Second brain tumors following central nervous system radiotherapy in childhood.

OBJECTIVE: The second tumour (ST) occurrence is a relatively uncommon late complication of radiotherapy but represents one of the most significant issues, especially in childhood oncology. We describe our experience with patients who developed second brain neoplasm following cranial irradiation in childhood. METHODS: We identified nine patients who received radiotherapy owing to central nervous system tumour in childhood and subsequently developed the second brain tumour. The full clinical and radiological documentation and histopathological reports were reviewed. Risk factors such as age at irradiation, latency period to ST diagnosis, radiotherapy doses and volumes and other therapy methods were evaluated. We correlated the ST location with the three levels of irradiation dose (high, >40 Gy; medium, 25-40 Gy; and low <25 Gy). RESULTS: Five meningiomas and four gliomas occurred as the ST after the mean time of 11.7 years after radiotherapy. The average age of children during irradiation was 4.6 years. The shorter latency time to the ST induction was found in children treated with chemotherapy (9 years vs 17.2 years). Seven STs developed in the area of high and moderate dose (>25 Gy), only two low-grade gliomas appeared in the low-dose region. CONCLUSION: Our data suggest that the STs usually develop in the brain tissues that received doses >25 Gy in patients irradiated at a young age. ADVANCES IN KNOWLEDGE: The low-dose volume seems not to be so significant for second brain neoplasm induction. Therefore, the modern intensity-modulated radiotherapy technique could be safely applied in paediatric patients.

Superiority of thyroid peroxidase DNA over protein immunization in replicating human thyroid autoimmunity in HLA-DRB1*0301 (DR3) transgenic mice.

Murine experimental autoimmune thyroiditis (EAT), characterized by thyroid destruction after immunization with thyroglobulin (Tg), has long been a useful model of organ-specific autoimmune disease. More recently, porcine thyroid peroxidase (pTPO) has also been shown to induce thyroiditis, but these results have not been confirmed. When (C57BL/6 x CBA)F(1) mice, recently shown to be susceptible to mouse TPO-induced EAT, were immunized with plasmid DNA to human TPO (hTPO) and cytokines IL-12 or GM-CSF, significant antibody (Ab) titres were generated, but minimal thyroiditis was detected in one mouse only from the TPO + GM-CSF immunized group. However, after TPO DNA immunization of HLA-DR3 transgenic class II-deficient NOD mice, thyroiditis was present in 23% of mice injected with TPO + IL-12 or GM-CSF. We also used another marker for assessing the closeness of the model to human thyroid autoimmunity by examining the epitope profile of the anti-TPO Abs to immunodominant determinants on TPO. Remarkably, the majority of the anti-TPO Abs was directed to immunodominant regions A and B, demonstrating the close replication of the model to human autoimmunity. TPO protein immunizations of HLA-DR3 transgenic mice with recombinant hTPO did not result in thyroiditis, nor did immunization of other mice expressing HLA class II transgenes HLA-DR4 or HLA-DQ8, with differential susceptibility to Tg-induced EAT. Moreover, our efforts to duplicate exactly the experimental procedures used with pTPO also failed to induce thyroiditis. The success of hTPO plasmid DNA immunization of DR3(+) mice, similar to our reports on Tg-induced thyroiditis and thyrotropin receptor DNA-induced Graves' hyperthyroidism, underscores the importance of DR3 genes for all three major thyroid antigens, and provides another humanized model to study autoimmune thyroid disease.

Evaluation of conformational epitopes on thyroid peroxidase by antipeptide antibody binding and mutagenesis.

