Production, characterization and in-vitro applications of single-domain antibody against thyroglobulin selected from novel T7 phage display library.

Single- domain antibodies (SdAbs) have been deployed in various biomedical applications in the recent past. However, there are no reports of their use in the immunoradiometric assays (IRMA) for thyroglobulin (Tg). Tg is the precursor molecule for the biosynthesis of thyroid hormones: thyroxine and triiodothyronine, which are essential for the regulation of normal metabolism in all vertebrates. Patients with differentiated thyroid cancer (DTC) require periodic monitoring of their serum thyroglobulin levels, as it serves as a prognostic marker for DTC. Here, we report a methodology to produce SdAbs against human-Tg, by a hybrid immunization/directed-evolution approach by displaying the SdAb gene-repertoire derived from a hyperimmune camel in the T7 phage display system. We have demonstrated the immunoreactivity of anti-Tg-SdAb (KT75) in immunoassays for thyroglobulin and measured its affinity by surface plasmon resonance (KD ~ 18 picomolar). Additionally, we have shown the quantitative-binding property of SdAb for the first time in IRMA for thyroglobulin. The serum Tg values obtained from SdAb-Tg-IRMA and in-house assay using murine anti-Tg-monoclonal antibody as tracer significantly correlated, r = 0.81, p < 0.05. Our results highlight the scope of using the T7 phage display system as an alternative for the conventional M13-phage to construct single-domain antibody display libraries.

Comparison of Serum Thyroglobulin Levels in Differentiated Thyroid Cancer Patients Using In-House Developed Radioimmunoassay and Immunoradiometric Procedures.

Thyroglobulin (Tg) is a proven tumor marker in the follow-up and post-operative management of patients with differentiated thyroid cancer (DTC). All assays for serum thyroglobulin (s-Tg) are based on immunoassays, however, the assay technique has a bearing on the variations seen in the estimations. We studied this using four in-house developed radioimmunoassays (RIA) and immunoradiometric assays (IRMA). Limit of detection, working range, recovery, dilution test, precision profiles and method comparison were evaluated. All four methods were used for the estimation of s-Tg in DTC patients and also compared for their performance using commercially available Tg IRMA kits from DiaSorin and Izotop. The s-Tg values measured by six different immunoassays showed very significant inter-method correlation (0.84-0.99, p < 0.001). However, among the in-house developed assays; the coated tube IRMA showed a better sensitivity and precision at the lower concentration range and hence, is preferable for the routine measurement of s-Tg in patients negative for Tg autoantibodies (TgAb). Although the second generation IRMAs offer practical benefits of having higher sensitivity, shorter turn-around time and convenience of automation, they, unfortunately, also have higher tendency for interference from both TgAb and heterophilic antibodies, if present in the sample. On the contrary, RIA is less prone to such interference and, hence, can be used in patients with TgAb. In order to effectively use this test, it is important that nuclear medicine physicians and endocrinologists understand these intrinsic technical limitations encountered during s-Tg measurement.

Activation of murine macrophages by G1-4A, a polysaccharide from Tinospora cordifolia, in TLR4/MyD88 dependent manner.

Macrophages are centrally placed in the innate immune system and their activation is crucial to the generation of appropriate immune response in the event of any pathogenic invasion, tumorigenesis or other human diseases. Many plant derived polysaccharides are known to activate macrophages. In the present study, effects of G1-4A, a polysaccharide derived from Tinospora cordifolia, on the activation of macrophages were investigated. Our data demonstrated the up regulation of expression of TNF-α, IL-ß, IL-6, IL-12, IL-10 and IFN-γ in RAW 264.7 cell line and peritoneal macrophages after G-14A treatment. Nitric oxide levels were also enhanced along with up-regulation of NOS2 expression in murine macrophages post G1-4A treatment. Further, G1-4A treatment up-regulated the surface expression of MHC-II and CD-86 in macrophages. Using siRNA against TLR4, MyD88 and anti-TLR4 blocking antibodies, we established that G1-4A activated macrophages by classical pathway in TLR4-MyD88 dependent manner. Additionally, G1-4A treatment activated p38, ERK and JNK MAPKs in macrophages. Using pharmaceutical inhibitors of above MAPKs we concluded that G1-4A activates the macrophages by activation of p38, ERK and JNK MAPKs in RAW264.7 macrophages. Thus our data suggests the activation of macrophages by classical pathway after treatment of G1-4A.

