A human ImmunoChip cDNA microarray provides a comprehensive tool to study immune responses.

DNA microarray technology has developed rapidly in recent years and has become an essential tool, providing novel approaches to biomedical research. In this paper, we describe a self-designed ImmunoChip cDNA array for immunological research. With a comprehensive selection of genes of interest, we can focus on key signalling pathways and molecular mechanisms at relatively low cost compared to commercial platforms which are usually targeted at global screening of gene expression. To validate the efficiency of the ImmunoChip, we studied T helper cell polarization to functionally distinct subsets (Th1 and Th2). We also developed a tool for quality control of cDNA microarrays that assesses the technical quality of an ImmunoChip. The information produced with the quality control tool is shown to be valuable for extracting correct information from cDNA microarrays. Gene expression measurements with ImmunoChip are in agreement with the results obtained using oligonucleotide microarrays and with published quantitative RT-PCR data. The ImmunoChip provides reliable measurements and gives new insights into various aspects of human immune responses.

[Effect of omega-3 polyunsaturated fatty acids of animal origin on changes in regulation of lipid peroxidation in patients with ischemic heart disease]. / Vplyv omeha-3 polinenasychenykh zhyrnykh kyslot tvarynnoho pokhodzhennia na zminy rehuliatsiï perekysnoho okyslennia lipidiv u khvorykh na ishemichnu khvorobu sertsia.

The study's results showed reliable decrease in lipid peroxidation activity according to value of general sum of light, induced by hydrogen peroxide of initiated hemoluminescence and malonic dialdehyde concentration (P<0.05). The activity of antioxidant enzymes in the second group as compared with the control one has changed as follows: catalase concentration of blood serum of patients with ischemic heart disease increased by 59.49% (P<0.05), superoxide dismutase concentration--decreased by 28.64% (P<0.05). The above mentioned changes show, that the treatment with Tecom medication, which consist of omega-3 polyunsaturated fatty acids of animal origin does not stress peroxid antioxidant system, does not affect cell membrane stability, does not lead to progression of ischemic heart disease course.

Highly specific tumor binding of a 213Bi-labeled monoclonal antibody against mutant E-cadherin suggests its usefulness for locoregional alpha-radioimmunotherapy of diffuse-type gastric cancer.

A monoclonal antibody (E-cadherin delta 9-1) directed against a characteristic E-cadherin mutation (in-frame deletion of exon 9), found in diffuse-type gastric cancer but not in any normal tissue, was conjugated with the high linear energy transfer alpha-emitter 213Bi and tested for its binding specificity in s.c. and i.p. nude mice tumor models. After intratumoral application in s.c. tumors expressing mutant E-cadherin, the 213Bi-labeled antibody was specifically retained at the injection site as shown by autoradiography. After injection into the peritoneal cavity, uptake in small i.p. tumor nodules expressing mutant E-cadherin was 17-fold higher than in tumor nodules expressing wild-type E-cadherin (62% injected dose/g versus 3.7% injected dose/g). 78% of the total activity in the ascites fluid was bound to free tumor cells expressing mutant E-cadherin, whereas in control cells, binding was only 18%. The selective binding of the 213Bi-labeled, mutation-specific monoclonal antibody E-cadherin delta 9-1 suggests that it will be successful for alpha-radioimmunotherapy of disseminated tumors after locoregional application.

In vivo detection of vascular adhesion protein-1 in experimental inflammation.

Vascular adhesion protein-1 (VAP-1) is an inflammation-inducible endothelial glycoprotein which mediates leukocyte-endothelial cell interactions. To study the pathogenetic significance of VAP-1 in inflammatory disorders, an in vivo immunodetection method was used to detect the regulation of luminally expressed VAP-1 in experimental skin and joint inflammation in the pig and dog. Moreover, VAP-1 was studied as a potential target to localize inflammation by radioimmunoscintigraphy. Up-regulation of VAP-1 in experimental dermatitis and arthritis could be visualized by specifically targeted immunoscintigraphy. Moreover, the translocation of VAP-1 to the functional position on the endothelial surface was only seen in inflamed tissues. These results suggest that VAP-1 is both an optimal candidate for anti-adhesive therapy and a potential target molecule for imaging inflammation.

