from the study. Cardiovascular autonomic tests according to Ewing, power spectrum analysis of RR variability (low- and high-frequency bands – LF and HF, LF/HF index), real-time beat-to-beat ECG signal monitoring with heart rate variability analysis and baroreflex function analysis were carried out in all patients. Time-domain parameters of heart rate variability (mean RR interval, SDNN, SDANN, SDNN index, rMSSD and pNN50%) were obtained from 24-h ECG monitoring. ALS patients had a significantly higher score of sympathetic (p <0.01) and parasympathetic (p <0.001) dysfunction, as well as of the overall score of autonomic dysfunction (p <0.001). LF/HF index was significantly increased; baroreflex sensitivity and time-domain parameters of heart rate variability were highly significantly decreased in ALS patients (p <0.001). Our results demonstrated impaired cardiac autonomic control in ALS with marked parasympathetic dysfunction and sympathetic predominance.

Epidemiological and clinical characteristics of ALS in Belgrade, Yugoslavia

We present the results of the first epidemiological study of ALS in Belgrade. The distribution of 58 newly discovered cases in a 7‐year survey period (1985–1991) showed that the average annual age‐adjusted incidence rate was 0.42 per 100,000 population (95% confidence interval, 0.18–0.83). The rate for males was 1.5 times higher than the rate for females. The greatest age‐specific average incidence rate was observed in patients between 60 and 64 (3.66 per 100,000 population; 95% confidence interval, 2.17–5.78). The actual age‐adjusted prevalence rate on December 31, 1991 was 1.07 per 100,000 (95% confidence interval, 0.71–1.71). The mean age at onset of the disease was 56.2 ± 9.8 and it ranged from 24 to 74. We studied the natural course of the disease through the mean duration and cumulative probability of survival. The mean duration of the disease was 27.7 ± 18.2 months. The cumulative probability of survival was 27% for the whole population in a 5‐year interval. Elderly patients and those with bulbar signs at onset had a poorer prognosis. Patients under 49 at onset and those with the spinal form of the disease survived longer.

Biotransformation of nitric oxide in the cerebrospinal fluid of amyotrophic lateral sclerosis patients

Abstract Recent findings indicate that nitric oxide (NO•) over-production might be an important factor in the pathogenesis of sporadic amyotrophic lateral sclerosis (SALS). We measured significantly higher concentrations of uric acid and thiol group-containing molecules (R–SH groups) in the cerebrospinal fluid (CSF) from SALS patients compared to controls. The above factors, together with a slightly increased free iron concentration found in the CSF, favour conditions necessary for the formation of the dinitrosyl iron complex, capable of NO• bio-transformation. Thus, we performed ex vivo saturation of CSF (from both SALS patients and controls) with NO•. A decrease in the level of R–SH was found. This was more pronounced in the CSF from SALS patients. In the CSF from SALS patients the production of nitrite and hydroxylamine was greater than that observed in the CSF from controls. Moreover, we also found increased Cu,Zn-SOD activity in the CSF from SALS patients (when compared to control subjects) but no activity corresponding to Mn-SOD in any CSF samples. As Cu,Zn-SOD can react with nitroxyl forming NO•, the conditions for a closed, but continuous, loop of NO• biotransformation are present in the CSF of ALS patients.

Survival and mortality of myotonic dystrophy type 1 (Steinert's disease) in the population of Belgrade

The purpose of this investigation was to determine survival and mortality in patients with myotonic dystrophy type 1 (DM1) in the Belgrade population within the period from 1983 to 2002. Data of a number of diagnosed DM1 patients with their demographic, clinical and genetic characteristics were gathered from hospital records in all neurologic institutions in Belgrade for the period 1983–2002. Death certificates were reviewed to determine the cause of death. Survival analysis by life table method and Cox proportional hazard model was performed. Within the observed period, in the population of Belgrade, 15 fatal outcomes among 101 patients with DM1 were registered. Average DM1 mortality rate was 0.5/1 000 000 (95% CI 0.3–0.8), and standardized mortality ratio (SMR) was 5.3. A significant inverse correlation was found between age at onset of DM1 and CTG repeats (P = 0.023). The cumulative probability of 15‐year survival for DM1 patients in Belgrade was 49 ± 5% (48 ± 2% for males and 50 ± 7% for females). Younger age at onset was a significant unfavorable prognostic factor (hazard ratio = 4.2; P = 0.012).

CTG repeat polymorphism in DMPK gene in healthy Yugoslav population.

