Evaluation of Radiolabeled Girentuximab In Vitro and In Vivo

Girentuximab (cG250) targets carbonic anhydrase IX (CAIX), a protein which is expressed on the surface of most renal cancer cells (RCCs). cG250 labeled with 177Lu has been used in clinical trials for radioimmunotherapy (RIT) of RCCs. In this work, an extensive characterization of the immunoconjugates allowed optimization of the labeling conditions with 177Lu while maintaining immunoreactivity of cG250, which was then investigated in in vitro and in vivo experiments. cG250 was conjugated with S-2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane tetraacetic acid (DOTA(SCN)) by using incubation times between 30 and 90 min and characterized by mass spectrometry. Immunoconjugates with five to ten DOTA(SCN) molecules per cG250 molecule were obtained. Conjugates with ratios less than six DOTA(SCN)/cG250 had higher in vitro antigen affinity, both pre- and postlabeling with 177Lu. Radiochemical stability increased, in the presence of sodium ascorbate, which prevents radiolysis. The immunoreactivity of the radiolabeled cG250 tested by specific binding to SK-RC-52 cells decreased when the DOTA content per conjugate increased. The in vivo tumor uptake was < 10% ID/g and independent of the total amount of protein in the range between 5 and 100 µg cG250 per animal. Low tumor uptake was found to be due to significant necrotic areas and heterogeneous CAIX expression. In addition, low vascularity indicated relatively poor accessibility of the CAIX target.

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Enlisting the Ixodes scapularis Embryonic ISE6 Cell Line to Investigate the Neuronal Basis of Tick—Pathogen Interactions

Pathogens ◽

10.3390/pathogens10010070 ◽

2021 ◽

Vol 10 (1) ◽

pp. 70

Author(s):

Lourdes Mateos-Hernández ◽

Natália Pipová ◽

Eléonore Allain ◽

Céline Henry ◽

Clotilde Rouxel ◽

...

Keyword(s):

Cell Line ◽

Peripheral Nerves ◽

Ixodes Scapularis ◽

Embryonic Cell ◽

Cell Subpopulation ◽

In Vivo Experiments

Neuropeptides are small signaling molecules expressed in the tick central nervous system, i.e., the synganglion. The neuronal-like Ixodes scapularis embryonic cell line, ISE6, is an effective tool frequently used for examining tick–pathogen interactions. We detected 37 neuropeptide transcripts in the I. scapularis ISE6 cell line using in silico methods, and six of these neuropeptide genes were used for experimental validation. Among these six neuropeptide genes, the tachykinin-related peptide (TRP) of ISE6 cells varied in transcript expression depending on the infection strain of the tick-borne pathogen, Anaplasma phagocytophilum. The immunocytochemistry of TRP revealed cytoplasmic expression in a prominent ISE6 cell subpopulation. The presence of TRP was also confirmed in A. phagocytophilum-infected ISE6 cells. The in situ hybridization and immunohistochemistry of TRP of I. scapularis synganglion revealed expression in distinct neuronal cells. In addition, TRP immunoreaction was detected in axons exiting the synganglion via peripheral nerves as well as in hemal nerve-associated lateral segmental organs. The characterization of a complete Ixodes neuropeptidome in ISE6 cells may serve as an effective in vitro tool to study how tick-borne pathogens interact with synganglion components that are vital to tick physiology. Therefore, our current study is a potential stepping stone for in vivo experiments to further examine the neuronal basis of tick–pathogen interactions.

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Tumor Uptake of Triazine Dendrimers Decorated with Four, Sixteen, and Sixty-Four PSMA-Targeted Ligands: Passive versus Active Tumor Targeting

Biomolecules ◽

10.3390/biom9090421 ◽

2019 ◽

Vol 9 (9) ◽

pp. 421 ◽

Cited By ~ 3

Author(s):

Jongdoo Lim ◽

Bing Guan ◽

Kien Nham ◽

Guiyang Hao ◽

Xiankai Sun ◽

...

