Enhancing efficacy of recombinant anticancer vaccines with prime/boost regimens that use two different vectors

BACKGROUND: The identification of tumor-associated antigens and the cloning of DNA sequences encoding them have enabled the development of anticancer vaccines. Such vaccines target tumors by stimulating an immune response against the antigens. One method of vaccination involves the delivery of antigen-encoding DNA sequences, and a number of recombinant vectors have been used for this purpose. To optimize the efficacy of recombinant vaccines, we compared primary and booster treatment regimens that used a single vector (i.e., homologous boosting) with regimens that used two different vectors (i.e., heterologous boosting). METHODS: Pulmonary tumors (experimental metastases) were induced in BALB/c mice inoculated with CT26.CL25 murine colon carcinoma cells, which express recombinant bacterial beta-galactosidase (the model antigen). Protocols for subsequent vaccination used three vectors that encoded beta-galactosidase--vaccinia (cowpox) virus, fowlpox virus, naked bacterial plasmid DNA. Mouse survival was evaluated in conjunction with antibody and cytotoxic T-lymphocyte responses to beta-galactosidase. RESULTS: Heterologous boosting resulted in significantly longer mouse survival than homologous boosting (all P<.0001, two-sided). Potent antigen-specific cytotoxic T lymphocytes were generated following heterologous boosting with poxvirus vectors. This response was not observed with any of the homologous boosting regimens. Mice primed with recombinant poxvirus vectors generated highly specific antibodies against viral proteins. CONCLUSIONS: The poor efficacy of homologous boosting regimens with viral vectors was probably a consequence of the induction of a strong antiviral antibody response. Heterologous boosting augmented antitumor immunity by generating a strong antigen-specific cytotoxic T-lymphocyte response. These data suggest that heterologous boosting strategies may be useful in increasing the efficacy of recombinant DNA anticancer vaccines that have now entered clinical trials.     

Accelerated (malignant) hypertension: a study of 121 cases between 1974 and 1996

DNA-based immunization is one of the most promising strategies to induce protective immunity against a variety of pathogens, presenting clear advantages as compared to the use of recombinant antigens. One of these advantages might be the ability to induce antibodies directed primarily against conformational determinants, as compared to immunization with recombinant proteins. To test this possibility, we have analyzed the antibody responses induced in mice by immunization with either recombinant soluble CD4 (rCD4) or by immunization with plasmid DNA-encoding CD4 (CD4-DNA). Mice immunized with CD4-DNA had lower titers of antibodies able to recognize rCD4 than mice immunized with rCD4. However, immunization with CD4-DNA induced antibodies reactive with the native cell surface CD4 molecule in all mice, whereas only two out of five mice immunized with rCD4 produced antibodies reactive with cell surface CD4, thus demonstrating that the genetic immunization approach may lead to an antibody response more consistent and superior at a qualitative level as compared to immunization with the corresponding recombinant protein. In addition, differences in the kinetics of appearance of antibodies directed against the native CD4 molecule were observed between mice immunized with CD4-DNA or rCD4. In the first case, antibodies reacting with cell surface CD4 were present 28 days after the first immunization, whereas mice immunized with rCD4 produced antibodies directed against the native molecule only following a booster injection. Finally, the two groups of mice produced antibodies with a different isotype distribution. No clear predominance of a specific IgG subclass was detected in the antibody population produced in response to DNA immunization. Conversely, mice immunized with rCD4 produced predominantly antibodies of the IgG1 isotype, indicating generation of a TH2 response. Together, results from this study indicate that the CD4 molecule endogenously produced following DNA immunization is expressed, at least partially, in a native conformation. This feature confers a major advantage to the DNA immunization approach as compared to immunization with the corresponding recombinant protein, which seems to elicit antibodies predominantly directed to epitopes uniquely expressed on the recombinant molecule.     