Improved method for preparing Ni(II) complex of (S)-tyrosine Schiff base and its use in the automated synthesis of O-(2'-[18F]fluoroethyl)-l-tyrosine using solid-phase extraction purification.

O-(2'-[18F]fluoroethyl)-l-tyrosine is reportedly suitable for PET-imaging of brain tumours. We report here the synthesis of Ni(II)-complex of Schiff's base (S)-[N-2-(N'-benzylprolyl)amino]-benzophenone,((S)BPB) and alkylated l-tyrosine precursor, Ni(II)-(S)-BPB-l-Tyr-O-CH2-CH2-OTs by an improved method. A fully-automated radio-synthesis including non-HPLC purification was developed with a radio-chemical yield of 24.6 ± 2% in 70 ± 2min (n = 5). Radiochemical and enantiomeric purity was > 98% and 94% respectively. Bio-distribution and micro-PET studies in C57BL/6 mice bearing B16F10 melanoma showed tumor to brain ratio of 3.36 and 3.62 respectively at 60min post-injection.

A Microarray Immunoassay for Serum Thyrotropin and Thyroglobulin Using Antibodies Immobilized on Track-Etched Membranes.

Serum thyroglobulin (Tg) and thyroid stimulating hormone (TSH) measurements have evolved as important analytes for monitoring the prognosis of patients with differentiated thyroid cancer, post-thyroidectomy. Individual analyte immunoassay is the current practice in clinical pathology, but the simultaneous assay for all relevant analytes for a given disease, can reduce assay costs, improve patient compliance and give the clinician more information for an unequivocal diagnosis. Microarray immunoassay (MI) can achieve this goal and, hence, we have developed and validated a immuno-radiometric MI for quantitation of serum TSH and Tg by using highly micro-porous polycarbonate (PC) track-etched membranes (TEM) to immobilize the monoclonal anti-TSH and polyclonal anti-Tg antibodies in ~1 mm diameter spots. Non-competitive immunoassays were performed using mixture of 125I labeled monoclonal anti-TSH and anti-Tg antibodies. Phosphorimager was used to quantify the bound radioactivity. TSH and Tg were detected with detection limit of 0.07 µIU/ml and 0.13 ng/ml respectively, which is lower than the clinically required cut-off level. The assay showed: acceptable intra-assay precision within 20 % and recovery in the range of 76-111.2 %. MI compared well with the established immunoradiometric assay (IRMA) with r = 0.98, p < 0.01 (n = 41). No cross-reactivity was seen between the immobilized antibodies. Although two hormones are addressed in this report, MI using PC TEM and isotopic/non-isotopic tracers has the potential for highly automated multiplexed analysis.

In-House Solid-Phase Radioassay for the Detection of Anti-thyroglobulin Autoantibodies in Patients with Differentiated Thyroid Cancer.

Thyroglobulin autoantibodies (TgAb) are estimated to detect potential interferences in thyroglobulin (Tg) immunoassays and also for the diagnosis of autoimmune thyroid disease. A user friendly and robust in-house solid-phase radioassay was standardized and parameters like sensitivity, reproducibility and stability were assessed. Further, it was validated and evaluated for the detection of autoantibodies in differentiated thyroid cancer (DTC) patients. Totally 301 samples received in our laboratory for routine serum Tg estimation were studied. The samples were analyzed for TgAb by the solid-phase radioassay developed in-house and compared with commercial anti-hTg IRMA kit (Immunotech, France). The control group comprised of 37 euthyroid males from our Centre. The intra- and inter-assay CVs for the two quality control samples (Control A = 104 ± 12.6 IU/mL and Control B = 1029 ± 114 IU/mL) were found less than or equal to 6.05 and 13.85 % respectively. Solid-phase radioassay showed a good agreement on comparison with Immunotech IRMA (r = 0.99). Using the proposed cut-off thresholds (in-house solid-phase radioassay 52 IU/mL and Immunotech IRMA 30 IU/mL), 5.4 % of the control subjects were positive for TgAb by both the methods. Prevalence of TgAb in DTC patients was 17.3 and 16.6 % using the Immunotech kit and in-house solid-phase radioassay respectively. The in-house solid-phase radioassay has the requisite sensitivity for the evaluation of TgAb comparable to commercial kit and also suitable for routine use as it is rapid, user friendly and economical.