Keyword(s):

Specific Binding ◽

Quantitative Imaging ◽

Molecular Size ◽

Tumor Uptake ◽

Competitive Binding Assay ◽

The Core ◽

Positron Emission ◽

Pentanedioic Acid

Various glutamate urea ligands have displayed high affinities to prostate specific membrane antigen (PSMA), which is highly overexpressed in prostate and other cancer sites. The multivalent versions of small PSMA-targeted molecules are known to be even more efficiently bound to the receptor. Here, we employ a well-known urea-based ligand, 2-[3-(1,3-dicarboxypropyl)-ureido] pentanedioic acid (DUPA) and triazine dendrimers in order to study the effect of molecular size on multivalent targeting in prostate cancer. The synthetic route starts with the preparation of a dichlorotriazine bearing DUPA in 67% overall yield over five steps. This dichlorotriazine reacts with G1, G3, and G5 triazine dendrimers bearing a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) group for 64Cu-labeling at the core to afford poly(monochlorotriazine) intermediates. Addition of 4-aminomethylpiperidine (4-AMP) and the following deprotection produce the target compounds, G1-(DUPA)4, G3-(DUPA)16, and G5-(DUPA)64. These targets include 4/16/64 DUPA groups on the surface and a DOTA group at the core, respectively. In vitro cell assay using PC3-PIP (PSMA positive) and PC3-FLU (PSMA negative) cells reveals that G1-(DUPA)4 has the highest PC3-PIP to PC3-FLU uptake ratio (10-fold) through the PSMA-mediated specific uptake. While G5-(DUPA)64 displayed approximately 12 times higher binding affinity (IC50 23.6 nM) to PC3-PIP cells than G1-(DUPA)4 (IC50 282.3 nM) as evaluated in a competitive binding assay, the G5 dendrimer also showed high non-specific binding to PC3-FLU cells. In vivo uptake of the 64Cu-labeled dendrimers was also evaluated in severe combined inmmunodeficient (SCID) mice bearing PC3-PIP and PC3-FLU xenografts on each shoulder, respectively. Interestingly, quantitative imaging analysis of positron emission tomograph (PET) displayed the lowest tumor uptake in PC3-PIP cells for the midsize dendrimer G3-(DUPA)16 (19.4 kDa) (0.66 ± 0.15%ID/g at 1 h. p.i., 0.64 ± 0.11%ID/g at 4 h. p.i., and 0.67 ± 0.08%ID/g at 24 h. p.i.). Through the specific binding of G1-(DUPA)4 to PSMA, the smallest dendrimer (5.1 kDa) demonstrated the highest PC3-PIP to muscle and PC3-PIP to PC3-FLU uptake ratios (17.7 ± 5.5 and 6.7 ± 3.0 at 4 h p.i., respectively). In addition, the enhanced permeability and retention (EPR) effect appeared to be an overwhelming factor for tumor uptake of the largest dendrimer G5-(DUPA)64 as the uptake was at a similar level irrelevant to the PSMA expression.

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Characterization of Endogenous Retroviruses in Sheep

Journal of Virology ◽

10.1128/jvi.77.20.11268-11273.2003 ◽

2003 ◽

Vol 77 (20) ◽

pp. 11268-11273 ◽

Cited By ~ 14

Author(s):

Nikolai Klymiuk ◽

Mathias Müller ◽

Gottfried Brem ◽

Bernhard Aigner

Keyword(s):

Endogenous Retrovirus ◽

Ovis Aries ◽

Endogenous Retroviruses ◽

Open Reading Frames ◽

In Vivo Experiments ◽

Pregnant Sheep ◽

Reading Frames

ABSTRACT Endogenous retrovirus (ERV) sequences have been found in all mammals. In vitro and in vivo experiments revealed ERV activation and cross-species infection in several species. Sheep (Ovis aries) are used for various biotechnological purposes; however, they have not yet been comprehensively screened for ERV sequences. Therefore, the aim of the study was to classify the ERV sequences in the ovine genome (OERV) by analyzing the retroviral pro-pol sequences. Three OERV β families and nine OERV γ families were revealed. Novel open reading frames (ORF) in the amplified proviral fragment were found in one OERV β family and two OERV γ families. Hybrid OERV produced by putative recombination events were not detected. Quantitative analysis of the OERV sequences in the ovine genome revealed no relevant variations in the endogenous retroviral loads of different breeds. Expression analysis of different tissues from fetal and pregnant sheep detected mRNA from both gammaretrovirus families, showing ORF fragments. Thus, the release of retroviruses from sheep cells cannot be excluded.