Alpha-emitting bismuth cyclohexylbenzyl DTPA constructs of recombinant humanized anti-CD33 antibodies: pharmacokinetics, bioactivity, toxicity and chemistry.

UNLABELLED: The alpha-particle-emitting radionuclides have several physical characteristics that make them attractive candidates for radioimmunotherapy: (a) high linear energy transfer; (b) short path lengths (50-80 microm); and (c) limited ability of cells to repair damage to DNA. This article describes the pharmacokinetic, bioactivity, toxicity and chemical characteristics of alpha-particle-emitting, 213Bi and 212Bi radiometal conjugated HuM195 (anti-CD33) constructs. Conjugation of HuM195 to SCN-CHX-A-DTPA resulted in the attachment of up to 10 chelating ligand molecules per antibody. RESULTS: Radiolabeling efficiency of the CHX-A-DTPA-HuM195 construct with 213Bi was 78%+/-10% (n = 46) after 10 min at specific activities of up to 1110 MBq/mg. The immunoreactivity of the 213Bi-labeled CHX-A-DTPA-HuM195 construct was 84%+/-10% (n = 28) and was independent of the specific activity. The bismuth-labeled CHX-A-DTPA-HuM195 construct was rapidly internalized into the cell in a time-dependent manner ranging from 50% at 1 h to 65% at 24 h. 205Bi/206Bi-labeled constructs were stable for at least 2 d in vitro in the presence of human serum at 37 degrees C. After injection into mice, there was no uptake or loss of bismuth to mouse tissues, which do not express CD33, or to the kidney, which has avidity for free bismuth. Mice injected intraperitoneally with doses of (213Bi)CHX-A-DTPA-HuM1 95 ranging from 18.5 to 740 MBq/kg showed no toxicity, but at 2590 MBq/kg, two of the three mice died within 2 wk and a third mouse showed significant reductions in white blood cell counts. Mice injected intravenously with doses of (213Bi)CHX-A-DTPA-HuM195 up to 370 MBq/kg exhibited little toxicity, but 666 MBq/kg was above the MTD for mice. Leukemia cell killing in vitro with bismuth-labeled HuM1 95 showed dose- and specific activity-dependent killing of CD33+ HL60 cells; approximately 50% killing was observed when two bismuth atoms (50 fM radiolabeled antibody) were initially bound onto the target cell surface. CONCLUSION: Alpha-emitting antibodies are among the most potent cytotoxic agents known, yet are specific and appear safe in vivo. The physical and biochemical characteristics of the 213Bi isotope and its generation, as well as the biochemistry of the 213Bi-labeled CHX-A-DTPA-HuM195 construct, make it possible to use the constructs safely and feasibly in humans at therapeutic levels.

Expression profile of vascular cell adhesion molecule-1 (CD106) in inflammatory foci using rhenium-188 labelled monoclonal antibody in mice.

Rhenium (Re)-188 is a generator (W-188/Re-188) produced high energy beta-emitter suitable for radionuclide therapy (T1/2 is 16.9 hrs and Emax 2.1 MeV (range 11 mm)). We have labelled monoclonal antibody (MAb) raised against vascular cell adhesion molecule-1 (VCAM-1) with Re-188 using glucoheptonate chelation technique and SnCl2 as reducing agent. The labelling efficiency, free perrhenate and reduced Re were controlled with thin layer chromatography and the purification of Re-188-MoAbs was performed using gel filtration. Our results indicate that Re-188-labelled antibodies remain in vitro stable and the labelling purity is > 90%. We also have applied these Re-188-MoAbs for detection of inflammatory disease in a mouse. The effective half-lives of organs of interest after an injection of Re-188-anti-VCAM1 were as follows: blood 5.2 hr, kidney 4.7 hr, and liver 9.6 hr. Re-188-anti-VCAM-1 was found to accumulate mainly in kidney and liver. One hour after the injection, the kidney contained in average as high as 12.5% and the liver 2.8 ID/g tissue. After 6 hr, the kidney contained 5.5% ID/g and the liver 2.6% ID/g. At 24 hr, the kidney uptake was 0.5% ID/g and the liver uptake 0.8% ID/g, respectively. The inflamed foci, subcutaneous lesions in the footpad skin, were visualized using gamma camera. From the distribution data the uptakes in the inflamed foci as follows: at 1 hr 2.18 (inflammation) and 1.72% ID/g (control), at 6 hr 1.42 (inflammation) and 0.85% ID/g (control), and at 24 hr 0.17 (inflammation) and 0.084% ID/g (control), respectively. Anti-VCAM-1 MAb showed better targeting as compared to control MoAbs in inflammation (caused by E.coli lipoplysaccaride). In conclusion, Re-188 is suitable for MAb labelling, and MAb against VCAM-1 may be used for detection of local inflammatory disease.

