Phosphorylation of a C-SRC-related protein in macrophages activated in vitro with lymphokine

Treatment of proteose peptone elicited peritoneal macrophages from C3H/HeN mice or the macrophage cell line B6MP102 with a T-cell lymphokine preparation induces cytotoxicity for SV3T3 tumor cells. The Triton X-100 (TX-100) insoluble fractions from activated macrophages possessed kinase activity for an endogenous 53 kDa phosphoprotein (pp53) which was markedly greater than extracts from untreated macrophages. Addition of the tyrosine phosphatase inhibitor, Na₃,VO₄ to the cytotoxicity assay also enhanced tumor cell lysis and Na₃VO₄ treated macrophages showed increased phosphorylation of pp53. Moreover, addition of Na₃VO₄ to the cytoskeleton kinase assay enhanced the phosphorylation of pp53 in a dose dependent manner. Pp53 was immunoprecipitated from the in vitro phosphorylated TX-100 insoluble fraction with monoclonal antibody to pp60^v-src. Anti-pp60^v-src also precipitated a 53 and a 60 kDa phosphoprotein from whole cell extracts and from TX-100 cytoskeleton extracts of macrophages phosphorylated as viable intact cells. Addition of a known tyrosine kinase inhibitor, quercetin, to the macrophage cytoskeleton kinase assay inhibited phosphorylation of pp53, and the in vitro phosphorylated pp53 was resistant to 1 N NaOH hydrolysis, indicating phosphorylation of tyrosine residues. Immune complex kinase assays of anti-pp60^c-src precipitated TX-100 insoluble macrophage fractions revealed strong phosphorylation for -casein which was inhibited by quercetin. These data suggest that macrophage pp53 is a c-src-related gene product that is inducible by stimuli that activate macrophages to cytotoxicity.     

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.     

Developmental stage of oligodendrocytes determines their response to activated microglia in vitro

Background  

Oligodendrocyte progenitor cells (OPCs) and mature oligodendrocytes are both lost in central nervous system injury and disease. Activated microglia may play a role in OPC and oligodendrocyte loss or replacement, but it is not clear how the responses of OPCs and oligodendrocytes to activated microglia differ.     

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.     

Bioaminergic neuromodulation of respiratory rhythm in vitro

Bioamines, such as norepinephrine and serotonin are key neurotransmitters implicated in multiple physiological and pathological brain mechanisms. Evolutionarily, the bioaminergic neuromodulatory system is widely distributed throughout the brain and is among the earliest neurotransmitters to arise within the hindbrain. In both vertebrates and invertebrates, monoamines play a critical role in the control of respiration. In mammals, both norepinephrine and serotonin are involved in the maturation of the respiratory network, as well as in the neuromodulation of intrinsic and synaptic properties, that not only differentially alters the activity of individual respiratory neurons but also the activity of the network during normoxic and hypoxic conditions. Here, we review the basic noradrenergic and serotonergic pathways and their impact on the activity of the pre-Bötzinger Complex inspiratory neurons and network activity.     

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.     

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.     

Osservazioni sulla crescita dell'epitelio polmonare in vitro

Summary Embryonic mouse lung was explanted at the 11th day of gestation and cultured in vitro; the growth of its epithelial tree was studied in relation to changes experimentally induced in the amount of the developing mesenchyme. The growth was estimated, according to the method suggested by Alescio (1965), by drawing the epithelial area with the camera lucida, measuring it by planimetry, and calculating the ratio between the area at successive culture times and the initial one. The ratios obtained were plotted against the time, and they showed a linear behaviour; the slopes of the regression lines express the growth rate in the different conditions.The epithelial area appeared to grow with greater speed than in the whole lung anlage, both in the right and in the left isolated cultured lung. A greater increase of growth rate was obtained when the quantitative relationship of the epithelium to the mesenchyme was changed, by combining the whole mesencyme with the epithelium of only one lung. The results agree with the hypothesis that the mesenchymal mantle of the lung exerts a stimulating action on the global growth of the epithelial tree.

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Ricerca eseguita mediante contributi del C.N.R., dell'U.S. Atomic Energy Commission (NYO-3355-4) e dell'Euratom (043-65-1 BIOI).

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Si ringrazia il Prof. I. Barrai per la sua collaborazione nella parte statistica del presente lavoro.     

Generating substrate bound functional chemokine gradients in vitro

Microcontact printing (mCP) is employed to generate discontinuous microscale gradients of active fractalkine, a chemokine expressed by endothelial cells near sites of inflammation where it is believed to form concentration gradients descending away from the inflamed area. In vivo, fractalkine is a transmembrane molecule extending its chemokine domain into the vascular lumen. Substrate bound in vitro gradients may thus closely resemble in vivo conditions. Direct mCP of sensitive proteins like fractalkine may cause partial protein denaturation and will not ensure correct orientation of the biologically active part of the molecules. Here, indirect mCP of a capture antibody recognizing a molecular tag on the target protein is successfully used to pattern tagged fractalkine in microscale gradient patterns. Fractalkine functions as an adhesion molecule for leukocytes. Cells expressing the fractalkine receptor are found to attach to the gradient structure at a density correlated with the fractional area covered by fractalkine. This indicates that the patterned fractalkine maintains its biological function. The method can be applied to in vitro studies of cell responses to the wide range of naturally surface-bound chemokines (haptotactic gradients). The use of a capture antibody facilitates control of the orientation of tagged molecules, thereby ensuring a high degree of bio-functionality through correct presentation and reduced protein denaturation.     

Protein-primed replication of bacteriophage PRD1 genome in vitro

A cell-free system has been developed from cells of an Escherichia coli strain, carrying cloned genes 1 and 8 of bacteriophage PRD1, that catalyzes protein-primed DNA synthesis. DNA synthesis in vitro is entirely dependent upon the addition of PRD1 DNA-protein complex as template, Mg²⁺, and four deoxyribonucleoside triphosphates. No in vitro DNA synthesis was observed when deproteinized PRD1 DNA was used as template. The origin and direction of PRD1 DNA replication in vitro was determined by restriction enzyme analysis of ³²P-labeled PRD1 DNA synthesized in this system. Replication starts at both ends of the linear PRD1 DNA template. Alkaline sucrose gradient centrifugation and agarose gel electrophoresis showed that full-length PRD1 DNA is synthesized in vitro. DNA synthesis in this system is inhibited by the drug aphidicolin. We also observed that dimethyl sulfoxide (DMSO) stimulates in vitro DNA synthesis, although it inhibits bacterial DNA polymerase.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

The in vivo and in vitro degradation of poly(glycolic acid) suture material as a function of applied strain

The in vivo and in vitro stability of a degradable suture material, poly(glycolic acid) has been shown to be dependent on the magnitude of a pre-imposed strain. The degradation, monitored by changes in the tensile load at break, was considerably enhanced by pre-straining the material to one half of the normal extension at break, using a novel implantable device.