Synergism between bovine papillomavirus type 4 and the flavonoid quercetin in cell transformation in vitro.

Bovine papillomavirus type 4 (BPV-4) morphologically transforms primary bovine cells in vitro only in the presence of an activated ras gene. The transformed cells are capable of anchorage-independent growth, but are not immortal and are incapable of inducing tumors in nude mice, suggesting that other events are needed to convert the cells to the fully transformed phenotype. We show here that treatment of the cells with a single dose of the flavonoid quercetin leads to full oncogenic transformation of cells transfected with BPV-4 and ras. Quercetin is one of the most potent mutagens found in bracken fern, the environmental cofactor in BPV-4-associated carcinogenesis of the upper alimentary canal of cattle. Our results point to quercetin as the probable in vivo cocarcinogen synergizing with BPV-4 in malignant progression.     

Pasteurella haemolytica leukotoxin inhibits mitogen-induced bovine peripheral blood mononuclear cell proliferation in vitro

In this study we demonstrate that partially purified Pasteurella haemolytica leukotoxin inhibits the proliferative response of bovine peripheral blood mononuclear cells (PBMC) to mitogens in vitro. Inhibition of PBMC proliferation did not appear to be due to cell death. Addition of a neutralizing anti-leukotoxin monoclonal antibody restored a normal proliferative response.     

Inhibition of the proteasome influences murine and human dendritic cell development in vitro and in vivo

Dendritic cells (DC) are the most potent antigen-presenting cells (APC) known today and are designated as nature′s adjuvant since they are the only antigen-presenting cell type capable of inducing naïve T cell responses in vivo. In order to become potent T cell stimulators DC have to mature. This mature DC phenotype is characterized amongst other characteristics by the up-regulation of co-stimulatory molecules such as CD40, CD80, CD86 and the cell surface expression of CD83. Inhibition of their expression blocks the immune responses in vitro and in vivo, and thus represents an interesting strategy to control undesired and/or over-activated immune responses such as in autoimmune disorders, transplant rejections and allergies. Here we investigated the in vitro and in vivo effects of the proteasome inhibitor Velcade^® in respect to DC phenotype and DC functions in murine and human DC. Interestingly, in vitro, DC maturation as well as DC-mediated T cell stimulation and cytokine production was impaired. Furthermore, administration of the inhibitor in vivo resulted in a reduced mature phenotype of ex vivo generated murine DC. Thus, inhibition of the proteasome interferes with DC maturation and subsequently with DC-mediated T cell stimulation events.     

Differences between transfer RNA methylase activity in human diploid fibroblasts during in vitro and in vivo aging

The change of the specific activity of S-adenosylmethionine: tRNA methyltransferase in cultures of human diploid fibroblasts at different passages has been measured and compared with that in the same type of cells derived from donors of different ages. Whereas the specific activity of tRNA methylase in the in vitro aged cells was found to decline gradually with increasing passage number of the culture, a different activity--age relationship was observed for this enzyme in cells derived from donors of different ages. The activity of tRNA methylase is high in the fetal cells and drops drastically in the "newborn" cells. After a further 10% decline, the activity of this enzyme reaches a steady low level in the postnatal cells from donors ranging in age from 3 months to 94 years. These findings cast doubt on the validity of the assumption that the results obtained from in vitro aging experiments reflect the biochemistry of aging in vivo. The "fetal" enzyme can methylate the "aged" tRNA but the "aged" enzyme cannot methylate the "fetal" tRNA. The fetal cells contain enzyme activities specific for the formation of m1A, m5C and m1G. These activities are low or deficient in "aged" cells. Control experiments showed that all of these results are due neither to the presence of inhibitor or stimulator in the extract nor to effects related to the population density, sex or growth rate of the culture.     

Acidity near eroding polylactide-polyglycolide in vitro and in vivo in rabbit tibial bone chambers

Eroding poly( -lactide-co-glycolide) (PDLLG) washers were observed chronically in vitro and in vivo following loading in a bone chamber tibial implant (BCl). Images were recorded using intravital microscopy of the implanted rabbit and direct pH measurements were obtained of the tissue in the bone chamber using a combination probe-reference microelectrode. While statistically significant pH differences were found between the control (unloaded) and experimental (PDLLG-bearing) BCls, they were only of the order of 0.2 pH units. This value proved to be physiologically insignificant as no statistically significant difference in bone defect healing, as indicated by angiogenesis, was detected. It was shown that the measured small pH changes during PDLLG washer erosion would result whether the buffer was phosphate-buffered saline replaced weekly or interstitial fluid subject to vascular exchanges.     