Autoantibodies to thyroid peroxidase (TPO) recognize predominantly conformational epitopes, which are restricted to two distinct determinants, termed immunodominant domain region (IDR) A and B. These dominant determinants reside in the region with structural homology to myeloperoxidase (MPO)-like domain and may extend into the adjacent complement control protein (CCP) domain. We have explored the location of these determinants on the MPO-like domain of the structural model of TPO, by identifying exposed hydrophilic loops that are potential candidates for the autoantigenic sites, generating rabbit antipeptide antisera, and competing with well characterized murine monoclonal antibodies (mabs) specific for these two IDRs. We recently defined the location of IDR-B, and here report our findings on the location of IDR-A and its relationship to IDR-B, defined with a new panel of 15 antipeptide antisera. Moreover, in combination with single amino acid replacements by in vitro mutagenesis, we have defined the limits of the IDR-B region on the TPO model. The combination of antisera to peptides P12 (aa 549-563), P14 (aa 599-617) and P18 (aa 210-225) inhibited the binding of the mab specific for IDR-A (mab 2) by 75%. The same combination inhibited the binding of autoantibodies to native TPO from 67 to 94% (mean 81.5%) at autoantibody levels of 5 IU. Fabs prepared from the antipeptide IgG and pooled in this combination were also effective in competition assays, thus defining the epitopes more precisely. IDR-A was found to lie immediately adjacent to IDR-B and thus the two immunodominant epitopes form an extended patch on the surface of TPO. Finally, by single amino acid mutagenesis, we show that IDR-B extends to residue N642, thus further localizing the boundary of this autoantigenic region on the structural model.

Human thyroid peroxidase: mapping of autoantibodies, conformational epitopes to the enzyme surface.

The enzyme, thyroid peroxidase (TPO), is a dominant antigen in thyroid autoimmune diseases. Autoantibodies recognised two major dominant conformational epitopes termed A and B. The epitopes have been defined by mAbs, but the amino acid residues which constitute these determinants remain unknown. Using a model of TPO, built from the structure of myeloperoxidase (MPO), we have synthesised peptides corresponding to exposed loops and generated rabbit antibodies to the peptides. Antisera to peptide sequence 599-617 (peptide 14) representing a highly protrusive loop on the TPO, showed the highest inhibition in 65 sera from patients positive with anti-TPO antibodies. The inhibition was by 15-80% (mean 41%), and no other antibody showed any inhibition. Binding of hFabs to the B determinant on TPO was inhibited by anti-peptide 14 antibodies more then 85%, but not Fabs to the A determinant. In conclusion, the peptide 14 defines a sequence taking part in building up the B major conformational epitope. None of generated anti-peptide antibodies alone inhibited the binding of human Fabs to the A epitope, however a combination of four anti-peptide antibodies (P1, P12, P14 and P18) inhibits Fabs binding to the A determinant by more then 60% and autoantibodies binding from 65% to 94%. Combination of antibodies reacting with peptides outside the surface defined by those four antipeptide antibodies did not give any inhibition of Fabs to TPO. The inhibition of Fabs and auto Abs to TPO by this combination of anti-peptide Abs is the result of steric hindrance as none of these Abs individually inhibited auto Abs' or Fabs' binding to TPO. The four peptides define an area on the enzyme surface where the A and B major conformational epitopes are localised.

Identification of an immunodominant region recognized by human autoantibodies in a three-dimensional model of thyroid peroxidase.

Autoimmune thyroid diseases (AITD) are characterized by the presence of autoantibodies to thyroid peroxidase (TPO). This response is dominated by autoantibodies to two conformational determinants, termed A and B, that have been defined by monoclonal antibodies but whose structures and location within TPO are unknown. We have modeled the three-dimensional structure of the extracellular region of TPO, raised antisera to prominent surface structures, and identified an epitope that we show to be a critical part of the B determinant. Antibodies to this epitope inhibit the binding to TPO of human autoantibodies in virtually all serum samples from 65 patients with AITD that were tested. This first description of a model of the three-dimensional structure and location of a major autoantigenic determinant within the TPO molecule may provide structural clues for identifying causative agents or developing novel therapeutic strategies.

Radiotherapy of central nervous system tumors in young children: benefits and pitfalls.

