Conversion of anti-tissue factor antibody sequences to chimeric antigen receptor and bi-specific T-cell engager format.

BACKGROUND: The efficacy of antibody-targeted therapy of solid cancers is limited by the lack of consistent tumour-associated antigen expression. However, tumour-associated antigens shared with non-malignant cells may still be targeted using conditionally activated-antibodies, or by chimeric antigen receptor (CAR) T cells or CAR NK cells activated either by the tumour microenvironment or following 'unlocking' via multiple antigen-recognition. In this study, we have focused on tissue factor (TF; CD142), a type I membrane protein present on a range of solid tumours as a basis for future development of conditionally-activated BiTE or CAR T cells. TF is frequently upregulated on multiple solid tumours providing a selective advantage for growth, immune evasion and metastasis, as well as contributing to the pathology of thrombosis via the extrinsic coagulation pathway. METHODS: Two well-characterised anti-TF monoclonal antibodies (mAb) were cloned into expression or transposon vectors to produce single chain (scFv) BiTE for assessment as CAR and CD28-CD3-based CAR or CD3-based BiTE. The affinities of both scFv formats for TF were determined by surface plasmon resonance. Jurkat cell line-based assays were used to confirm the activity of the BiTE or CAR constructs. RESULTS: The anti-TF mAb hATR-5 and TF8-5G9 mAb were shown to maintain their nanomolar affinities following conversion into a single chain (scFv) format and could be utilised as CD28-CD3-based CAR or CD3-based BiTE format. CONCLUSION: Because of the broad expression of TF on a range of solid cancers, anti-TF antibody formats provide a useful addition for the development of conditionally activated biologics for antibody and cellular-based therapy.

A unique approach to screen for blood donors lacking high-prevalence antigen Inb of the Indian blood group system.

The Inb antigen of the Indian blood group system is a high-prevalence antigen. The presence of alloanti-Inb in a recipient may pose a problem in finding compatible blood for transfusion. The aim of this study was to screen blood donors for Inb and to include individuals found to be In(b-) in our rare donor registry. To save resources, a unique study design was constructed. Blood group O donors were tested for Inb because their red blood cell (RBC) units could serve recipients across all ABO groups. EDTA blood samples were used for serologic and genomic testing. These samples were first tested serologically for Ina, and samples typed as In(a+) were then tested both serologically and molecularly for Ina and Inb to find homozygous IN*01/01 [i.e., the predicted In(b-) phenotype]. A cost-conservative approach in using recycling of antibody was adopted to economize available resources. Of 6300 donors, 196 donor samples typed as In(a+) and were also found to be In(b+) when tested by serologic and genomic methods. Although none of the donors typed as In(b-), the statistical analysis suggests the expected prevalence for this rare phenotype to be 0.02 percent among the total number of donors tested. In conclusion, this report presents a unique cost-conservative approach using limited reagents to screen a large number of donors for the rare In(b-) phenotype.The Inb antigen of the Indian blood group system is a high-prevalence antigen. The presence of alloanti-Inb in a recipient may pose a problem in finding compatible blood for transfusion. The aim of this study was to screen blood donors for Inb and to include individuals found to be In(b­) in our rare donor registry. To save resources, a unique study design was constructed. Blood group O donors were tested for Inb because their red blood cell (RBC) units could serve recipients across all ABO groups. EDTA blood samples were used for serologic and genomic testing. These samples were first tested serologically for Ina, and samples typed as In(a+) were then tested both serologically and molecularly for Ina and Inb to find homozygous IN*01/01 [i.e., the predicted In(b­) phenotype]. A cost-conservative approach in using recycling of antibody was adopted to economize available resources. Of 6300 donors, 196 donor samples typed as In(a+) and were also found to be In(b+) when tested by serologic and genomic methods. Although none of the donors typed as In(b­), the statistical analysis suggests the expected prevalence for this rare phenotype to be 0.02 percent among the total number of donors tested. In conclusion, this report presents a unique cost-conservative approach using limited reagents to screen a large number of donors for the rare In(b­) phenotype.