Comparison of the ability of wild type and stabilized human IgG4 to undergo Fab arm exchange with endogenous IgG4 in vitro and in vivo

Fab arm exchange by a stabilized anti-IL-31 IgG₄S228P monoclonal antibody (mAb) was studied using physiologically relevant antibody concentrations and thiol exchange conditions, and directly compared to that of matched wild type IgG₄ (IgG₄wt) and IgG₁ control antibodies. In vitro arm exchange between the test mAbs and a purified IgG₄wt exchange partner was monitored using capillary isoelectric focusing and a size-exclusion peak shift assay. Arm exchange between the test mAbs and IgG exchange partners with unknown specificity was monitored using only the shift assay. Studies were performed using single isotype human and mouse mAbs, unfractionated human, mouse, and cynomolgus monkey IgG, and human serum as the sources of the exchange partners. In vitro studies using human serum demonstrated that anti-IL-31 IgG₄S228P did not undergo significant Fab arm exchange with endogenous human IgG₄ whereas anti-IL-31 IgG₄wt underwent rapid and extensive Fab arm exchange. The in vitro results were corroborated by in vivo studies in which mice were injected with a mixture of either form of the test mAb and an excess of non-specific human IgG₄ exchange partner.     

Bartonella quintana invades and multiplies within endothelial cells in vitro and in vivo and forms intracellular blebs

Bartonella quintana, the aetiologic agent of trench fever, has recently been implicated in culture-negative endocarditis and bacteraemia amongst homeless people. B. quintana is a fastidious slow-growing organism. A tissue culture system of human endothelial cells was developed in which B. quintana grew intracellularly. Observation of the different steps during infection of these cells demonstrated that the bacteria adhered to and penetrated the cells by phagocytosis. During the preadherence stage, most bacteria exhibited surface appendages that resembled those described for Salmonella typhimurium and which may mediate specific interactions between the eucaryotic cell and the bacterium. Soon after the engulfment step, the bacterium appeared in a cell vacuole where it multiplied, giving the typical aspect of morulae which has also been reported with Ehrlichiae or Chlamydiae. In older cultures, the coexistence of bacteria and huge quantities of vesicle-like structures in the same vacuole were noted. These vesicle-like structures were also found with agar-grown bacteria and were identified as membrane blebs. Microscopic observation of heart valves from B. quintana endocarditis patients demonstrated the intracellular location of B. quintana in vivo. This intracellular location of B. quintana should now be considered in further studies on the pathogenesis of the diseases it causes.     

SDS-PAGE analysis of the protein layers adsorbing in vivo and in vitro to bone substituting materials

The composition of the protein layer adsorbed to the bone substituting materials, hydroxyapatite, β-whitlockite, titanium and aluminium, in vivo (intramuscularly in guinea pig) and in vitro, was investigated using SOS-gel electrophoresis (SDS-PAGE). After in vivo implantation for 1 d mainly proteins with molecular weights between 10 000 and 20 000 were adsorbed. After 3 months the biolayer of the implanted biomaterials also contained proteins with molecular weights 35 000, 45 000, 60 000 and 200 000. No large qualitative differences in protein composition of the biolayers on the various implanted materials were found.

