Aban M. Samuel

Differentiated thyroid carcinomas in children and adolescents

An analysis of differentiated thyroid carcinomas in children and adolescents revealed that the incidence was 3.05% of total number of patients with differentiated thyroid cancers in all age groups. There was a female preponderance. The incidence of papillary, follicular and papillary with follicular elements was equal. There were no papillary carcinomas observed in children younger than 10 years. The predominant mode of presentation was a solitary nodule of thyroid and some of them had associated cervical adenopathy. A considerable number presented with only cervical adenopathy. The incidence of nodal metastases was 50% at time of presentation and lung involvement was present in 15% of children at the time of diagnosis. Radioiodine treatment was given in 70% of children. Ablation was achieved in 86% of patients given two doses of radioiodine (200 millicuries). The more resistant cases were those with lung and nodal metastases. There was complete ablation in 100% with only residual thyroid tissue, 83% in those with associated nodal metastases, and 57% in those with lung involvement. Average duration of follow‐up was 10.3 years (range, 2 to 19 years). Recurrence rate or relapse was observed in 8.5% and was in the regional nodes. There was no recorded mortality due to the disease.

BRAIN METASTASES IN WELL-DIFFERENTIATED CARCINOMA OF THE THYROID

Fifteen patients (4 males and 11 females) developed brain metastases from well-differentiated thyroid cancer within 1 month to 14 years of the initial diagnosis. One patient presented with a brain tumor. Except for 3 patients with unique brain metastases, all the others had extensive metastases in nodes, lungs and bones in various combinations. Brain metastases generally appeared after the onset of metastases at other sites. The histology of the brain tumor matched the primary pathology in the 6 operated cases. The treatment was surgery and external radiation in 6 cases, and radioiodine or chemotherapy in the others. Survival in general was less than 6 months after the diagnosis of brain metastases. The prognosis is poor once the onset of brain metastases is evident.

Noninvasive scintigraphic method to quantify unstimulated secretions from individual salivary glands

PURPOSE Historically salivary gland function has been associated with maintenance of oral hygiene The difference in secretory behavior of parotid and submandibular glands has previously been shown. The purpose of this study was to establish a noninvasive technique for quantification of unstimulated (resting state) secretion of saliva based on the tracer output theory. PROCEDURES A total of 14 99mTc-pertechnetate salivary function studies were performed under Gamma camera. The time activity curves were subjected to a two step background subtraction protocol (area normalised background subtraction, followed by a graphical method for background correction). Individual salivary glands were modeled as Organ Curve = Input - Output. From these Uptake rates and unstimulated salivary gland fractional output rates (FOR) were calculated. MAIN RESULTS Parotid as well as submandibular glands have identical Uptake rates for the tracer. A distinct pattern was noted in submandibular glands as against parotid glands. Submandibular glands showed a steady rise in total quantity of unstimulated secretion. The FOR for submandibular glands was about three times higher than parotid glands (P< 0.000001). The observed distribution of FOR for parotid and submandibular glands FOR showed that parotid-FOR was normally on the lower side whereas submandibular-FOR showed a wide range of distribution which was multimodal in nature. PRINCIPAL CONCLUSION A unique approach has been presented for quantification of unstimulated salivary secretion. The method is simple and noninvasive.

Detection of antibodies to defined M. tuberculosis antigen (38 kDa) in cerebrospinal fluids of patients with tuberculous meningitis.

Antibodies to the 38 kDa antigen of M. tuberculosis which is serospecific to the tuberculosis complex group of organisms was studied in CSF samples of patients with tuberculosis meningitis. Patients were classified into four groups, viz. post-mortem-proved, culture-proved, clinically suspected and tuberculoma. Anti-38 kDa antibody was detected by ELISA and was positive in 60%, 80% 62.5% and 0%, respectively in the four groups. Controls showed a false-positive detection of 5%. Follow-up of patients was done up to 6 weeks and antibody levels dropped in all the patient groups.

Polymerase chain reaction in the diagnosis of tuberculosis. Comparison of two target sequences for amplification

Amplification of a 340 bp sequence of the 38 kDa protein gene of Mycobacterium tuberculosis by the polymerase chain reaction has been developed. The sensitivity of this PCR was shown to be 10 fg both by agarose gel electrophoresis and Southern blot hybridisation being equivalent to 2-3 organisms and highly specific to M. tuberculosis and excluding even M. tuberculosis H37Ra and Mycobacterium bovis BCG. Sputum samples from patients with pulmonary tuberculosis gave a positivity rate of 45%. PCR was also performed using pt8 and pt9 primers which amplified a 541 bp sequence of IS6110. 41% of the above samples gave positive amplification. Three samples that were positive for 38 kDa sequence were negative for IS6110.