Primary diffuse large B-cell lymphoma of the mediastinum: outcome following high-dose chemotherapy and autologous hematopoietic cell transplantation

ESAT-6 is an important T-cell antigen recognized by protective T cells in animal models of infection with Mycobacterium tuberculosis. In an enzyme-linked immunosorbent assay (ELISA) with overlapping peptides spanning the sequence of ESAT-6, monoclonal antibody HYB76-8 reacted with two peptides in the N-terminal region of the molecule. Assays with synthetic truncated peptides allowed a precise mapping of the epitope to the residues EQQWNFAGIEAAA at positions 3 to 15. Hydrophilicity plots revealed one hydrophilic area at the N terminus and two additional areas further along the polypeptide chain. Antipeptide antibodies were generated by immunization with synthetic 8-mer peptides corresponding to these two regions coupled to keyhole limpet hemocyanin. Prolonged immunization with a 23-mer peptide (positions 40 to 62) resulted in the formation of antibodies reacting with the peptide as well as native ESAT-6. A double-antibody ELISA was then developed with monoclonal antibody HYB76-8 as a capture antibody, antigen for testing in the second layer, and antipeptide antibody in the third layer. The assay was suitable for quantification of ESAT-6 in M. tuberculosis antigen preparations, showing no reactivity with M. bovis BCG Tokyo culture fluid, used as a negative control, or with MPT64 or antigen 85B, previously shown to cross-react with HYB76-8. This capture ELISA permitted the identification of ESAT-6 expression from vaccinia virus constructs containing the esat-6 gene; this expression could not be identified by standard immunoblotting.     

Extensive reinnervation of the hippocampus by embryonic basal forebrain cholinergic neurons grafted into the septum of neonatal rats with selective cholinergic lesions

The immunodominant surface antigen of Toxoplasma gondii, surface antigen 1 (SAG1), was expressed in Escherichia coli as a fusion protein containing a majority of the SAG1 protein supplied with six histidyl residues in the N-terminal end. The recombinant protein was purified on a Ni-chelate column and then on a fast-performance liquid chromatography column and was in a nonreduced condition. It was recognized by T. gondii-specific human immunoglobulin G (IgG) and IgM antibodies as well as by a mouse monoclonal antibody (S13) recognizing only nonreduced native SAG1. Antibodies induced in mice by the recombinant SAG1 recognized native SAG1 from the T. gondii RH isolate in culture. Recombinant SAG1 is suitable for use in diagnostic systems for detecting anti-SAG1-specific IgG and IgM antibodies.     

FlaA, a putative flagellar outer sheath protein, is not an immunodominant antigen associated with Lyme disease

FlaA was recently found to be associated with flagellar filaments of Borrelia burgdorferi. We tested whether antibodies to this protein are a good indicator of infection, as antibodies to FlaA proteins in other spirochetal infections show an increase in titer. Although overproduction of intact FlaA was highly toxic to Escherichia coli, truncated proteins which lacked the N-terminal signal sequence could be successfully overexpressed. Immunoblotting with sera from mammalian hosts infected with B. burgdorferi indicated that FlaA is not an immunodominant antigen in Lyme disease. However, sera from two patients reacted with both recombinant and native FlaA protein, suggesting that B. burgdorferi FlaA was antigenic and expressed in vivo.     

American Heart Association Report on the Public Access Defibrillation Conference, December 8-10, 1994. American Heart Association Taskforce on Automatic External Defibrillation

Experimental conditions (pH 6.5, 24 h reaction, peptide:biotin ratio 1:5) were defined for preferential incorporation of the biotin molecule in the N-terminal alpha-amino group of peptides. This strategy could be helpful in numerous applications when an entire peptide chain must remain accessible for antibody or receptor binding. We illustrate this advantage in a solid-phase enzyme immunoassay designed to detect antibodies specific for bovine beta-lactoglobulin present in rabbit or human sera. This test involves synthetic peptides biotinylated in different positions and immobilized on a solid phase. The use of biotin/streptavidin interactions permitted more efficient detection of specific anti-peptide antibodies than solid phases prepared using conventional passive-adsorption techniques. The highest levels of antibody binding were measured when biotinylation occurred at the N-terminal extremity of immobilized peptides.     

Molecular analysis of the Gal/GalNAc adhesin of Entamoeba histolytica

Attachment of Entamoeba histolytica to colonic epithelium and a variety of other target cells is mediated by a galactose/N-acetyl D-galactosamine (Gal/GalNAc) inhibitable adhesin. Seven monoclonal antibodies specific for nonoverlapping epitopes of the 170 kDa subunit have been shown to have distinct effects on adherence. Four of these monoclonal antibodies inhibit or have no effect on amebic adherence while two others enhance amebic adherence. The epitopes recognized by these seven monoclonal antibodies have been mapped to the extracellular cysteine rich region of the 170 kDa subunit. The conformational nature of the epitopes was examined by testing monoclonal antibody reactivity with isolated regions of the 170 kDa subunit expressed as fusion proteins in E. coli and also with denatured native adhesin. These analyses suggested that three of monoclonal antibodies recognized conformational epitopes while the remaining four recognized linear epitopes. The mapping of these monoclonal antibodies have identified functionally important regions of the Gal/GalNAc adhesin and have also shown that recombinant Gal/GalNAc adhesin, when expressed in E.coli, retained at least some of its native conformation.     