Photoluminescent thermometer probes: temperature measurements in microwave fields.

Based on luminescence, the photoluminescent thermometer can in principle function in electromagnetic field coupling and perturbation. Several prototypal probes that have been constructed and tested demonstrate the feasibility of the concept. Temperature resolution of approximately 0.3 degrees C has thus far been achieved. The probes are needle-shaped, sturdy and less than 1 millimeter in size. This thermometer system has excellent potential application in monitoring and controlling tissue temperatures when microwaves are used to induce hyperthermia for cancer treatment.

In vitro biocontrol activity of halotolerant Streptomyces aureofaciens K20: A potent antagonist against Macrophomina phaseolina (Tassi) Goid.

A halotolerant actinobacterial strain isolated from salinity affected soil of Eastern Indo-Gangetic plains (IGP), Uttar Pradesh, India, was characterised for its antagonistic potential against Macrophomina phaseolina by dual-culture assay. It was shown to effectively inhibit the growth of M. phaseolina with an inhibition zone of 27 ± 1.33 mm. Further the actinobacterial strain was evaluated for its plant growth promoting (PGP) properties and its ability to produce biocontrol related extracellular enzymes viz. amylase, protease, cellulase, chitinase, gelatinase and urease. The results revealed that the actinobacterial strain had PGP potential along with positive assay for amylase, chitinase and urease. The interaction study between antagonist strain and fungal pathogen, performed by scanning electron microscopy technique revealed that the actinobacterium was able to damage fungal mycelia may be due to chitinase, establishing its role as a potential antagonist against M. phaseolina. The actinobacterial isolate was characterised by 16S rDNA gene sequencing, and was identified as Streptomyces genera. The identified gene sequence was deposited to NCBI GenBank with an accession number KP331758.

Oxetane locked thymidine in the Dickerson-Drew dodecamer causes local base pairing distortions -- an NMR structure and hydration study.

The introduction of a North-type sugar conformation constrained oxetane T block, 1-(1',3'-O-anhydro-beta-D-psicofuranosyl) thymine, at the T(7) position of the self-complementary Dickerson-Drew dodecamer, d[(5'-C(1)G(2)C(3)G(4)A(5)A(6)T(7)T(8)C(9)G(10)C(11)G(12)-3')](2), considerably perturbs the conformation of the four central base pairs, reducing the stability of the structure. UV spectroscopy and 1D NMR display a drop in melting temperature of approximately 10 degrees C per modification for the T(7) oxetane modified duplex, where the T(7) block has been introduced in both strands, compared to the native Dickerson-Drew dodecamer. The three dimensional structure has been determined by NMR spectroscopy and has subsequently been compared with the results of 2.4 ns MD simulations of the native and the T(7) oxetane modified duplexes. The modified T(7) residue is found to maintain its constrained sugar- and the related glycosyl torsion conformations in the duplex, resulting in staggered and stretched T(7).A(6) and A(6).T(7) non-linear base pairs. The stacking is less perturbed, but there is an increased roll between the two central residues compared to the native counterpart, which is compensated by tilts of the neighboring base steps. The one dimensional melting profile of base protons of the T(7) and T(8) residues reveals that the introduction of the North-type sugar constrained thymine destabilizes the core of the modified duplex, promoting melting to start simultaneously from the center as well as from the ends. Temperature dependent hydration studies by NMR demonstrate that the central T(7).A(6)/A(6).T(7) base pairs of the T(7) oxetane modified Dickerson-Drew dodecamer have at least one order of magnitude higher water exchange rates (correlated to the opening rate of the base pair) than the corresponding base pairs in the native duplex.

Iodine deficiency disorders among school children of Dakshin Dinajpur District, West Bengal.