Objectives– Myotonic dystrophy type 1 (DM1) is caused by large expansions of cytosine‐thymine‐guanine (CTG)‐repeats in myotonic dystrophy protein kinase (DMPK)‐gene. This gene is highly polymorphic in healthy individuals. It has been proposed that expanded alleles originated from the group of large sized normal alleles. If this is correct, one should expect a positive correlation between the frequency of large sized normal alleles and a prevalence of this disorder in a population. In this paper we determined the distribution of alleles of DMPK gene in healthy Yugoslav population. Material and methods– A sample of 235 healthy individuals of Yugoslav origin have been genotyped for the alleles of DMPK locus. Results– We found 22 different alleles, ranging in size from 5 to 29 repeats. Among 470 chromosomes studied, 41 chromosomes had more than 18 repeats (8.72%). Conclusions– Relatively high frequency of large sized normal alleles found in our population, suggest that prevalence of DM1 in Yugoslavia should not be different from the prevalence in other European populations.

Intergenerational changes of CTG repeat depending on the sex of the transmitting parent in myotonic dystrophy type 1.

Sir, Myotonic dystrophy type 1 (DM1) is an autosomal dominant, multisystemic disease caused by expansion of a CTG repeat, located in the 3¢ untranslated region of dystrophia myotonica protein kinase (DMPK) gene in the chromosome region 19q13.3. Normal DMPK alleles contain between five and 37 copies of the CTG repeat, but this number is greatly increased in DM1 patients. DM1 is the locus that appears to show the most dramatic instability, often with very high levels of somatic mosaicism and very large intergenerational differences. To date, most studies have concentrated on the comparison of blood DNA from patients within families, and the parent–child differences observed defined as intergenerational differences (Harley et al., 1992; Ashizawa et al., 1993; O’Hoy et al., 1993; Martorell et al., 2000). Size of CTG repeat expansion usually increases upon intergeneration transmission and underlies the phenomena of genetic anticipation. However, the reduction in size of the DM1 trinucleotide repeat mutation during transmission is relatively rare. The aim of this study was to analyse the role of parents sex in intergenerational changes of CTG repeats in patients with DM1. Fifty-four DM1 patients (31 males and 23 females), aged between 19 and 51 years (mean ± SD: 36 ± 15), were studied. Diagnosis was based on clinical, electromyographic and genetic examination. Genomic DNA was isolated from white blood cells using proteinase K/SDS digestion and phenol-chloroform extraction. All subjects were studied by both polymerase chain reaction (PCR) and southern blot analyses. All samples showed a series of discrete bands representing somatic mosaicism of diseaseassociated alleles seen in the blood of DM1 patients. The size of each discrete band (expanded allele) was determined according to DNA molecular weight marker, using scatter plot on which band size of DNA marker was plotted against the length to which it migrated in the gel. For each patient the progenitor, the average, and the largest allele was estimated from the lower boundary of the series of discrete bands observed in allele distribution in lymphocytes beyond which rare alleles were detected in repeated analyses. PCR, as previously described (Brook et al., 1992), was performed for precise determination of wild type (wt) DMPK alleles size. Products were resolved on 6% denaturing polyacrylamide gels, detected by silver staining, and a number of CTG repeats was determined using a DNA sequencing ladder. Out of 54 investigated DM1 patients, 30 inherited the DM1 mutation from their mothers and 24 from their affected fathers. Analysis of variance showed that the mean of the smallest CTG expansion (progenitor allele) was smaller in patients with paternal DM1 mutation inheritance (194 ± 113 CTG repeats) than in those with maternal inheritance (292 ± 123 CTG repeats), P < 0.05. The same was found when comparing the means of the average expansions. Conversely, there was no significant influence of parents sex on the value of the largest CTG expansion (P > 0.05), Table 1. We analysed intergenerational changes of the smallest CTG repeats (progenitor alleles) in 20 parent– child pairs (16 mother–child and four father–child pairs). All 16 mother–child pairs exhibited increased CTG repeat expansion in the children (from 63 to 159 CTG repeats in the mothers and 129–596 CTG repeats in the children). A 22-yearold woman exhibited typical clinical signs for juvenile-adult type of DM1 and DMPK expansion range in blood cells from 429 to 900 repeats. As she has a risk of having children affected with congenital type of DM1, she was referred for prenatal diagnosis. CVS sample was taken during gestation week 10, and isolated DNA was tested for DM1 mutation using SP/LR PCR. The CVS sample was contaminated with maternal DNA. Repeated prenatal diagnosis from DNA isolated from amniotic fluid cells revealed the presence of expansion in the range of 2000–3000 CTG repeats. This pregnancy was terminated. From four father–child pairs, we found increased CTG repeat expansion in three pairs, but in one paternal transmission we found reduction (contraction) in size of the CTG repeats (from 113 in the father to 96 in the child). This study shows that maternal transmission of the DM1 mutation results in a greater number of CTG repeats than does paternal transmission. This suggests a greater instability of mutant alleles in female meiosis and predominantly