FS stress induces long-lasting memory facilitation: involvement of cholinergic pathways

A gene coding for epsilon-toxin was isolated from a field isolate of Clostridium perfringens type D by PCR amplification and was cloned under the control of T5 promoter fused with six-histidine tag at the amino terminal end. Escherichia coli cells harbouring this construct expressed high levels of the recombinant protein in the form of inclusion bodies. The protein was purified using single step affinity chromatography on a Ni(2+)-nitrilotriacetic acid (NTA) agarose column. Upon immunization of rabbit with the purified recombinant protein, high antibody titre was detected. The antibodies raised against the recombinant protein were able to recognize the recombinant as well as the native toxin. Anti epsilon-toxin monoclonal antibody was able to detect the recombinant protein in a Western blot. N-terminal sequence of the recombinant protein matched with the known sequence of the toxin. At the shake flask level, up to 20 mg of pure epsilon-prototoxin was produced per litre of culture.     

Characterization of the neutrophil molecules identified by quinine-dependent antibodies from two patients

BACKGROUND: Two patients with episodic pancytopenia and renal failure associated with quinine (Qn) ingestion were previously found to have Qn-dependent antibodies that reacted with red cells, platelets, and neutrophils. The purpose of these studies was to characterize the neutrophil antigens recognized by Qn-dependent antibodies from these two patients. STUDY DESIGN AND METHODS: The neutrophil molecules recognized by the Qn-dependent antibodies in the sera from the two patients were analyzed by immunoprecipitation using 125I-labeled neutrophils. Neutrophils from 13 different donors were tested. RESULTS: The Qn-dependent antibodies from Patient 1 immunoprecipitated a 60-kDa molecule on neutrophils from seven donors and an 85-kDa molecule on neutrophils from three donors. The Qn-dependent antibodies from Patient 2 reacted with a 32-kDa molecule on neutrophils from 5 donors, a 60-kDa molecule on neutrophils from 9 donors, and an 85-kDa molecule on neutrophils from 10 donors. Neutrophil-specific antigen NB1 is also located on a 60-kDa glycoprotein (GP). While the antibody in serum from Patient 1 did not show specificity for NB1, the antibody from Patient 2 detected the 60-kDa molecule on NB1-positive neutrophils from 9 of 11 donors tested and did not detect the 60-kDa molecule on NB1-negative neutrophils from 2 donors. In a monoclonal antibody immobilization of granulocyte antigens assay, the Qn-dependent antibody from both patients reacted with the 60-kDa molecule carrying NB1. The Qn-dependent antibody from a third patient, Patient 3, was previously found to react with an 85-kDa GP and the 60-kDa NB1 GP. To determine if the Qn-dependent antibodies from Patients 2 and 3 recognized the same 85-kDa GP, neutrophils were treated with serum from Patient 3 plus Qn to remove the 85-kDa GP. Then, serum from Patient 2 plus Qn no longer immunoprecipitated the 85-kDa GP. CONCLUSION: The antigens recognized by Qn-dependent neutrophil antibodies were located on molecules of 85, 60, and 32 kDa. Qn-dependent antibodies from two patients reacted with the same 85-kDa GP and those from three patients reacted with the same 60-kDa GP. The 60-kDa molecule recognized by the Qn-dependent antibodies carried the NB1 antigen.     