BACKGROUND: The prognosis for young children suffering from brain tumors is, as many authors report, generally poor. The probability of late complications in young children is also high, and their quality of life is significantly worse than that of older children. This study presents the experience of one center in management of central nervous system tumors in children in aged 1-3 years with radiation therapy. PROCEDURE: From 1981 to 1990, 52 children (aged 1-3 years) with CNS tumors were treated at the First Radiotherapy Department in the Center of Oncology in Warsaw, Poland. These patients represented 18% of the total number of 293 children with brain and spine tumors treated in this period. The radiation treatment policy for young children was similar to that applied to older children, but total doses and doses per fraction were lower and did not exceed 50 Gy to the tumor site and 30 Gy to the CNS axis. RESULTS: Overall 5-year survival was 52%, and disease-free survival was 50%. These results were similar to those obtained in older children (53% and 50%, respectively). Serious mental retardation (IQ < 70) was observed in 30% of young children in comparison to 7% of older patients, but in 62% these symptoms were noted even before the start of radiotherapy. CONCLUSIONS: The results obtained in this series did not confirm the hypothesis of more aggressive biology of CNS tumors in younger children. The proportion of children with serious psychological disturbances in this group is apparently high, although in most cases these symptoms were observed before the start of radiotherapy and were a result of tumor mass effects.

Distinct immunological and biochemical properties of thyroid peroxidase purified from human thyroid glands and recombinant protein produced in insect cells.

The biosynthesis of thyroid hormone from thyroglobulin is catalysed by thyroid peroxidase (TPO), an integral membrane protein. TPO is also a major autoantigen in autoimmune thyroid disease and autoantibodies to TPO are markers for disease activity. Large quantities of purified TPO are essential for elucidating its structure and understanding its role in disease activity. We describe the high yield purification of full-length recombinant human TPO from baculovirus infected insect cells and compare it to purified native TPO from human thyroid glands. In contrast to native human TPO, the human TPO produced in insect cells as a recombinant protein was insoluble and resistant to solubilisation in detergents. Reversible substitution of lysine residues with citraconic anhydride led to increased solubility of the recombinant TPO, allowing high-yield purification by monoclonal antibody chromatography. The purified enzyme preparation was shown to be TPO by its reactivity with monoclonal and polyclonal antibodies by enzyme linked immunosorbent assay and Western blotting. Both the human and recombinant purified TPO preparations also react with sera from patients with autoimmune thyroid disease, although the binding of conformational dependent autoantibodies was considerably lower to the recombinant TPO than to the native TPO. This suggests that the recombinant TPO may differ in some aspects of its tertiary structure. The purified recombinant TPO was devoid of enzyme activity, in contrast to the enzymatically active, purified human TPO preparations. Both preparations contained comparable amounts of haem (R(z)=0.269), but a shift in the Soret band of recombinant TPO (402 nm) from that of natural TPO (409 nm) indicates that the lack of enzymatic activity of the recombinant enzyme may be due to changes in the protein backbone surrounding the haem. Both the purified native and recombinant TPO, under non-denaturing conditions, show evidence of high molecular mass oligomers, although the latter preparation is prone to a greater degree of aggregation. In conclusion, our studies indicate that recombinant TPO generated in insect cells is conformationally distinct from the native TPO, is insoluble and enzymatically inactive, consistent with the difficulties associated with its purification and crystallisation.

Antiphospholipid antibodies and lipoprotein (a) in women with recurrent fetal loss.

OBJECTIVE: To estimate the occurrence of lupus anticoagulant, both isotypes of anticardiolipins, IgG and IgM, and lipoprotein (a) in women with recurrent fetal loss. METHOD: 43 women with two or more fetal losses were included in this open, prospective study. They were divided into a group of 32 nullipara who have had only miscarriages and a group of 11 women who besides the fetal losses were able to give birth to at least one live newborn. Two control groups were also added: 35 women who were never pregnant and 15 parous who did not have any miscarriage. The presence of lupus anticoagulant was assessed by the Staclot LA clotting test. IgG and IgM isotypes of anticardiolipins and lipoprotein (a) were determined using the immunoenzymatic methods. RESULTS: Lupus anticoagulant was discovered in only one of 23 examined patients. Abnormally high values of IgG isotype of anticardiolipins were found in two subjects of each control group and in two nullipara with miscarriages only. The increased level of IgM isotype was noted in approx. 50% of patients with recurrent fetal loss, but at least one successful delivery and in control subjects who were never pregnant. In contrast, the frequency of the increased level of this antibody was diminished by half in the remaining two groups: women who delivered a live child (children) without any miscarriage and women who had only miscarriages. Elevated levels of lipoprotein (a) were found in approx. 30% of non-pregnant control subjects and in the patients of both groups; the abnormally high concentration of this lipoprotein was encountered in only 13% of women who gave birth to live newborn(s) and did not have any miscarriage. CONCLUSION: Of all four determined variables, only abnormally high values of lipoprotein (a) may have some prognostic significance in women predisposed to the recurrent fetal loss.