Iodine deficiency disorders (IDD) are major public health problems in India, including West Bengal. Existing programme to control IDD needs to be continuously monitored through recommended methods and indicators. Thus we undertook the study to assess the prevalence of goiter, status of urinary iodine excretion (UIE) level and to estimate iodine content of salts at the household level in Dakshin Dinajpur district, West Bengal. We conducted a community-based, cross-sectional study in 2004; among 2250 school children, aged 8-10 years. The '30 cluster' sampling methodology and indicators for assessment of IDD, as recommended by the joint WHO/UNICEF/ICCIDD consultation, were used for the study. Goitre was assessed by standard palpation technique, UIE was analyzed by wet digestion method and salt samples were tested by spot iodine testing kit. Of the 2250 children, 419 (18.6%) had goitre (95% CI = 17.0 - 20.2%). Total goitre rate (TGR) was not significantly different in respect of gender, age and religion. Visible goitre rate was 2.5%. Median urinary iodine excretion level was 16 mcg/dL (normal: > or = 10 mcg/dl.) and 16.5% children had value less than 5 mcg/dL. Only 67.4% of the salt samples tested had adequate iodine content of > or = 15 ppm, with significant difference between Hindus and Muslims (chi2 = 12.68, d.f. = 1, p < 0.01). TGR of 18.6% indicate the district is still endemic for IDD, but median urinary iodine within normal range reflects no current iodine deficiency. The district is in the transition phase from iodine-deficient to iodine-sufficient. Measures are to be sustained for successful transition towards elimination.

Hyperthermia thermometry evaluation: criteria and guidelines.

Results of the evaluation of thermometry devices used during hyperthermia treatments at 14 different clinics in the USA are presented. Measurements were made by the Hyperthermia Physics Center (HPC, a national hyperthermia quality assurance program under NCI contract No. N01-CM-37512) according to a protocol. Our sample included thermocouples, fiberoptic thermometers, and high lead resistance thermistors. We found that only some but not all of the thermometers of each kind performed within the +/- 0.2 degrees C acceptability criteria of accuracy. The precision, stability, and response times achieved with each type of thermometer are presented. A summary of perturbations and artifacts typical for each system is presented together with suggested precautions to avoid them during clinical usage. We conclude that although the technology used with each thermometer system is capable of producing a temperature accuracy of 0.2 degrees C, this accuracy is clinically achievable only with a concerted effort and a constant alertness on the part of the investigator. Based on the combined experience of this survey, the clinical investigators we visited, and published reports, we present certain guidelines and procedures that can help to reduce the inaccuracies and improve the reliability of temperature data obtained in clinical hyperthermia trials.

Abdomino-pelvic hyperthermia and intraperitoneal carboplatin in epithelial ovarian cancer: feasibility, tolerance and pharmacology.