Radiochemical studies, pre-clinical investigation and preliminary clinical evaluation of 170Tm-EDTMP prepared using in-house freeze-dried EDTMP kit.

The objective of the present work is to formulate 170Tm-EDTMP using an in-house freeze-dried EDTMP kit and evaluate its potential as a bone pain palliation agent. Patient dose of 170Tm-EDTMP was prepared with high radiochemical purity using the lyophilized kit at room temperature within 15min. Pre-clinical evaluation in normal Wistar rats revealed selective skeletal accumulation with extended retention. Preliminary clinical investigation in 8 patients with disseminated skeletal metastases exhibited selective uptake in the bone and retention therein for a long duration.

G1-4A, a Polysaccharide from Tinospora cordifolia Inhibits the Survival of Mycobacterium tuberculosis by Modulating Host Immune Responses in TLR4 Dependent Manner.

Rapid emergence of drug resistance in Mycobacterium tuberculosis (MTB) is a major health concern and demands the development of novel adjunct immunotherapeutic agents capable of modulating the host immune responses in order to control the pathogen. In the present study, we sought to investigate the immunomodulatory effects of G1-4A, a polysaccharide derived from the Indian medicinal plant Tinospora cordifolia, in in-vitro and aerosol mouse models of MTB infection. G1-4A treatment of MTB infected RAW264.7 macrophages significantly induced the surface expression of MHC-II and CD-86 molecules, secretion of proinflammatory cytokines (TNF-α, IL-ß, IL-6, IL-12, IFN-γ) and nitric oxide leading to reduced intracellular survival of both drug sensitive (H37Rv) as well as multi drug resistant strains (Beijing and LAM) of MTB, which was partially attributed to G1-4A induced NO production in TLR4-MyD88 dependent manner. Similarly, bacillary burden was significantly reduced in the lungs of MTB infected BALB/c mice treated with G1-4A, with simultaneous up-regulation of the expression of TNF-α, INF-γ and NOS2 in the mouse lung along with increased levels of Th1 cytokines like IFN-γ, IL-12 and decreased levels of Th2 cytokine like IL-4 in the serum. Furthermore, combination of G1-4A with Isoniazid (INH) exhibited better protection against MTB compared to that due to INH or G1-4A alone, suggesting its potential as adjunct therapy. Our results demonstrate that modulation of host immune responses by G1-4A might improve the therapeutic efficacy of existing anti-tubercular drugs and provide an attractive strategy for the development of alternative therapies to control tuberculosis.

A Novel Folate-Targeted Nanoliposomal System of Doxorubicin for Cancer Targeting.

Targeted drug delivery systems for cancer improves anti-tumor efficacy and reduces systemic toxicity by restricting availability of cytotoxic drugs within tumors. Targeting moieties, such as natural ligands (folic acid, transferrin, and biotin) which are overexpressed on tumors, have been used to enhance liposome-encapsulated drug accumulation within tumors and resulted in better control. In this report, we explored the scope of targeting ligand folic acid, which is incorporated in liposome systems using folic acid-modified cholesterol (CPF), enabled highly selective tumor-targeted delivery of liposome-encapsulated doxorubicin and resulted in increased cytotoxicity within tumors. Folate-tagged poloxamer-coated liposomes (FDL) were found to have significantly higher cellular uptake than conventional poloxamer-coated liposomes (DL), as confirmed by fluorometric analysis in B16F10 melanoma cells. Biodistribution study of the radiolabeled liposomal system indicated the significantly higher tumor uptake of FDL as compared to DL. Anti-tumor activity of FDL against murine B16F10 melanoma tumor-bearing mice revealed that FDL inhibited tumor growth more efficiently than the DL. Taken together, the results demonstrated the significant potential of the folate-conjugated nanoliposomal system for drug delivery to tumors.

Identification of a novel class of quinoline-oxadiazole hybrids as anti-tuberculosis agents.