Plasmodium vivax: epitope mapping of monoclonal antibodies against the N-terminal region of the merozoite surface protein 1

Plasmodium vivax is the most widely distributed human malaria with an estimate of 35 million cases per year. The deduced amino acid sequence comparisons of the Merozoite Surface Protein 1 (MSP1) from several plasmodial species, including that of P. vivax (PvMSP1), revealed the existence of highly conserved blocks and polymorphic blocks. We had previously shown that sequences within conserved blocks from the N-terminal region of the PvMSP1 were poorly immunogenic in natural human infections. These results suggest that these regions code for important and unknown structural and/or functional features and thus they could potentially be tested as a sub-unit PvMSP1 vaccine. In the present study, a battery of monoclonal antibodies (Mabs) was produced against the N-terminal region of the PvMSP1 in an attempt to determine whether these N-terminal ICBs contained all the epitopes exposed on the native molecule. The results suggest that the most terminal ICB2 and ICB3 blocks are not exposed on the surface of the PvMSP1 native molecule and clearly eliminate the possibility of considering the N-terminal domains as unique components of a sub-unit PvMSP1 vaccine candidate.     

Heart rate variability, norepinephrine and ECG changes in subarachnoid hemorrhage patients

We have studied Pbs21, a major ookinete surface protein of Plasmodium berghei, for the development of a model transmission blocking immunogen. In the mouse, recombinant Pbs21 expressed in the Escherichia coli expression system (EcrPbs21) is not as effective in inducing transmission blocking antibodies as native Pbs21 (nPbs21), possibly because of differences in post-translational processing between EcrPbs21 and nPbs21. In an attempt to improve the efficacy of the recombinant molecule, we describe here the use of a baculovirus expression vector system in the silkworm Bombyx mori. Following an injection of recombinant baculovirus containing Pbs21 cDNA, B. mori larvae produced recombinant Pbs21 (BmrPbs21) with a molecular weight indistinguishable from nPbs21. Fifty micrograms of BmrPbs21 could be purified from the hemolymph of each infected larva using affinity chromatography. Immunization of Balb/c mice with BmrPbs21 induced high anti-BmrPbs21 and anti--ookinete antibodies but low anti-EcrPbs21 antibody. In contrast, EcrPbs21 induced high anti--EcrPbs21 antibody but low anti-BmrPbs21 and anti-ookinete antibodies. This suggests that most B-cell epitopes on nPbs21 are conformational and that many of the linear epitopes in EcrPbs21 are not normally exposed in nPbs21. Oocyst formation in Anopheles stephensi mosquitoes, which fed on mice immunized with purified BmrPbs21 and infected with P. berghei, was blocked by 85.5-97.1%. These results suggest that the baculovirus-silkworm system produces useful quantities of recombinant Pbs21 which in limited studies is structurally and immunogenically indistinguishable from the native molecule.     

Glycoprotein E2 of bovine viral diarrhea virus expressed in insect cells provides calves limited protection from systemic infection and disease

Calves were vaccinated with a C-terminally truncated baculovirus expression product of E2 from the Singer strain of bovine viral diarrhea virus. The expressed E2 was glycosylated and retained antigenic authenticity. After induction of viral neutralizing antibody, the calves were challenge exposed with either the homologous Singer strain of virus or with the heterologous 890 strain of virus. Vaccine-induced antibody titer of > or = 2 protected calves from clinical signs of disease induced by homologous viral challenge exposure. An antibody titer of > or = 512 reduced replication of homologous challenge virus to a level which did not induce an appreciable increase in serologic titer of viral neutralizing antibody. Vaccine-induced antibody titer of < or = 4096 did not protect calves from systemic spread of virus or from disease after challenge exposure with heterologous bovine viral diarrhea virus.     

Antigenic characterization of Brazilian bovine viral diarrhea virus isolates by monoclonal antibodies and cross-neutralization

Nineteen Brazilian isolates of bovine viral diarrhea virus (BVDV) were characterized antigenically with a panel of 19 monoclonal antibodies (mAbs) (Corapi WV, Donis RO and Dubovi EJ (1990) American Journal of Veterinary Research, 55: 1388-1394). Eight isolates were further characterized by cross-neutralization using sheep monospecific antisera. Analysis of mAb binding to viral antigens by indirect immunofluorescence revealed distinct patterns of reactivity among the native viruses. Local isolates differed from the prototype Singer strain in recognition by up to 14 mAbs. Only two mAbs--one to the non-structural protein NS23/p125 and another to the envelope glycoprotein E0/gp48--recognized 100% of the isolates. No isolate was recognized by more than 14 mAbs and twelve viruses reacted with 10 or less mAbs. mAbs to the major envelope glycoprotein E2/gp53 revealed a particularly high degree of antigenic variability in this glycoprotein. Nine isolates (47.3%) reacted with three or less of 10 E2/gp53 mAbs, and one isolate was not recognized by any of these mAbs. Virus-specific antisera to eight isolates plus three standard BVDV strains raised in lambs had virus-neutralizing titers ranging from 400 to 3200 against the homologous virus. Nonetheless, many antisera showed significantly reduced neutralizing activity when tested against heterologous viruses. Up to 128-fold differences in cross-neutralization titers were observed for some pairs of viruses. When the coefficient of antigenic similarity (R) was calculated, 49 of 66 comparisons (74.24%) between viruses resulted in R values that antigenically distinguish strains. Moreover, one isolate had R value suggesting that it belongs to a distinct serologic group. The marked antigenic diversity observed among Brazilian BVDV isolates should be considered when planning diagnostic and immunization strategies.     