PURPOSE: To investigate the feasibility, toxicity, and pharmacokinetics of intraperitoneal (i.p.) carboplatin (CB) with concomitant abdomino-pelvic hyperthermia (HT) in advanced ovarian cancer patients. METHODS AND MATERIALS: Patients with residual disease mainly confined to the peritoneal cavity after platinum based chemotherapy received an initial course of i.p. CB for baseline pharmacokinetics followed by three cycles of i.p. CB with concomitant regional hyperthermia. The goal of HT was to achieve at least 45 min of intraperitoneal temperature > 42 degrees but < 50 degrees C while maintaining normal tissue temperatures < 43 degrees C and systemic body temperatures < 38 degrees C. No analgesic premedication was used. Thermometry was recorded by multisensor fiberoptic probes placed within the peritoneal cavity, bladder, vagina, and oral cavity. RESULTS: Thirteen patients received a total of 31 sessions. Our intraperitoneal temperature goal could not be achieved because of patient intolerance. At best, we could maintain intraperitoneal temperatures > 40 degrees C, for more than 40 min in 7 of 31 sessions. The average values of thermal variables were T90 = 40 degrees C, TAVE = 41 degrees C, TMIN = 38.2 degrees C, and TMAX = 42.9 degrees C. The mean maximum systemic temperature was 38 degrees C. Acute thermal toxicities requiring early interruption of hyperthermia were systemic temperature exceeding 38 degrees C (11 of 31), abdominal pain or generalized distress (20 of 31), and vomiting (2 of 31). Hematological toxicities were not increased by hyperthermia. Pharmacokinetics were consistent with enhanced clearance of CB by HT. Lower radio frequencies (< 75 MHz) achieved better heat deposition in the peritoneal cavity than higher frequencies (> 75 MHz). Two of the 13 patients (a Stage III and a Stage IV patient) are alive with no evidence of disease at 40 and 43 months from treatment. CONCLUSIONS: Intraperitoneal temperatures in the range of 40 degrees C maintained for approximately 40 min can be achieved within the described setting. The probability of successful induction of therapeutic intraperitoneal temperatures appears to be higher when frequencies below 75 MHz are used. Patients who are potentially platinum sensitive and have minimal residual disease could potentially benefit from the combined treatment under the conditions studied. However, this temperature-time range appears inadequate against platinum resistant disease, and/or bulky residual pelvic disease. Alternative approaches such as whole body hyperthermia and carboplatin are warranted to overcome some of the obstacles observed.

Hyperthermia quality assurance guidelines.

These Hyperthermia Quality Assurance guidelines are a result of a joint workshop of the Hyperthermia Committee of the American College of Radiology and the Hyperthermia Physics Center, which is the national quality assurance program under Contract No. N01-CM-37512 with the National Cancer Institute. Hyperthermia technology presently lacks the kind of standardization in equipment, treatment procedures, patient monitoring, and treatment documentation available in radiotherapy. Therefore, preventing unacceptable variability in treatment data demands a strong commitment to in-house quality control procedures and to centralized quality assurance reviews in cooperative multi-institutional trials. This paper presents a set of test procedures necessary to ensure proper operation of equipment, suggests a frequency for such tests, and also includes guidelines on quality control procedures to be used during treatment to improve the safety, effectiveness, and reproducibility of hyperthermia treatments. A set of forms are presented to indicate the minimum data, albeit incomplete, that must be collected for acceptable documentation of treatment. These guidelines should be valuable not only to the new entrants in the field but also to those participating in multi-institutional cooperative hyperthermia trials. They have been approved by the Hyperthermia Committees of American College of Radiology, American Society for Therapeutic Radiology and Oncology, Radiation Therapy Oncology Group and the American Association of Physicists in Medicine.

Circumvention of multidrug resistance by a quinoline derivative, MS-209, in multidrug-resistant human small-cell lung cancer cells and its synergistic interaction with cyclosporin A or verapamil.

PURPOSE AND METHODS: To develop a clinically useful approach to circumvent P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) in MDR human small-cell lung cancer (SCLC), we examined the ability of a novel quinoline compound, MS-209, to reverse MDR by inhibition of P-gp function in combination with other MDR-reversing drugs using a cytotoxicity assay. RESULTS: We established MDR human SCLC cells by culture in medium with gradually increasing concentrations of adriamycin (ADM). Compared with the parental human SCLC cells, SBC-3, the MDR variant SBC-3 cells obtained (SBC-3/ADM) were highly resistant to various chemotherapeutic agents due to P-gp expression. MS-209 reversed the resistance to ADM and vincristine (VCR) of SBC-3/ADM and H69/VP cells in a dose-dependent manner. Moreover, MS-209 in combination with cyclosporin A (CsA) or verapamil (VER) synergistically enhanced the antitumor effects of ADM and VCR on SBC-3/ADM cells. MS-209 restored ADM incorporation and this effect was enhanced by CsA and VER, suggesting that these synergistic effects were due to competitive inhibition of P-gp function. CONCLUSION: MS-209 in combination with CsA or VER might increase the efficacy of these chemotherapeutic agents against MDR human SCLC cells.