A series of novel quinoline-oxadiazole hybrid compounds was designed based on stepwise rational modification of the lead molecules reported previously, in order to enhance bioactivity and improve druglikeness. The hybrid compounds synthesized were screened for biological activity against Mycobacterium tuberculosis H37Rv and for cytotoxicity in HepG2 cell line. Several of the hits exhibited good to excellent anti-tuberculosis activity and selectivity, especially compounds 12m, 12o and 12p, showed minimum inhibitory concentration values<0.5µM and selectivity index>500. The results of this study open up a promising avenue that may lead to the discovery of a new class of anti-tuberculosis agents.

Mycobacterium tuberculosis H37Rv infected THP-1 cells induce epithelial mesenchymal transition (EMT) in lung adenocarcinoma epithelial cell line (A549).

Chronic infections of Mycobacterium tuberculosis (MTB) cause oxidative stress, TLR activation and production of inflammatory cytokines and thus can create an environment reinforcing tumorigenesis, progression and metastasis. Epidemiological studies have established a relation between lung cancer and tuberculosis but cellular mechanism is still poorly understood. In present study, we have shown for the first time that MTB infection in human monocytic cell line (THP-1) enhances invasion and induces EMT characteristics in lung adenocarcinoma cell line (A549) during co-culture. After co-culture with MTB infected THP-1 cells A549 cells exhibited morphological and molecular signatures of EMT. During co-culture, expression of inflammatory cytokines like TNF-α, IL-1ß and IL-6 was enhanced in the microenvironment of A549 cells in comparison to single culture of A549 cells. Using pharmacological inhibitors of JNK (SP-600125) and p38 MAPK (SB-203580), we demonstrated the involvement of JNK and p38 MAPK in MTB induced EMT induction in A549 cells. To the best of our knowledge this is the first report demonstrating the role of MTB infection in induction of metastasis associated EMT in lung cancer.

Antifolate Activity of Plant Polyphenols against Mycobacterium tuberculosis.

With the view of exploring phytochemicals as Mycobacterium tuberculosis (Mtb) dihydrofolate reductase inhibitors, known plant polyphenols from various classes were subjected to detailed docking studies. From this in-silico screening, seven polyphenols were selected and tested against Mtb H37 Rv in whole cell assays. The phytochemicals exhibited potential activity ranging from 3 to 183 µm. These molecules were then tested against the pathogenic and human enzymes in a high-throughput microtitre assay. Epigallocatechin gallate showed the best activity and selectivity. The in-silico analysis was in agreement with the assay results. Of these 7 polyphenols, 5 exhibiting minimum inhibitory concentration values of ≤15 µm were tested for synergistic activity with first line drug Ethambutol and second line folate inhibitor para-amino salicylic acid. Epigallocatechin gallate, Magnolol and Bakuchiol exhibited moderate synergistic association by lowering the minimum inhibitory concentration of these drugs. These simple phytochemicals could hence be considered as leads for further studies, or for preparation of semi-synthetic derivatives to be used in combination therapy, for increased anti-tuberculosis activity after validation in-vivo.

Early gestation screening of pregnant women for iodine deficiency disorders and iron deficiency in urban centre in Vadodara, Gujarat, India.

Pregnancy is a special condition where many metabolic changes may occur because of increased requirement of essential micronutrients such as iron and iodine. Foetal thyroid starts producing its own thyroid hormones after 12 weeks of gestation. Therefore, the first trimester is very crucial for meeting thyroid hormone requirements of the mother and foetus. Iodine deficiency and iron deficiency may affect mental and physical growth of the foetus. Hence, it is very important to establish a programme on the screening of pregnant women for thyroid dysfunction tests along with established iron status assessment. Thus, the study was aimed to screen the pregnant women for iodine deficiency disorders and iron deficiency during early gestation, situational analysis on thyroid insufficiency and iron deficiency in pregnant women (gestational age <15 weeks) in urban Vadodara, Gujarat. n = 256 healthy pregnant women with uncomplicated singleton pregnancy were selected. The thyroid hormone was estimated by RIA, UIE using simple microplate technique and haemoglobin (Hb) concentration by acid hematin method. Median thyrotropin (TSH), free thyroxine (FT4), total thyroxine (TT4) and UIE concentrations were 1.88 µIU/ml, 0.83 ng/dl, 10.24 µg/dl and 297.14 mcg/l, respectively. There was a significant correlation between TSH, FT4 and month of gestation. Mean Hb concentration was 9.27 ± 1.09 g/dl. The prevalence of iodine insufficiency (based on UI) was 16.79% and iron deficiency was 91%. Screening programme for iodine deficiency during early gestation should be implemented along with the existing programme of haemoglobin estimation at first prenatal visit. This would help prevent damage to the developing brain and growth of the foetus and also to trace at-risk pregnant women.