Prevalence of high IgM anticardiolipins in patients with ischemic stroke.

Lupus anticoagulant (LA), IgG and IgM isotypes of anticardiolipins (aCL), lipoprotein (a), and the resistance to activated protein C were determined in patients with ischemic stroke. The raised concentration of the aCL-IgM isotype was noted in 42% of patients with this type of stroke, and it was in contrast with an 8% frequency of an increased level of aCL-IgG isotype in these cases. The high level of lipoprotein (a) was found with similar frequency in stroke patients and in age-matched control subjects. It is concluded that the elevated concentration of IgM isotype of anticardiolipin antibodies can be regarded as significant in the ethiological work-up in elderly stroke patients.Copyright Lippincott-Raven Publishers

Human thyroid peroxidase (TPO) isoforms, TPO-1 and TPO-2: analysis of protein expression in Graves' thyroid tissue.

Thyroid peroxidase (TPO) is the key enzyme involved in the biosynthesis of thyroid hormones and is an important autoantigen in autoimmune thyroid disease. Different messenger RNA species coding for TPO are present in thyroid tissue, including the species coding for a 933-amino acid protein (termed TPO-1) and a second in which exon 10 is deleted and which is 57 residues shorter (termed TPO-2). However, it is not known whether the smaller, TPO-2 isoform is expressed as a protein in thyroid cells. In SDS-PAGE under reducing conditions, TPO appears in the thyroid microsome and purified protein preparations as a closely migrating double band of approximately 105 (larger form) and 100 kilodaltons (smaller form). We investigated the presence of the isoform TPO-2 polypeptide in Graves' thyroid tissue using rabbit antisera to three different synthetic peptides from exon 10 (specific for TPO-1) and a polyclonal rabbit and monoclonal anti-TPO antibody (both of which are specific for the two forms of TPO). The larger and smaller forms of TPO were purified by electroelution after gel electrophoresis of highly purified natural TPO from Graves' thyroid microsomes. Both of the purified forms of TPO react with all three anti-exon 10 peptide antibodies, the polyclonal anti-TPO and the monoclonal antibody anti-TPO. This shows that both forms of TPO contain exon 10-encoded polypeptide of TPO-1. Interestingly, the proportion of the larger and smaller forms of TPO varied in different Graves' thyroid microsome preparations. To investigate the presence of the smaller TPO-2 isoform in the purified natural TPO preparation, affinity depletion of TPO-1 using the anti-exon 10 peptide antibodies was carried out. The binding of anti-exon 10 peptide antibodies to the immunodepleted TPO-1 fraction was considerably diminished in comparison to binding of polyclonal anti-TPO, suggesting the presence of small amounts (< 10%) of TPO-2 expressed as a protein in thyroid cells. Our results extend previous observations by showing that the alternatively spliced form of TPO, in which exon 10 is excised, is expressed at low levels in Graves' thyroid tissue. Furthermore, we confirm that both the larger and smaller forms of TPO observed on gel electrophoresis contain TPO-1, suggesting that the difference is caused by posttranslational modifications. The presence of small amounts of TPO-2 in Graves' thyroid glands argues for its role in thyroid function, which remains to be clarified.

Analysis of immunoglobulin G kappa antithyroid peroxidase antibodies from different tissues in Hashimoto's thyroiditis.