Pharmacokinetics and dosimetry of iodine-123 labelled PE2I in humans, a radioligand for dopamine transporter imaging.

The iodine-123 labelled selective ligand N-(3-iodoprop-2E-enyl)-2-beta-carbomethoxy-3beta-(4-methylphenyl) nortropane ([123I]PE2I) was evaluated as a probe for in vivo dopamine transporter imaging in the human brain. Six healthy subjects were imaged with a high-resolution single-photon emission tomography scanner. Striatal radioactivity peaked at 1 h after injection. The background radioactivity was low. The volume of distribution in the striatum was 94+/-24 ml/ml. The results were compared with those of [123I]beta-CIT imaging. There was no significant uptake of [123I]PE2I in serotonin-rich regions such as the midbrain, hypothalamus and anterior gingulus, suggesting that in vivo binding is specific for the dopamine transporter. One main polar metabolite of [123I]PE2I was found in plasma, and the parent plasma concentration decayed rapidly. Radiation exposure to the study subject is 0.022+/-0.004 mSv/MBq (effective dose). The preliminary results suggest that [123I]PE2I is a selective SPET ligand for imaging striatal dopamine transporter density.

Iodine-123 labeled nor-beta-CIT as a potential tracer for serotonin transporter imaging in the human brain with single-photon emission tomography.

Iodine-123 labelled 2beta-carbomethoxy-3beta-(4-iodophenyl) (nor-beta-CIT) is an analogue of beta-CIT, which has high affinity to the serotonin transporter. Initial single-photon emission tomography (SPET) studies with [123I]nor-beta-CIT were performed in five healthy volunteers. In addition, its metabolism in plasma was investigated with gradient high performance liquid chromatography. [123I]nor-beta-CIT was prepared by a method which gave a specific radioactivity of more than 180 GBq/micromol. Unchanged [123I]nor-beta-CIT in plasma accounted for 43% and 19% of total radioactivity after 30 and 180 min, respectively. The dynamic SPET studies demonstrated a high and rapid uptake of radioactivity in the brain (6%/ID at 30 min). Highest accumulation was observed in the striatum, the mid-brain and the thalamus. The specific binding in the mid-brain was 33% higher compared with that of [123I]beta-CIT. The high radioactivity in the mid-brain is assumed to represent the accumulation of [123I]nor-beta-CIT in the serotonin transporter-rich regions, which indicates that [123I]nor-beta-CIT might be a potential tracer for visualization of serotonin transporter sites in the human brain with SPET.

Lymph flow dynamics in exercising human skeletal muscle as detected by scintography.

1. The effects of dynamic and isometric muscle contractions on the lymph flow dynamics in human skeletal muscle were studied with a scintographic method. 2. Radioactively labelled human serum albumin (99mTc-HSA) was injected bilaterally into the vastus lateralis muscles of eight men (n = 16), four of whom had had an endurance training background. The subjects performed 100 submaximal contractions in 10 min as (i) dynamic knee extensions (CONS), (ii) isometric contractions with the knees at full extension (IMExt), or (iii) isometric contractions with knees fixed at 90 deg angle flexion (IMFlex). The exercises were separated by 65 min periods in supine rest. The level of radioactivity at the injection site was monitored by a gamma-camera, and the clearance rate of radioactivity (CR) was calculated as the fractional decrease during the periods of interest (CR unit = % min-1). 3. The clearance rate was low during the rest periods (0.04 +/- 0.05% min-1), though higher in the trained than in the sedentary subjects (0.06 +/- 0.05 vs. 0.03 +/- 0.03% min-1; P = 0.008). Exercise increased the clearance rate three- to sixfold, to 0.16 +/- 0.16% min-1 during CONS, 0.20 +/- 0.15% min-1 during IMExt and 0.09 +/- 0.11% min-1 during IMFlex. There were no differences between the subject subgroups. 4. The higher clearance rate during IMExt than during IMFlex (P = 0.02) demonstrates the importance of muscle deformations on lymph propulsion and experimentally confirms the current concepts of lymph formation and propulsion in voluntarily active skeletal muscle. It is suggested that lymph propulsion by working muscle is most efficient when the muscle is able to shorten close to its minimum length.