Model to analyze radiographic factors in mammography.

A regression model and analysis is presented to determine the effect of various radiographic factors on mammographic breast exposures. The analysis is based on data collected jointly by the six Regional Centers for Radiological Physics (CRPs) at some sixty installations throughout the United States. A "stepwise regression analysis" of this data projects that the choice between xerox and screen-film receptors is the most critical factor affecting breast exposure followed by choice of HVL and target material. The analysis also shows that, although, for a given receptor categroy, the breast surface exposure varied over a wide range for the diverse techniques acceptable to radiologists, the receptor exposures varied over a rather narrow range of +/- 12%.

Microwave hyperthermia. Principles and quality assurance.

This article discusses some of the basic principles involved in the deposition of microwave power in the tissue. Insight is offered into the characteristics of the instrumentation, its limitations, and the necessary quality control needs.

Activation of tumor-associated macrophages by thymosin alpha 1.

It was shown earlier that the progressive growth of a transplantable T-cell lymphoma of spontaneous origin, designated as Dalton's lymphoma (DL), in a murine host is associated with an inhibition of macrophages (TAM) along with an involution of thymus. However, it remained unclear if a decline in the level of thymic peptides in DL-bearing host, due to thymic regression, has any implications in the inhibited responses of TAM. Therefore, the present investigation was under taken to study whether the TAM of DL-bearing host can be activated to tumoricidal state by peptides of thymic origin. It was observed that intraperitoneal administration of thymosin alpha 1 to DL-bearing mice resulted in activation of TAM. Such TAM were found to produce enhanced amount of interleukin-1 (IL-1), tumor necrosis factor (TNF), reactive oxygen intermediates (ROI), nitric oxide (NO) and showed an increased abilities of pinocytosis, phagocytosis, antigen presentation and tumor cytotoxicity. The TAM were found to be directly responsive to thymosin alpha1 as in vitro treatment with thymosin alpha 1 could activate TAM to tumoricidal state. Treatment of TAM with thymosin alpha 1 also enhanced their LPS responsiveness for an augmented state of activation. The findings of this study demonstrate for the first time that the TAM of a T cell lymphoma can be activated to tumoricidal state by thymosin alpha 1.

Non-interventional background corrections for scintigrams.

A method is presented for the interpolation of optimally 'smooth' background correction matrices within arbitrarily shaped boundaries. Practical means of implementing this technique are discussed, along with series expansions for the special cases of square and round boundaries. This class of interpolation methods has the advantages over previous techniques of providing a unique mathematical solution, connecting exactly to count rates on the boundary, and containing no singularities. Clinical scintigraphic images and mathematically generated surfaces are used to test the different cases of the method, and it is shown that confining the boundary to a circle produces the most rapid convergence with the minimum deviation from ideal surfaces. Under common clinical conditions, these techniques lend themselves well to automated, non-interventional computer processing.

Management of colonic perforation during percutaneous nephrolithotomy in horseshoe kidney.

Few cases of colonic injury during percutaneous nephrolithotomy (PCNL) have been reported in orthotopic kidneys and none in horseshoe kidney, and the management protocol has not been standardized. A plain film on postoperative day 1 following PCNL showed contrast medium in the descending colon, leading to the diagnosis of colonic injury in a 53-year-old male patient with horseshoe kidney and multiple bilateral stones. He was asymptomatic and was treated successfully by minimally invasive techniques. An asymptomatic patient with a colonic injury following PCNL can be treated by minimal manipulations. Computed tomography imaging is necessary prior to percutaneous surgery on a horseshoe kidney.

Effect of thymosin-alpha1 on the production of nitric oxide by tumor-associated macrophages.