Rational drug design based synthesis of novel arylquinolines as anti-tuberculosis agents.

A series of novel arylquinoline derivatives was designed retaining significant pharmacophoric features and three dimensional geometry of bedaquiline. In silico ADME study was performed to assess drug likeness and toxicity profiles of the designed molecules. The compounds were evaluated for activity against Mycobacterium tuberculosis H37Rv using Resazurin Microtitre Assay (REMA) plate method and cytotoxicity in VERO C1008 cell line. Several of the synthesized compounds exhibited good antituberculosis activity and selectivity, especially compounds, 12i (MIC: 5.18 µM and MIC/CC50: 152.86) and 12l (MIC: 5.59 µM and MIC/CC50: 160.57). The study opens up a new platform for the development of arylquinoline based drugs for treating tuberculosis.

BRAF V600E mutation in papillary thyroid carcinoma: significant association with node metastases and extra thyroidal invasion.

B-Raf (BRAF) is the strongest activator in the downstream of MAP kinase signaling. The somatic point mutation of BRAF gene (V600E) is the most common and specific event in papillary thyroid carcinoma (PTC). However, its prevalence is variable among different studies and its association with clinico-pathological features is controversial. This study tests the prevalence of BRAF (V600E) mutation in thyroid cancer patients in Indian subcontinental population. We analyzed 140 thyroid tumor specimens for BRAF gene mutation at codon 600 using mutant-allele-specific amplification, single-strand conformation polymorphism, Mutector assay, and DNA sequencing of the PCR-amplified exon 15. BRAF mutation at codon 600 was detected in 46 of 86 PTC patients (53.4%) from Indian subcontinental cohort. Frequency of mutation varied across the subtypes of PTCs. BRAF (V600E) mutation was more common in the conventional PTC (38 out of 62; 61%) than in the follicular variant of PTC (2 out of 17; 11.7%). None of the 8 follicular thyroid adenomas, 14 follicular thyroid carcinomas, 16 medullary thyroid carcinomas, and 16 benign hyperplasia patients showed any exon 15 mutation. We found significant correlation between BRAF mutation status and extra-thyroidal invasion, lymph node metastasis, and tumor stage. However no correlation was observed with gender, age, and tumor size of the patients. Thus our findings suggest that BRAF (V600E) is a prevalent genetic alteration in adult sporadic PTCs in Indian cohort and it may be responsible for the progression of classic variant of PTC to metastatic and poorly differentiated subtype and likely to have significant impact on its diagnostic and prognostic management.

Radioprotective effect of Ocimum sanctum and amifostine on the salivary gland of rats after therapeutic radioiodine exposure.

The current study investigated the radioprotective effect of Ocimum sanctum on the salivary gland of rats administered radioiodine ((131)I) and compared its efficacy with a known radioprotectant, amifostine. The experimental rats were divided in four groups and sacrificed in three different batches at 1, 3, and 6 months of time interval after 18.5 MBq/100g (i.p.) (131)I exposure. Six months duration batch received (131)I exposure twice with the gap of 3 months. Two groups of experimental rats were presupplemented with O. sanctum (40 mg/kg for 5 days, orally) and amifostine (200 mg/kg, s.c) before (131)I exposure separately. Increased Technetium-99m-pertechnetate ((99m)TcO(4)(-)) uptake at 30 minutes post injection in salivary glands of only (131)I exposed rats may imply delay in clearance at 6 months of exposure in comparison to their counterparts sacrificed at 1 month. Parotid gland histology showed atrophy with lipomatosis in only (131)I exposed rats at 3 and 6 months of duration. O. sanctum and amifostine presupplemented and subsequently exposed to (131)I rats at 3 and 6 months duration exhibited comparable histopathology with controls. Our study indicates possible radioprotective effect of O. sanctum and amifostine against high-dose (131)I exposure.

Fluorine-18 radiopharmaceuticals beyond [18F]FDG for use in oncology and neurosciences.