Radiolabeled anti-CD33 monoclonal antibody M195 for myeloid leukemias.

M195, a mouse monoclonal antibody reactive with the early myeloid antigen CD33, has been shown to target leukemia cells in patients and to reduce large leukemic burdens when labeled with 131I. A complementarity-determining region-grafted, humanized version (HuM195) has demonstrated similar targeting of leukemia cells without immunogenicity. We have studied two applications of therapy with 131I-M195. First, to intensify therapy prior to bone marrow transplantation (BMT), we combined 131I-M195 with busulfan and cyclophosphamide. Fifteen patients received first BMT for relapsed or refractory acute myelogenous leukemia or accelerated or blastic chronic myelogenous leukemia; four received second BMT for relapsed chronic or accelerated chronic myelogenous leukemia. Doses of 131I-M195 ranged from 120 to 230 mCi/m2. Few toxicities could be attributed to 131I-M195 therapy, and all patients engrafted. Eighteen patients achieved complete remission. Among those patients receiving first BMT, three have remained in unmaintained remission for 18+ to 29+ months. Six patients relapsed, including one with isolated central nervous system disease 32 months after BMT. Ten patients died in complete remission of transplant-related complications. Second, we studied whether 131I-M195 could reduce minimal residual disease and prolong remission and survival durations safely in patients with relapsed acute promyelocytic leukemia after they attained remission with all-trans-retinoic acid. Seven patients were treated with either 50 or 70 mCi/m2 131I-M195. Toxicity was limited to myelosuppression. As a measure of minimal residual disease, we monitored PML/RAR-alpha mRNA by reverse transcription PCR. Six patients had positive reverse transcription PCR assays prior to receiving 131I-M195; two converted transiently to negative. Median disease-free survival and overall survival of the seven patients were 8 (range, 3-14.5) months and 28 (range, 5.5-43+) months, respectively. This regimen compares favorably with others for relapsed acute promyelocytic leukemia. In an effort to avoid nonspecific cytotoxicity associated with 131I in future trials for minimal residual disease, we have conjugated short-range, alpha particle-emitting radioisotopes to HuM195 using a bifunctional chelate, 2-(p-isothiocyanatobenzyl)-cyclohexyldiethyl-enetriaminep entaacetic acid, with high efficiency and specific activities. 212Bi-HuM195 has demonstrated dose- and specific activity-dependent killing of HL60 cells in vitro. Injection of 213Bi-HuM195 into healthy BALB/c mice produced no effects on weight or viability.

Impact of the high tyrosine fraction in complementarity determining regions: measured and predicted effects of radioiodination on IgG immunoreactivity.

Although iodine-131 is the most widely used radionuclide for radioimmunotherapy, direct radiolabeling methods yield decreased immunoreactivity of the antibody as a function of increased iodine incorporation. We have studied the amino acid sequences of a therapeutic IgG (HuM195), and in particular its complementarity determining regions (CDR), in order to correlate the iodination of tyrosine residues in the antigen binding site with changes in immunoreactivity. The CDR contained an overabundance of tyrosines relative to an expected random distribution of amino acids. In contrast, lysine residues that can be used for ligand attachment were evenly distributed throughout the IgG. HuM195 was first trace labeled with 111In and then labeled with stable 127I at various specific activities. The immunoreactivity of each product was determined using the 111In tracer. The immunoreactivity measured after varying levels of iodination fit a theoretical curve that was generated based on the assumption that a single iodine incorporation anywhere on a tyrosine residue in a CDR destroys the immunoreactivity of the antibody. Similar theoretical curves for antibody fragments (Fab, Fv) suggest an even faster decrease in immunoreactivity with increasing iodination. A review of the sequences of other therapeutic IgG shows that a similar overabundance of tyrosine residues is found in the CDRs. Using enzyme digestion, the distribution of iodine on different parts of the antibody was also studied. The iodinated residues were distributed non uniformly throughout the IgG, with the heavy chain variable region tyrosines having a higher propensity for iodine incorporation than tyrosines in the other regions of the IgG. The common abundance of tyrosine in the CDR of IgG and its correlation with loss of function have important implications for therapeutic use of high specific activity radioiodinated monoclonal antibodies or fragments.