The present investigation was conducted to study the effect of thymic peptide: thymosinalpha1 (thyalpha1) on the activation of tumor associated mphi (TAM) obtained from mice bearing a transplantable T cell lymphoma of spontaneous origin designated as Dalton's lymphoma, to produce nitric oxide (NO). It was found that in vivo administration of aqueous thymic extract obtained from thymus of normal mice or thyalpha1 could activate the TAM to produce enhanced amount of NO which was further augmented on in vivo treatment of these TAM by LPS. These observations suggest that thyalpha1 could prime TAM for activation by second signal of LPS. The study also presents evidence that tumor cell elaborate factors that enhance the effect of thyalpha1 on TAM for production of NO. This is the first study to show that thyalpha1 can activate TAM directly even in the absence of LPS, and may, therefore, have clinical significance.

Hyperthermia: principles and quality assurance.

The methods of energy deposition, the power absorbtion properties of biological tissues and the basic components of a typical hyperthermia system are described. In addition, the clinical requirements of hyperthermia treatment are discussed. A perspective on treatment planning and quality control is presented.

Leigh's syndrome.

A 15-month-old female child presented with sudden onset cough and hyperventilation along with evidence of metabolic acidosis. She had past history of recurrent vomiting, episodes of abnormal posturing, difficulty in deglutition and regression of milestones since 12 months of age. CT scan of the brain revealed hypodense lesions in bilateral basal ganglia and on MRI there were T2 hyperintensities in bilateral lentiform nuclei, caudate nuclei, thalamus, red nuclei and dentate nuclei. Biochemical examination revealed persistently elevated serum lactate levels with high lactate/pyruvate ratio. Resuscitative measures were of no avail and the child succumbed to the illness on the second day of admission. Neuropathological examination at autopsy demonstrated marked spongiosis, focal necrosis, endothelial proliferation, reactive astrogliosis and extensive demyelination involving bilateral basal ganglia, midbrain and spinal cord which were typical of Leigh's sub acute necrotizing encephalomyelopathy.

P.C. Sen Memorial best paper award on rural health practice: A study on visual acuity and vitamin A deficiency among primary school students in Naxalbari Village, Darjeeling district of West Bengal.

331 primary school students studying from Nursery classes to Class IV in 2 randomly selected schools in Naxalbari Village in Darjeeling district of West Bengal were tested for visual acuity (VA), Vitamin A deficiency (VAD) and Bitot's spot. 329 students could be tested for visual acuity whereas all 331 students were tested for VAD and Bitot's spot. The prevalence of abnormal Visual Acuity (VA) (VA < 6/9 in any eye) in this study group was 3.65% and it was highest in 7-8 years age group in both the sexes contributing to 75% of the total students having abnormal VA. All these children fell within 50th percentile of weight and height for their respective age and sex. Hindu & ST children accounted for 91.67% & 50% abnormal VA respectively; however, when presence of abnormal VA was compared with its absence between 2 sexes, Hindu and Muslim students and in different castes, no statistically significant differences were found (p > .05). Students of Class-I and Class-II contributed 25% and 50% respectively and together accounted for 75% of abnormal VA. Prevalence of VAD was 8.16%. Among sufferers of VAD Hindus (81.48%) and ST (51.85%) were the main contributors; the differences between presence and absence of VAD in 2 sexes, among 3 religious groups were not statistically significant (p > .05); however, differences among ST and NonSCST groups, and again SC, ST and Non-SCST groups were statistically significant (p < .05). Prevalence of Bitot's spot was 3.63%. Among the students having Bitot's spot, females (58.33%) outnumber the males (41.67%); but the difference between presence and absence of Bitot's spot in 2 sexes was not statistically significant (p > .05). 83.33% each of Hindus and STs had Bitot's spot. No SC and no Muslim student had this spot; the differences between presence and absence of Bitot's spot between Hindu and Christian students were statistically significant (p < .01); similarly when the findings were considered between ST and NonSCST students the difference was found to be statistically highly significant (p < .001).