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A primary study on genes with selected mutations by in vitro passage of Chlamydia muridarum strains

Pathogens and Disease ◽

10.1093/femspd/ftz017 ◽

2019 ◽

Vol 77 (3) ◽

Author(s):

Zhou Zhou ◽

Na Liu ◽

Yingzi Wang ◽

Arthur Wirekoh Emmanuel ◽

Xiaoxing You ◽

...

Keyword(s):

Single Gene ◽

Point Mutations ◽

Primary Study ◽

Chlamydia Muridarum ◽

In Vivo Experiments ◽

High Incidence ◽

Identification And Characterization

ABSTRACTObjectiveThis study is to investigate the functions of newly discovered genes in Chlamydia muridarum (C. muridarum) strains with single gene differences.MethodsUsing whole genome sequencing and plaque formation assays, C. muridarum parental and passaging strains were established, and the isogenic clones expressing certain genotypes were isolated. Strains with single gene differences were obtained. Based on prediction, the valuable strains with single gene differences of tc0412, tc0668 or tc0237 were subjected to the in vitro and in vivo experiments for biological characterization and virulence analysis.ResultsInsertional -472840T mutation of the tc0412 gene (T28T/B3 type) matching with the nonmutant tc0668 gene and tc0237 gene with point mutations G797659T (Q117E) might slow the growth of Chlamydia due to the lack of a plasmid. The nonmutant tc0668 in the strain might induce a high incidence of hydrosalpinx in mice, while tc0668 with a G797659T point mutation was significantly attenuated. Compared with the nonmutant tc0237, the strains containing mutant tc0237 were characterized by reduced centrifugation dependence during infection.ConclusionThe identification and characterization of these genes might contribute to the comprehensive understanding of the pathogenic mechanism of Chlamydia.

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Emerging Vascular Applications of Magnetic Resonance Imaging: A Picture is Worth More than a Thousand Words

Vascular ◽

10.2310/6670.2006.00062 ◽

2006 ◽

Vol 14 (6) ◽

pp. 366-371 ◽

Cited By ~ 3

Author(s):

Tamara N. Fitzgerald ◽

Akihito Muto ◽

Fabio Akimaro Kudo ◽

Jose Mario Pimiento ◽

Robert Todd Constable ◽

...

Keyword(s):

Magnetic Resonance ◽

Three Dimensional ◽

Quantitative Information ◽

Data Set ◽

Vascular Intervention ◽

In Vivo Experiments ◽

Blood Flow Dynamics

Vascular applications of magnetic resonance (MR) imaging are reviewed, with emphasis on algorithms that use nonpictorial information contained in the MR data set. Current clinical vascular practice generally limits use of MR angiography and three-dimensional vessel images to qualitative pictorial rendering without routinely using the available quantitative information contained within the MR data. This review is dedicated to recent advances that include characterization of vessel histology, assessment of carotid plaque vulnerability, characterization of blood flow dynamics, quantitative analysis of disease severity, and prediction of vascular intervention outcome. Examples from histologic preparation, in vitro and in vivo experiments, are discussed, with an emphasis on potential clinical applications and advances in acquisition technology.

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Discovery and characterization of a fourth class of guanidine riboswitches

Nucleic Acids Research ◽

10.1093/nar/gkaa1102 ◽

2020 ◽

Vol 48 (22) ◽

pp. 12889-12899

Author(s):

Felina Lenkeit ◽

Iris Eckert ◽

Jörg S Hartig ◽

Zasha Weinberg

Keyword(s):