[The ultrastructural characteristics of the duodenal mucosa in peptic ulcer patients undergoing treatment with EHF-range electromagnetic radiation]. / Ul'trastrukturnye osobennosti slizistoi obolochki dvenadtsatiperstnoi kishki u bol'nykh iazvennoi bolezn'iu pri lechenii élektromagnitnym izlucheniem KVCh-diapazona.

An effect was studied of electromagnetic radiation of the millimetric wave band (EHF EMR) on ultrastructural processes of the mucosa and healing of ulcer in 10 patients with duodenal ulcer. Biopsy specimens were taken from the base of the margin of the ulcer and in the area 1 cm from the margin of the ulcer defect before the treatment, after 5 and 10 sessions of EHF EMR to acupuncture points. EHF EMR was found to exert a stimulating effect on the processes of ulcer regeneration. Seen after session 5 are layers of proliferating poorly differentiated enterocytes, activation of immune and autoimmune processes, normalization of the hemomicrovessel endothelial cell structure, filling in the ulcer defect with a newly formed granulation tissue in which, among fibroblasts, macrophages, mast cells and lymphocytes, there prevail plasmic cells with an augmented secretion of immunoglobulins, increase in proliferation of epitheliocytes, with their differentiation slowing down, and a complete epithelialization of the ulcer following ten treatment procedures.

A rapid, single vessel method for preparation of clinical grade ligand conjugated monoclonal antibodies.

A rapid, single vessel method for the preparation of clinical grade chelate conjugated monoclonal antibodies has been developed. By use of an Amicon concentrator with reservoir, each of the steps necessary for the preparation of the conjugated drug may be performed in a single vessel. Advantages include reduced risk of metal, pyrogen and bacterial contamination; buffer exchanges are achieved rapidly and efficiently using a continuous dilution method. The radiolabeling efficiency, the radiochemical purity, the total immunoreactivity and the affinity of the final product have been evaluated in the production of CHXA-DTPA-chelate conjugated HuM195. The characteristics compare favorably to those achieved using our conventional synthetic methods.

Accumulation of 99mTc-low-density lipoprotein in human malignant glioma.

Low-density lipoprotein (LDL) uptake in gliomas was studied to find out if LDL has potential as a drug carrier of boron, especially for boron neutron capture therapy. Single photon emission tomography (SPET) was performed 2 h and 20 h after intravenous injection of autologous 99mTc-labelled LDL in four patients with untreated and five patients with recurrent glioma. 99mTc-LDL uptake was compared with the uptake of 99mTc-labelled human serum albumin (HSA), an established blood pool marker. The intra- and peritumoral distributions of radioactivity in the SPET images were not identical for radiolabelled LDL and HSA. The mean LDL tumour to brain ratio, determined from transversal SPET slices at 20 h post injection, was 1.5 in untreated and 2.2 in recurrent gliomas; the corresponding ratios for HSA were 1.6 and 3.4. The brain to blood ratio remained constant at 2 h and 20 h in both types of tumours. These data are not consistent with highly selective, homogeneous uptake of LDL in gliomas. However, the different tumoral distribution and rate of uptake of 99mTc-LDL, as compared with 99mTc-HSA, indicate that the uptake of LDL is different from that of HSA and that further studies on the mechanism of LDL uptake in glioma are warranted.

A low pH 111In-bleomycin complex: a tracer for radiochemotherapy of head and neck cancer.