Antibodies (Ab) to thyroid peroxidase (TPO) are common in patients with autoimmune thyroid disease and may play a role in disease pathogenesis. We have prepared immunoglobulin G kappa (IgG kappa) and IgG lambda phage display combinatorial libraries from the cervical (thyroid-draining) lymph nodes of 2 Hashimoto's thyroiditis patients and from the thyroid of 1 patient. After selection with purified recombinant human TPO, up to 10 high affinity IgG kappa clones from each tissue source were analyzed further. No IgG lambda Fab were detected in the patient with the highest TPO Ab titer. Sequence analysis of the clones showed restricted heavy and light chain usage, similar to that in previously published TPO-reactive Fabs. This was despite the substantially larger sizes of the initial libraries, the use of lymph node tissue to generate libraries, and the analysis of the repertoire in patients with Hashimoto's thyroiditis rather than Graves' disease. There was overall similarity in sequences obtained from lymph node and thyroid libraries, with higher levels of somatic hypermutation in the former. The Fab inhibited binding of serum TPO Ab from five patients by 55-95%. These data together with those from previous reports indicate that although there is no unique Ab gene usage, there is the recurrent presence of certain variable regions in the high affinity TPO Ab response.

Purification and crystallisation of the autoantigen thyroid peroxidase from human Graves' thyroid tissue.

Milligram quantities of the human membrane autoantigen thyroid peroxidase (TPO) have been purified to a high degree of homogeneity by a combination of detergent solubilisation, monoclonal antibody affinity, and ion exchange chromatography, from pooled Graves' disease thyroid glands. The purified TPO of greater than 90% purity was enzymatically active as judged by its ability to oxidise guaiacol. Crystals of TPO have been grown from solutions of the protein solubilised in sodium deoxycholate, in the presence of ammonium sulphate. The crystals exhibited birefringence under polarised light, indicative of molecular order. Crystallisation of this large, membrane autoantigen represents the first step in delineating the complete three-dimensional structure of a human autoantigen involved in destructive thyroiditis.

Characterisation of the antibody response to the extracellular region of recombinant thyrotropin receptor.

Autoantibodies to the human thyrotropin receptor (TSH-R) are pathogenic in a number of autoimmune thyroid diseases including Graves' disease. We have characterised polyclonal antisera to TSH-R for antibodies which may mimic those present in autoimmune thyroid disease. For immunisations, recombinant extracellular region of human TSH-R which does not interact with its ligand TSH was used. The induced antibodies react with the full length membrane receptor in transfected mammalian cells by flow cytometry showing the presence of antibody capable of recognising the native functional receptor. The properties of the generated antibodies have been compared after two injections or following a multiple immunisation protocol with the receptor in adjuvant. High titre antisera were readily generated after the short injection protocol and further immunisations did not lead to any change in antibody titers. Analysis of the epitopes recognised using synthetic peptides confirmed previous observations that the immunodominant determinants localise to the amino and the carboxyl terminal part of the extracellular region of the receptor. Antisera from both rabbits contain TSH blocking antibody as assessed by inhibition of TSH mediated cAMP stimulation. There was an increase in TSH binding inhibitory immunoglobulin (TBII) activity with multiple injections. Furthermore, the increase in TBII activity was not related to spreading of the antibody response to new determinants on TSH-R. Our results support previous observations on the difficulties in reproducing, by adjuvant immunisation with recombinant TSH-R preparations, the fine specificity of antibodies to TSH-R present in autoimmune disorders such as Graves' disease or primary myxoedema.

Majority of thyroid peroxidase autoantibodies in patients with autoimmune thyroid disease are directed to a single TPO domain.

Murine monoclonal antibodies (mAb) produced against native human thyroid peroxidase (TPO) are powerful tools for analyzing the autoantibody (Aab) epitopes on TPO. Binding sites of thirteen mAbs cover all or most antigenic regions on TPO. We determined the competition between Aabs from 75 AITD patients and 13 mAbs in binding to TPO. Autoantibodies recognize predominantly the TPO area close or identical to mAb#9 epitope. All sera tested inhibited this mAb binding by 92.9 +/- 14.8 (mean +/- SD), range from 69-100%. AITD patients' sera with low Aabs titer up to 1/2,000 inhibited mAb#9 binding to TP0 by 85 +/- 11.5% (mean +/- SD) and did not influence remaining mAbs binding to TPO. With elevated Aab levels the inhibition of other mAbs binding was higher, but never exceeded 35%. The amount of Aabs yielding 50% inhibition of mAbs binding was lowest for mAb#9. In order to obtain this degree of inhibition for other mAbs 5 to 25 times more Aabs were needed. Our results demonstrate that the majority of autoantibodies in sera of patients with AITD recognize a single immunodominant region on the TPO mapped by mAb#9. They account for about 80-90% of serum TPO autoantibodies. The autoimmune response to other regions on TPO molecule is directed to several other epitopes, but represents quantitatively a minority of autoantibodies. This response intensifies with increasing Aabs level in the serum.