Radioimmunoscintigraphic approach for the in vivo detection of tuberculomas—A preliminary study in a rabbit model

Radioimmunoscintigraphic approach could provide a viable non-invasive alternative for the diagnosis of deeply situated tuberculomas. We have evaluated this in a rabbit model constructed to give a characteristic localized tubercular lesion, with complimentary morphological, histological and antigenic profiles. 131I-anti Myobacterium bovis (BCG) antibody was shown to localize at the lesion, where as 131I-bovine serum albumin and 99mTc-red blood cell scans were negative. The clearance of intradermally-injected tuberculous antigen could be traced into ascending lymph nodes using 131I-anti M. bovis (BCG) antibody.

Use of the Recombinant 38-kDa Antigen of Mycobacterium tuberculosis as an Immunogen for Specific Antisera Production

The Mycobacterium tuberculosis 38‐kDa protein antigen is one of the secreted immunodominant antigens showing high immunogenicity at B‐cell and T‐cell levels. Although monoclonal antibodies to this antigen have been produced, specific polyclonal antisera is required for standardization of specific immunodiagnostic assays. This protein has been overexpressed and purified from recombinant Escherichia coli using an inducible vector system. During each stage of expression and purification, the recombinant protein was used to immunize mice and rabbits by several methods: 1) as overexpressed protein present as inclusion bodies in recombinant E. coli; 2) embedded in a polyacrylamide gel; 3) fixed to a solid‐phase nitrocellulose membrane and 4) emulsified with an adjuvant. All strategies yielded specific antisera as determined by enzyme‐linked immunosorbent assay (ELISA) and immunoblot analyses. The results obtained, both quantitative (ELISA) and qualitative (immunoblot) demonstrate that the purified recombinant antigen retains its antigenicity and immunogenicity throughout the various steps in the process of expression and purification and serves as a potent antigen for production of specific antisera to be used in immunoassays.

Mycobacterium tuberculosis 38kDa antigen and its encoding gene-experience in diagnostic applications.

ConclusionOur experience has revealed that the detection of 38 kDa antigen or antibody to the antigen in various fluids is useful in diagnosis of various mainfestations of tuberculosis. The PCR developed for 340bp sequence of its encoding gene also shows a high degree of sensitivity and specificity. Thus the 38 kDa antigen/antibody combination or the PCR are ideal for development of kits for diagnosis. These immunoassays to be successful, isolation of the 38 kDa antigen in large quantities is essential. Using recombinant DNA technology and expression inE. coli this has been achieved. However, such recombinant antigen does not have the same immunological properties as the native antigen and hence not suitable in immunodiagnosis. To fully realise the potential of the MoAb defined antigens such as the 38 kDa antigen, 19 kDa antigen and others it is essential to devise alternative vector-host systems that help in glycosylation, do not accumulate as inclusion bodies and can be isolated with less damage.

Study of anti-idiotype antibodies to the monoclonal antibody HGT3a and its relation to the 38 kDa antigen of Mycobacterium tuberculosis.

The hypervariable regions of the immunoglobulins which function as the antigen binding sites are capable of provoking an antibody response and are referred to as anti-idiotypic antibodies. Antisera were raised in rabbits against the idiotypes of a mouse monoclonal antibody HGT3a which binds only to the 38 kDa antigen of the M. tuberculosis complex group of organisms. Idiotype specificity in these antisera was determined by dot ELISA, Western blot and solid phase inhibition assays. In vivo administration of this rabbit anti-idiotypic antibody to Swiss mice evoked an anti-anti-idiotypic antibody response, further confirming the internal antigen mimicry by the anti-idiotypic antibodies of the 38 kDa antigenic epitope and its potential use as a surrogate antigen. Antibody response to the anti-idiotypic antibodies in the sera of patients with pulmonary tuberculosis showed significant correlation with the antibody response to the 38 kDa antigen studied in the same clinical samples indicating a close similarity of the 38 kDa antigen of M. tuberculosis and the rabbit anti-idiotypic antibody produced against MoAb HGT3a.

Increased sensitivity of triiodothyronine antibodies for radioimmunoassay after removal of endogenous antigen by simple laboratory procedures

Conventionally produced antibodies against triiodothyronine (T3) are known to possess high amounts of endogenously produced T3 associated with them. We felt that such antibodies would work better for T3 radioimmunoassay (RIA) after prior removal of the antigen. With this in view, we attempted dissociation and subsequent removal of T3 from antisera by two different methods, viz. dialysis and alcohol extraction. It was possible to remove T3 to an extent of 77% by alcohol extraction and 60% by dialysis. Resultant antisera fail to demonstrate any increase in the titre. However, when standard curves were generated using these antisera, the assays became more sensitive and it was possible to detect T3 in concentrations as low as 6.25 pg. The affinity constants of these antisera calculated from the respective Scatchard plots were found to have increased after both dialysis treatment was well as alcohol extraction. This was thought to be due to rendering some of the high affinity binding sites on the antibodies free of antigen after treatment. Serum T3 levels were measured in 68 patients with various thyroid status using both treated as well as untreated antiserum. The difference between the average values of serum T3 concentration estimated using various antisera before and after the treatment was not statistically significant. Our results suggested that a simple procedure like stripping of antigen from antibodies could be of help for acquiring high affinity and high sensitivity antibodies for this purpose.