Bleomycin (BLM) is a well known natural antibiotic. It is toxic to dividing cells and has been used for the treatment of several forms of cancer. BLM has been labeled with various cations, but most of them have turned out be unstable in in-vivo experiments. In-BLM demonstrated high bone marrow uptake, but using 111In-bleomycin complex (BLMC) formed at low pH, the low in vivo stability and high bone marrow seeking behavior of the molecule could be avoided. The idea of using BLMC in combined radiotherapy and chemotherapy is intriguing. In this study we examined the effects of 111In-A'2a-c-BLMC in the treatment of 31 head and neck cancer patients. Findings were compared with those of surgery, and pre-operative radiology. The injected activity was 85-110 MBq, and the specific activity was approximately 100 MBq/mg. The half-life of 111In activity in serum varied from 1.5 to 3.1 hours. Maximum activity in the urine was achieved in all patients within 3 hours, and the average half-life in urine was 2 hours. In most patients 50% was excreted within 3 hours, in some 70%; in all patients > 95% of the activity was excreted within 22 hours. In surgical samples from 24 patients the best tumor-to-tissue ratios were: fat 60:1, bone 17:1, muscle 12:1, blood 3.6:1. All patients were examined on the injection day with ultrasonography of the neck. Using 111In-BLMC we missed a few small lymph nodes in 2 patients, but there were no false positive findings.(ABSTRACT TRUNCATED AT 250 WORDS)

Radioimmunotherapy with 90Y-labeled monoclonal antibodies in a nude mouse ovarian cancer model.

Tumor stroma contains much fibrin, and so monoclonal antifibrin antibody can accumulate in tumors. We treated nude mice bearing human ovarian carcinoma xenografts with 90Y-labeled monoclonal antifibrin antibody Fab fragments administered intratumorally. The survival time vs. a control group was significantly prolonged and tumor growth rate was decreased. Another group of animals was treated with 90Y-labeled OC 125-monoclonal antibody; these mice received the antibodies intratumorally, intraperitoneally or intravenously. The survival time was longest in the intratumorally treated group. There was no significant difference in survival between 90Y-labeled OC 125 and antifibrin in the intratumorally treated animal groups. The tissue activity distribution studies revealed that bone marrow is the critical organ. Intratumorally injected monoclonal 90Y-antifibrin antibodies were retained at least 36 h (up to 50% of injected activity per gram tumor tissue) in the xenograft after one treatment, causing cell death. Beta-camera imaging and immunohistochemistry were performed for studies of the correlation between 90Y activity and fibrin distribution in tumor specimens. These results were in concordance. In conclusion, intratumoral administration seems suitable for radioimmunotherapy, with an antibody that targets stromal structures. The accumulation can be successfully monitored by a beta-camera.

[The effect of microwave resonance therapy and hypnotherapy on the links in the pathogenesis of peptic ulcer]. / Vliianie mikrovolnovoi rezonansnoi terapii i gipnoterapii na nekotorye zven'ia patogeneza iazvennoi bolezni.

Results of a study are reported of a study of the concentration of biologically active substances such as histamine, serotonin, noradrenaline, adrenalin, corticotropin and indices of gastric secretion in ulcer disease patients at the stage of exacerbation receiving microwave resonance therapy and hypnotherapy. The obtained results evidence that hypnotherapy and microwave resonance therapy are pathogenetic methods of treatment of patients with ulcer disease. Combined treatment proved more effective.

[The effect of hypnotic suggestion and millimeter-range electromagnetic radiation on the clinical and endoscopic indices in peptic ulcer patients]. / Vliianie gipnosuggestii i élektromagnitnogo izlucheniia millimetrovogo diapazona na klinicheskie i éndoskopicheskie pokazateli u bol'nykh iazvennoi bolezn'iu.

The authors studied the effect of microwave resonance therapy, hypnosuggestion and their combinations on the dynamics of clinico-endoscopic indices in 182 patients with duodenal ulcer. It was established that these methods were highly effective allowing to control rapidly the pain syndrome, to achieve complete healing of the ulcer in 70-95% of cases within 14.8-16.7 days. The results were best when the two methods were combined.