Positron emission tomography (PET) is a rapidly expanding clinical modality worldwide thanks to the availability of compact medical cyclotrons and automated chemistry for the production of radiopharmaceuticals. There is an armamentarium of fluorine-18 ((18)F) tracers that can be used for PET studies in the fields of oncology and neurosciences. However, most of the (18)F-tracers other than 2-deoxy-2-[18F]fluoro-D-glucose (FDG) are in less than optimum human use and there is considerable scope to bring potentially useful (18)F-tracers to clinical investigation stage. The International Atomic Energy Agency (IAEA) convened a consultants' group meeting to review the current status of (18)F-based radiotracers and to suggest means for accelerating their use for diagnostic applications. The consultants reviewed the developments including the synthetic approaches for the preparation of (18)F-tracers for oncology and neurosciences. A selection of three groups of (18)F-tracers that are useful either in oncology or in neurosciences was done based on well-defined criteria such as application, lack of toxicity, availability of precursors and ease of synthesis. Based on the recommendations of the consultants' group meeting, IAEA started a coordinated research project on "Development of (18)F radiopharmaceuticals (beyond [(18)F]FDG) for use in oncology and neurosciences" in which 14 countries are participating in a 3-year collaborative program. The outcomes of the coordinated research project are expected to catalyze the wider application of several more (18)F-radiopharmaceuticals beyond FDG for diagnostic applications in oncology and neurosciences.

The possibility of a fully automated procedure for radiosynthesis of fluorine-18-labeled fluoromisonidazole using a simplified single, neutral alumina column purification procedure.

A novel fully automated radiosynthesis procedure for [(18)F]Fluoromisonidazole using a simple alumina cartridge-column for purification instead of conventionally used semi-preparative HPLC was developed. [(18)F]FMISO was prepared via a one-pot, two-step synthesis procedure using a modified nuclear interface synthesis module. Nucleophilic fluorination of the precursor molecule 1-(2'-nitro-1'-imidazolyl)-2-O-tetrahydropyranyl-3-O-toluenesulphonylpropanediol (NITTP) with no-carrier added [(18)F]fluoride followed by hydrolysis of the protecting group with 1M HCl. Purification was carried out using a single neutral alumina cartridge-column instead of semi-preparative HPLC. The maximum overall radiochemical yield obtained was 37.49+/-1.68% with 10mg NITTP (n=3, without any decay correction) and the total synthesis time was 40+/-1 min. The radiochemical purity was greater than 95% and the product was devoid of other chemical impurities including residual aluminum and acetonitrile. The biodistribution study in fibrosarcoma tumor model showed maximum uptake in tumor, 2h post injection. Finally, PET/CT imaging studies in normal healthy rabbit, showed clear uptake in the organs involved in the metabolic process of MISO. No bone uptake was observed excluding the presence of free [(18)F]fluoride. The reported method can be easily adapted in any commercial FDG synthesis module.

Evaluation of serum vascular endothelial growth factor (VEGF) and microvessel density (MVD) as prognostic indicators in carcinoma breast.

PURPOSE: Vascular endothelial growth factor (VEGF) is a potent angiogenic peptide. A great deal of interest has been paid to the predictive value of neoangiogenesis represented by microvessel density (MVD), on clinical progression and prognosis of several types of tumors. Serum VEGF levels may therefore be clinically useful for the prediction of increase in tumor growth, metastasis or recurrence spread in individual patients. METHODS: A total of 265 cases of breast lesions were studied to note the importance of Serum VEGF as a prognostic marker in cases of breast carcinoma. The expressed serum VEGF levels and microvessel density (MVD) were assessed quantitatively and were correlated with tumor grade, tumor necrosis, stromal reaction and nodal metastasis. RESULTS: Serum VEGF was increased in patients with lesions of breast and the levels of serum VEGF in malignant lesions were significantly increased when compared to benign lesions. It was also noted that the levels of serum VEGF increased with increasing grades of malignancy. MVD showed a significant correlation in the early stages of the malignant tumors, where there was no necrosis, but in tumors associated with necrosis and hemorrhage MVD failed to show significant correlation. CONCLUSION: Hence, serum vascular endothelial growth factor can be used as a more reliable, non-invasive adjunctive diagnostic criteria in the assessment of the grade and hence, the prognosis of malignant tumors of the breast.