In vitro incubation with human serum resulted in binding of proteins with estimated molecular weights of 30 000, 60 000 (albumin), 200 000 and > 200 000. It is suggested that the differences between in vivo and in vitro protein adsorption are due to proteolysis occurring in vivo in the vicinity of the implanted material.     

c-Met-targeted chimeric antigen receptor T cells inhibit hepatocellular carcinoma cells in vitro and in vivo

c-Met is a hepatocyte growth factor receptor overexpressed in many tumors such as hepatocellular carcinoma(HCC).Therefore,c-Met may serve as a promising target for HCC immunotherapy.Modifying T cells to express c-Met-specific chimeric antigen receptor(CAR)is an attractive strategy in treating c-Met-positive HCC.This study aimed to systematically evaluate the inhibitory effects of 2^nd-and 3^rd-generation c-Met CAR-T cells on hepatocellular carcinoma(HCC)cells.Here,2^nd-and 3^rd-generation c-Met CARs containing an anti-c-Met singlechain variable fragment(scFv)as well as the CD28 signaling domain and CD3ζ(c-Met-28-3ζ),the CD137 signaling domain and CD3ζ(c-Met-137-3ζ),or the CD28 and CD137 signaling domains and CD3ζ(c-Met-28-137-3ζ)were constructed,and their abilities to target c-Met-positive HCC cells were evaluated in vitro and in vivo.All c-Met CARs were stably expressed on T cell membrane,and c-Met CAR-T cells aggregated around c-Met-positive HCC cells and specifically killed them in vitro.c-Met-28-137-3ζCAR-T cells secreted more interferon-gamma(IFN-γ)and interleukin 2(IL-2)than c-Met-28-3ζCAR-T cells and c-Met-137-3ζCAR-T cells.Compared with c-Met low-expressed cells,c-Met CAR-T cells secreted more cytokines when co-cultured with c-Met high-expressed cells.Moreover,c-Met-28-137-3ζCAR-T cells eradicated HCC more effectively in xenograft tumor models compared with the control groups.This study suggests that 3^rd-generation c-Met CAR-T cells are more effective in inhibiting c-Met-positive HCC cells than 2^nd-generation c-Met CAR-T cells,thereby providing a promising therapeutic intervention for c-Met-positive HCC.     

Structural Requirements for SP-D Function in vitro and in vivo: Therapeutic Potential of Recombinant SP-D

Surfactant protein D has multiple functions in innate immunity in the lung. The generation of SP-D knock-out mice has revealed a central role for this protein in the control of lung inflammation. Accumulating evidence in mouse models of infection and inflammation indicates that truncated recombinant forms of surfactant protein D are biologically active in vivo. This review addresses the structural requirements for recognised activities of SP-D in vitro and in vivo, with emphasis on evidence arising from studies with transgenic mice and mouse models of inflammatory lung disease. The potential of truncated recombinant forms of surfactant protein D as novel therapy for infectious and inflammatory disease is discussed.     

The normal breast microenvironment of premenopausal women differentially influences the behavior of breast cancer cells in vitro and in vivo

Background  

Breast cancer studies frequently focus on the role of the tumor microenvironment in the promotion of cancer; however, the influence of the normal breast microenvironment on cancer cells remains relatively unknown. To investigate the role of the normal breast microenvironment on breast cancer cell tumorigenicity, we examined whether extracellular matrix molecules (ECM) derived from premenopausal African-American (AA) or Caucasian-American (CAU) breast tissue would affect the tumorigenicity of cancer cells in vitro and in vivo. We chose these two populations because of the well documented predisposition of AA women to develop aggressive, highly metastatic breast cancer compared to CAU women.     

Neurosteroid regulation of inhibitory synaptic transmission in the rat hippocampus in vitro

The effect of the neurosteroid dehydroepiandrosterone sulfate on inhibitory synaptic transmission was studied in area CA1 of the rat hippocampus using an in vitro hippocampal slice preparation. Synaptic responses elicited by stimulation of Schaffer collateral fibers were recorded extracellularly as population spikes in the somatic region and as synaptic field potentials in the dendritic region. Bath application of dehydroepiandrosterone sulfate (10 μM) enhanced the synaptically evoked somatic population spike with no effect on the dendritic synaptic potential. Isolation of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor-mediated component of the synaptic response by addition of antagonists of N-methyl- -aspartate and GABA receptors to the perfusion saline demonstrated that dehydroepiandrosterone sulfate had no effect on this component of the dendritic synaptic potential. In contrast, dehydroepiandrosterone sulfate antagonized GABA receptor-mediated inhibitory effects in the somatic region, resulting in an augmentation of the somatic population spike amplitude. Paired-pulse facilitation was unaltered by dehydroepiandrosterone sulfate, thus arguing against possible presynaptic sites of dehydroepiandrosterone sulfate's actions.These results indicate that dehydroepiandrosterone sulfate can alter synaptic transmission in the hippocampus through selective postsynaptic actions on inhibitory synaptic transmission. A synaptic effect of dehydroepiandrosterone sulfate is consistent with a neuromodulatory role for this neurosteroid in the central nervous system, and may contribute to the reported effects of dehydroepiandrosterone sulfate on cognitive processes such as learning and memory.     