Biological Significance ◽

Protein Domains ◽

Structural Class ◽

New Class ◽

In Vivo Experiments ◽

Fourth Class ◽

Metabolite Binding

Abstract Riboswitches are RNAs that specifically sense a small molecule and regulate genes accordingly. The recent discovery of guanidine-binding riboswitches revealed the biological significance of this compound, and uncovered genes related to its biology. For example, certain sugE genes encode guanidine exporters and are activated by the riboswitches to reduce toxic levels of guanidine in the cell. In order to study guanidine biology and riboswitches, we applied a bioinformatics strategy for discovering additional guanidine riboswitches by searching for new candidate motifs associated with sugE genes. Based on in vitro and in vivo experiments, we determined that one of our six best candidates is a new structural class of guanidine riboswitches. The expression of a genetic reporter was induced 80-fold in response to addition of 5 mM guanidine in Staphylococcus aureus. This new class, called the guanidine-IV riboswitch, reveals additional guanidine-associated protein domains that are extremely rarely or never associated with previously established guanidine riboswitches. Among these protein domains are two transporter families that are structurally distinct from SugE, and could represent novel types of guanidine exporters. These results establish a new metabolite-binding RNA, further validate a bioinformatics method for finding riboswitches and suggest substrate specificities for as-yet uncharacterized transporter proteins.

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Virulence of Leishmania infantum Is Expressed as a Clonal and Dominant Phenotype in Experimental Infections

Infection and Immunity ◽

10.1128/iai.69.12.7365-7373.2001 ◽

2001 ◽

Vol 69 (12) ◽

pp. 7365-7373 ◽

Cited By ~ 33

Author(s):

Yves Jean-François Garin ◽

Annie Sulahian ◽

Francine Pratlong ◽

Pascale Meneceur ◽

Jean-Pierre Gangneux ◽

...

Keyword(s):

Leishmania Infantum ◽

Virulent Strain ◽

Biological Marker ◽

In Vivo Experiments ◽

Dominant Phenotype ◽

Virulence Phenotype ◽

Parasite Populations

ABSTRACT Human Leishmania infantum infection results in a spectrum of clinical expressions ranging from cutaneous to either asymptomatic or fatal visceral disease. In this context, characterization of parasite virulence appears to be relevant as a biological marker of intrinsic parasitic factors that can affect the pathology of leishmaniasis. Since parasite populations in naturally infected hosts are likely to be composed of multiclonal associations, we first explored the biodiversity of parasite virulence at the intrastrain level in vitro and in vivo by using 11 clones isolated from three strains previously known to express different virulence phenotypes in mice. Subsequently, we studied the course of infection in mice inoculated simultaneously or successively with strains or clones showing various virulence phenotypes. Analysis of in vitro growth characteristics showed no differences among clones from the different parental strains. By contrast, in vivo experiments evidenced a marked intrastrain heterogeneity of virulence to mice. One out of five clones obtained from a virulent strain showed a typical virulence phenotype, while the remaining four clones had low-virulence profiles, as did the six clones isolated from two low-virulence strains. In mixed multiclonal infections, the virulence phenotype was expressed as a dominant character over the associated low-virulence clones. After a challenge with either a homologous or a heterologous strain or clone, virulence phenotypes were conserved and expressed as in naive mice independently from the preexisting population. These results strongly suggest that parasite virulence in L. infantum visceral leishmaniasis is clonal and dominant in nature.

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Functional and Structural Analysis of HrcA Repressor Protein from Caulobacter crescentus

Journal of Bacteriology ◽

10.1128/jb.186.20.6759-6767.2004 ◽

2004 ◽

Vol 186 (20) ◽

pp. 6759-6767 ◽

Cited By ~ 12

Author(s):

Michelle F. Susin ◽

Humberto R. Perez ◽

Regina L. Baldini ◽

Suely L. Gomes

Keyword(s):