Reservations and preferences among procurement professionals concerning the donation of specific organs and tissues

The aim of this study was to analyze the ability of an alloantibody from a patient with severe von Willebrand disease (vWD) to interfere with the vWF domain for FVIII, to inhibit factor VIII (FVIII), and to compare it with a rabbit polyclonal antibody. The vWF domain for binding to FVIII was assayed by a method previously described but using recombinant FVIII (r-FVIII, Kogenate), which contains no vWF, instead of Hemofil M (HM). Rabbit or human antibodies towards FVIII (FVIII-Ab) were analyzed using microtiter wells with immobilized r-FVIII through a monoclonal anti-FVIII antibody and an ELISA method. IgG from plasma of a patient with hemophilia A and FVIII inhibitor was used as a positive control. Normal human and rabbit IgGs were included as negative controls. Human vWD alloantibody IgG and the rabbit anti-vWF antibody IgG reacted with immobilized normal vWF, inhibiting its binding to r-FVIII in a dose-dependent manner, which suggests that it is specific. Normal human IgG fraction, as well as nonspecific rabbit IgG, did not interfere with this binding at all. The monoclonal antibody used in this assay to immobilize vWF did not alter this interaction at all. Human vWD alloantibody IgG and the rabbit antibody against vWF showed a partial inhibitory activity to plasma FVIII as well as r-FVIII. The inhibition reached a plateau with residual FVIII activity. FVIII-Ab were not detected in human alloantibody or in rabbit antibody preparations. In contrast, hemophiliac FVIII inhibitor showed FVIII-AB. This human vWD alloantibody behaves like polyclonal heterologous antibodies, and their inhibition of FVIII seems to be nonspecific due to a steric hindrance mechanism provided that both have no FVIII antibodies.     

Flow cytometric analysis of microsporidia belonging to the genus Encephalitozoon

Flow cytometry was used in the identification of human microsporidia belonging to the genus Encephalitozoon. Microsporidian spores of Encephalitozoon hellem, E. cuniculi, and E. intestinalis were propagated in axenic cultures of monkey kidney E6 cells, purified with Percoll, and exposed to homologous and heterologous rabbit antiserum and monoclonal antibody prepared against E. hellem spores. After reaction to goat anti-rabbit immunoglobulin G (IgG) or goat anti-mouse IgG conjugated to fluorescein isothiocyanate, fluorescence histograms from gated data on light-scatter profiles showed that rabbit anti-E. hellem serum was reactive to E. hellem spores but also had cross-reactivity to spores of E. cuniculi and E. intestinalis. On the other hand, fluorescence histograms showed that rabbit anti-E. cuniculi and rabbit anti-E. intestinalis sera were reactive with homologous spores only. Monoclonal antibody prepared against E. hellem reacted only with spores of E. hellem. Neither the polyclonal antibodies nor the monoclonal antibodies reacted with Cryptosporidium parvum oocysts. Fluorescence histograms of spores treated with 10% formalin also showed reactivity, but the number of events in the most intense peaks of fluorescence was fewer (7 to 42%, depending on species) than the number of events in the most intense peaks of fluorescence for nontreated spores. By flow cytometry, formalin-treated and nontreated spores of Encephalitozoon were identified to the species level by using gated data on light-scatter profiles and analyzing the fluorescence histograms from the indirect immunofluorescence of the spores. Once a procedure is established for the isolation of Encephalitozoon spores from clinical specimens, identification of spores by flow cytometry may be useful not only for diagnosis but also for epidemiologic studies.     