Replication of Tomato bushy stunt virus RNA in a plant in vitro system

An ideal system to investigate individual determinants of the replication process of (+)-strand RNA viruses is a cell-free extract that supports viral protein and RNA synthesis in a synchronized manner. Here, we applied a translation/replication system based on cytoplasmic extracts of Nicotiana tabacum cells to Tomato bushy stunt virus (TBSV) RNA. In vitro translated TBSV proteins p33 and p92 form viral replicase, which, in the same reaction, accomplishes the entire replication cycle on exogenous TBSV DI or full-length RNA. Tests of mutant TBSV RNAs confirmed the template specificity of the in vitro replication reaction. Complementation experiments ascertained the significance of an earlier identified TBSV host factor. Interestingly, formation of the viral replicase occurs also in the absence of concurrent protein synthesis demonstrating that translation and RNA replication are not functionally linked in this system. Our studies with cell-free extracts of a plant host thus confirmed earlier findings and enabled novel insights into the TBSV RNA replication process.     

Improving antibody affinity through in vitro mutagenesis in complementarity determining regions

High-affinity antibodies are widely used in diagnostics and for the treatment of human diseases. However, most antibodies are isolated from semi-synthetic libraries by phage display and do not possess in vivo affinity maturation, which is triggered by antigen immunization. It is therefore necessary to engineer the affinity of these antibodies by way of in vitro assaying. In this study, we optimized the affinity of two human monoclonal antibodies which were isolated by phage display in a previous related study. For the 42A1 antibody, which targets the liver cancer antigen glypican-3, the variant T57H in the second complementarity-determining region of the heavy chain (CDR-H2) exhibited a 2.6-fold improvement in affinity, as well as enhanced cell-binding activity. For the I4A3 antibody to severe acute respiratory syndrome coronavirus 2, beneficial single mutations in CDR-H2 and CDR-H3 were randomly combined to select the best synergistic mutations. Among these, the mutation S53P-S98T improved binding affinity (about 3.7 fold) and the neutralizing activity (about 12 fold) compared to the parent antibody. Taken together, single mutations of key residues in antibody CDRs were enough to increase binding affinity with improved antibody functions. The mutagenic combination of key residues in different CDRs creates additive enhancements. Therefore, this study provides a safe and effective in vitro strategy for optimizing antibody affinity.     

Lesion-induced transient suppression of inhibitory function in rat neocortex in vitro

The structural and functional consequences of a local thermolesion were examined in rat neocortex with electrophysiological in vitro techniques and immunocytochemistry. Age-matched untreated and sham-operated animals served as controls and were analysed in the same way. The lesions consisted of a core of coagulated tissue 2–3 mm in diameter and reached ventrally into the deep cortical layers. After two days reactive astrocytes and after nine days a dense gliosis were observed in the immediate vicinity. Modifications in the intrinsic membrane characteristics and the synaptic network properties were investigated with intra- and extracellular recorcling techniques after survival times of one to eight days. Neurons recorded in the surrounding of lesions in neocortical slices revealed a significantly more depolarized resting membrane potential and a higher neuronal input resistance. In comparison to cells in control slices, maximal discharge rates to injection of depolarizing current pulses of neurons close to a focal lesion were not significantly altered and intrinsic burst firing was never observed. However, between postlesion days 1 and 5, neurons in the surroundings of lesions showed a transient increase in synaptic excitability. This hyperactivity was most clearly pronounced at a distance of 2–3 mm from the centre of the lesion (i.e. about 1–1.5 mm away from the lesion border) and characterized by long-duration field potential responses and multiphasic long-lasting excitatory postsynaptic potentials to orthodromic stimulation of the afferent input. This lesion-induced hyperexcitability was associated with a significant reduction in the peak conductance of the Cl⁻ -dependent fast inhibitory postsynaptic potential and the K⁺-dependent long-latency inhibitory postsynaptic potential, suggesting that the intracortical GABAergic system was functionally impaired. The decrease in synaptic inhibition was associated with prolonged N-methyl-d-aspartate receptor-mediated activity, which could be reversibly blocked byd-amino-phosphonovaleric acid. In addition, neurons recorded in the vicinity of the lesion responded to an orthodromic synaptic stimulus with a long-lasting burst.