Heat Shock ◽

Caulobacter Crescentus ◽

Specific Binding ◽

Regulatory Region ◽

Single Amino Acid ◽

Repressor Protein ◽

Mutant Proteins ◽

In Vivo Experiments

ABSTRACT A large number of bacteria regulate chaperone gene expression during heat shock by the HrcA-CIRCE system, in which the DNA element called CIRCE serves as binding site for the repressor protein HrcA under nonstress conditions. In Caulobacter crescentus, the groESL operon presents a dual type of control. Heat shock induction is controlled by a σ32-dependent promoter and the HrcA-CIRCE system plays a role in regulation of groESL expression under physiological temperatures. To study the activity of HrcA in vitro, we purified a histidine-tagged version of the protein, and specific binding to the CIRCE element was obtained by gel shift assays. The amount of retarded DNA increased significantly in the presence of GroES/GroEL, suggesting that the GroE chaperonin machine modulates HrcA activity. Further evidence of this modulation was obtained using lacZ transcription fusions with the groESL regulatory region in C. crescentus cells, producing different amounts of GroES/GroEL. In addition, we identified the putative DNA-binding domain of HrcA through extensive protein sequence comparison and constructed various HrcA mutant proteins containing single amino acid substitutions in or near this region. In vitro and in vivo experiments with these mutated proteins indicated several amino acids important for repressor activity.

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Metabolism of inorganic selenium in rumen bacteria

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y68-038 ◽

1968 ◽

Vol 46 (2) ◽

pp. 229-232 ◽

Cited By ~ 41

Author(s):

M. Hidiroglou ◽

D. P. Heaney ◽

K. J. Jenkins

Keyword(s):

Vitamin E ◽

Selenium Supplementation ◽

Rumen Bacteria ◽

Microbial Protein ◽

Rumen Microorganisms ◽

In Vivo Experiments ◽

Inorganic Selenium

In vitro and in vivo experiments demonstrated that rumen bacteria were capable of metabolizing inorganic 75Se and incorporating the element into the microbial protein. The fixation of 75Se into bacteria in vitro was inversely proportional to the previous dietary intake of selenium by the host sheep. In sheep fed a purified diet low in selenium and vitamin E, selenium supplementation caused a marked alteration of the rumen microorganisms. Characterization of the 75Se-containing compounds in the rumen bacteria protein hydrolysates revealed the presence of 75Se-selenomethionine and 75Se-selenomethionine selenoxide.

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F127/Cisplatin Microemulsions: In Vitro, In Vivo and Computational Studies

Applied Sciences ◽

10.3390/app11073006 ◽

2021 ◽

Vol 11 (7) ◽

pp. 3006

Author(s):

Saman Sargazi ◽

Mohammad Reza Hajinezhad ◽

Mahmood Barani ◽

Mahwash Mukhtar ◽

Abbas Rahdar ◽

...

Keyword(s):

Liver Enzymes ◽

Colorimetric Assay ◽

Morphological Changes ◽

Effective Strategies ◽

In Vivo Experiments ◽

Mtt Colorimetric Assay ◽

Male Wistar Rats

The development of effective strategies for local administration of chemotherapeutic drugs, thus minimizing the adverse side effects to patients, is one of the key challenges in biomedicine and cancer research. This work reports the formulation and characterization of PluronicF127 microemulsions to enhance the bioavailability of Cisplatin (Cis). The size of Cis microemulsion was about 12.0 nm, as assessed by dynamic light scattering analysis. In vitro cytotoxic activity of free Cis and F127/Cis microemulsions were studied on malignant (C152 and MCF7) and normal (HUVEC) cells via tetrazolium (MTT) colorimetric assay. Cell morphology was also monitored. In vitro assessments revealed thatF127/Cis microemulsions induced cytotoxicity/morphological changes to a lesser extent than free Cis. Regarding in vivo experiments, F127/Cis microemulsions were injected intraperitoneally at 7 and 14 mg/kg doses into adult male Wistar rats to assess histologic and biochemical changes. In this case, the bulk Cis group caused severe histopathological changes and significant increases in serum liver enzymes and serum kidney function markers. The group treated with the 14 mg/kg dose of F127/Cis microemulsions also showed severe fatty changes and significant increases in serum liver enzymes, blood urea nitrogen, and creatinine levels. The group treated with the low dose of nano-Cis showed a significant increase in serum liver enzymes levels accompanied by mild fatty changes of the liver. Theoretical surveys were performed to get an understanding of the interplay between F127 and Cis. Results reveal that hydrogen bonding (HB) interactions with F127have an influence on the molecular properties of Cis and may playa role in the lower toxicity of F127/Cis in comparison to free Cis.

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