Epitope-specific antibody and suppression of autoantibody responses against a hybrid self protein

This study addresses the relationship of epitope-specific Ab responses and alternative autoantibody responses in a model system in which an antigenized self protein serves as the carrier for a defined heterologous B cell epitope. Ubiquitin, a nonimmunogenic self protein, was engineered to present heterologous B and T cell epitopes in the recombinant molecule. Fusion to the C terminus introduced a universal T cell epitope from a Mycobacterium tuberculosis Ag. The B cell epitope was created by inserting a 12-residue loop sequence of HIV-1 gp120 at a surface-exposed position of ubiquitin. These modifications preserved the ubiquitin fold, allowing a new conformational epitope to be presented among native self epitopes. Mice immunized with the hybrid protein bearing only the mycobacterial T cell epitope elicited a strong autoantibody response to native ubiquitin. In contrast, antisera elicited against hybrid ubiquitin presenting the HIV B cell epitope reacted specifically with the foreign epitope but not with native ubiquitin. Absence of autoantibody in the response was attributed to poor competition of autoreactive B cells for limiting T cell help. Both types of responses were associated with Th responses to defined epitopes of the ubiquitin hybrid protein. These results may have implications for a tolerance mechanism dependent on B-T cell cooperation.     

The NH2-terminal region of the sickle hemoglobin beta chain. II. Characterization of monospecific antibodies

We have previously shown that antibodies specific for hemoglobin S could be fractionated by absorption of an antiserum to hemoglobin S to Sepharose containing a synthetic oligopeptide. betaS (1-13), corresponding to the first 13 amino acid residues of the beta chain of hemoglobin S. We report here that this antibody population, anti-betaS (1-13), shows considerable restriction of heterogeneity in isoelectric focusing studies and monospecificity on velocity ultracentrifugation in the presence of hemoglobin S. The binding of various hemoglobin species to anti-betaS (1-13) was studied using a double antibody radioimmunoassay with [14C]carbamoylated hemoglobin S. Carbonmonoxy-, oxy-, met-, and cyanmethemoglobin S reacted equally with the antibody, but deoxyhemoglobin (with or without organic phosphates) reacted differently. Hemoglobin A and several hemoglobin mutants with alterations in the NH2-terminal region of the beta chain did not displace labeled hemoglobin S from anti-betaS (1-13). BETAS chains reacted with the antibody, but less well than hemoglobin S, while betaA and alpha chains, and globins did not react with the antibody. The synthetic peptide, betaS (1-13), used for fractionation, reacted with the antibody about 300-fold less efficiently than hemoglobin S. BetaS (3-13) was even less reactive, while smaller peptides which included the valine residue at position 6 displaced little of the tracer [14C]carbamoylated hemoglobin S at concentrations as high as 10(-2) M. We interpret these results to indicate that this method of immunoabsorption has produced a monospecific subfraction of antibodies which is specific for the NH2-terminal region of the beta chain of hemoglobin S in its native conformation.     

Neutralizing antibodies to bovine thyrotropin in immunized patients with thyroid cancer

Bovine thyrotropin (bTSH) was administered to 17 patients who had thyroid carcinoma. Anti-bTSH antibodies in the patients' sera were detected by three methods: 1) cross-reaction of sera in a homologous bTSH RIA, 2) [125I]bTSH binding to the patients' sera using charcoal to separate bound from free fractions, and 3) gel filtraton to detect [125I]bTSH bound to anti-bTSH in patients' sera. Immunoreactive anti-bTSH antibodies were thus demonstrated in 14 patients. These patients showed specific binding of their sera to [125I]bTSH with the charcoal separation of free from bound hormone. A high titer of antiserum (1:10,000) was found in those patients whose sera reacted strongly in the bTSH RIA (greater than 50 mu U/ml). The binding capacity of the serum of 1 patient was estimated as 2,600 mu U/ml serum. Ten of the patients' sera which showed immunoreactivity to bTSH neutralized bTSH activity in the McKenzie mouse bioassay but did not neutralize the activity of human TSH in this bioassay. Repeated administration of bTSH to 14 patients resulted in development of immunoreactive and neutralizing anti-bTSH antibodies. Development of immunological resistance to bTSH appears inevitable in patients who receive repeated injections of this hormone. Because of the loss of effectiveness of bTSH by antibody formation, the repeated diagnostic and therapeutic use of bTSH is not recommended.