The lesion-induced disturbance in the balance between the excitatory and inhibitory system may not only have profound influences on the mechanisms of intracortical information processing, but may also lead to the expression of epileptiform activity and long-term functional deficits.     

Donor variation in in vitro HIV-1 susceptibility of monocyte-derived macrophages

Primary human cells from different donors vary in their susceptibility to in vitro infection with HIV-1. In order to perform genetic analysis to identify host factors that affect HIV-1 susceptibility, it is important that a clear phenotype is defined. Here, we report a standardized method to study variation for in vitro HIV-1 infection in monocyte-derived macrophages (MDM) from large numbers of individuals. With this assay, HIV-1 susceptibility of MDM from 489 different donors shows more than 3 log variation and a good correlation with the 32 base pair deletion in the CCR5 co-receptor (ccr5 Δ32 genotype) of the donors. However, in 7 of 12 donors completely resistant to infection with CCR5-using HIV-1, this was not explained by the ccr5 Δ32 genotype, showing evidence that other host factors are likely to influence HIV-1 replication in MDM. Infections with VSV-G pseudotyped HIV-1 indeed confirmed the existence of post-entry level restrictions in MDM.     

In vitro and in vivo evaluation of polyhydroxybutyrate and of polyhydroxybutyrate reinforced with hydroxyapatite

Polyhydroxybutyrate (PHB) is a polyester made by many microorganisms under conditions of nitrogen deficiency, and is produced commercially in bulk by biotechnology. It has been suggested that PHB-based materials (copolymers and composites) could be suitable for medical applications and may be biodegradable. This paper presents some findings regarding the degradation and biological properties of polyhydroxybutyrate and composites reinforced with particulate hydroxyapatite. It has been established that the strength and stiffness of these materials reduce on in-vitro environment exposure in phosphate-buffered saline at 37°C for periods up to 4 months, and that the degradation rate is a function of composition and processing conditions. It has also been demonstrated that materials based on PHB produce a consistent favourable bone tissue adaptation response with no evidence of an undesirable chronic inflammatory response after implantation periods up to 12 months. Bone is rapidly formed close to the material and subsequently becomes highly organized, with up to 80% of the implant surface lying in direct apposition to new bone. The materials showed no conclusive evidence of extensive structural breakdown in vivo during the implantation period of the study.     

Functional and pharmacological properties of human neocortical neurons maintained in vitro

The availability of neocortical tissue obtained during brain surgery has allowed for detailed studies of the membrane and synaptic properties of neurons maintained in vitro in a slice preparation. Many of the findings obtained in these studies are summarized here. The majority of the basic electrophysiological properties appear to be similar when human and rodent neurons are compared. However, some notable exceptions regarding specific membrane properties have been reported. Since the majority of the material used in these studies is obtained from epileptic patients, several neuroscientists have tried to determine whether this tissue retains any sign of epileptogenicity when analyzed in vitro. Abnormal synaptic activity was only seen in a fraction of neurons near identified anatomical foci, including tumors, or within neocortical areas that displayed abnormal electrographic activity in situ. This cellular activity included both the presence of all-or-none and graded synaptic bursts. Epileptiform activity comparable to that seen in rodent tissue has been obtained in vitro using several pharmacological procedures including the disinhibition and the Mg²⁺-free model. In conclusion, electrophysiological and pharmacological studies of the human neocortex obtained during surgery have so far been unsuccessful in isolating any definite cellular mechanism that may account for the expression of the epileptiform activity in situ. Nevertheless, these studies have provided valuable information on the cellular and synaptic properties of human neocortex under normal conditions, and following experimental procedures capable of increasing; neuronal excitability.