Hyperfractionated radiotherapy for brain stem tumours in children.

From 1988 to 1992 in the Centre of Oncology, Warsaw, 42 children with brain stem tumours were treated with hyperfractionated radiotherapy (HFRT). Two-year survival in nine (27%) patients was obtained. The HFRT treated group was compared with the historical, conventionally irradiated group with the same diagnosis. The hyperfractionated radiotherapy was well tolerated, but did not improve survival rate in comparison with conventionally treated group.

Iodine induced splitting of peptide bonds in human thyroglobulin.

Human thyroglobulin pretreated with iodine (1mM) at alkaline pH is split to small molecular weight fragments after reduction with dithiothreitol. Iodine pretreatment alone did not induce any changes in the thyroglobulin molecular weight.

Iodine induced alteration in immunological and biochemical properties of thyroglobulin.

The influence of iodine-iodide solution on the biochemical and immunological properties of human thyroglobulin (hTg) were studied. Human Tg preincubated with the iodine-iodide solution is split to small molecular mass fragments after disulphide bridge reduction with dithiothreitol. The peptide bond cleavage by iodine pretreatment and reduction is possibly linked with the coupling reaction of diiodotyrosyl residues. Pretreatment of hTg with iodine-iodide solution at 1-10 microM decreased the binding of autoantibodies to hTg. The iodine-iodide induced inactivation of hTg autoepitopes is pH dependent and is possibly caused by iodination of tyrosyl residues present in the epitope structure.

Radiotherapy of midbrain and brainstem tumours in children.

From 1984 to 1987 in the First Radiotherapy Department in the Centre of Oncology in Warsaw, 11 children with midbrain (group I) and 14 with brainstem (group II) tumours were treated. In 4 cases diagnostic biopsy was performed and in 21 diagnosis was established by CT scan. All children were treated with megavoltage radiotherapy with a Co-60 unit. The initial radiotherapy treatment volume was determined from CT scan and was subsequently adapted to include whole brain or whole cranio-spinal axis, depending on the response to treatment. Improvement or stabilization of disease in 23/25 (92%) of cases was observed. Total survival, longer than 3 years in 14/25 (56%) was observed, while 9/11 (82%) survived greater than 3 years (NED) in group I, and in 4/14 (28%) in group II. Ninety two percent of living children have normal school life, with minimal or no neurological defects.

The influence of iodine on the immunological properties of thyroglobulin and its immunological complexes.

Several papers described different immunological properties of thyroglobulin (Tg) after iodination. The influence of iodine-iodide solution on the immunological properties of hTg and its immunological complexes with autoantibodies (aAbs) were studied. Human Tg coated to polystyrene plates, incubated for 30 min with iodine-iodide solution at concentration from 1 to 200 microM at pH 9.0 lost its ability to bind aAbs. Preincubation with iodine (2 microM), decreased aAbs binding by 50%. Tg epitope inactivation induced by iodine depended on the buffer pH and the presence of carbonate ions. The binding of rabbit Tg-antibodies to iodine pretreated Tg was only slightly changed. Thyroglobulin preincubation with iodine solutions decreased aAbs binding from all tested sera (67) of patients with autoimmune thyroid disease (AITD). Excess of iodide (0.2 M KJ) or equimolar concentration of diiodotyrosine protects the Tg molecule from iodine induced inactivation. Immunological complexes of Tg with aAbs dissociate at low iodine concentrations. The results suggest that a product of iodine disproportionation reaction induces changes in the Tg molecule and Tg-aAb's complexes leading to complex dissociation or epitope inactivation.