In vitro and in vivo iodination of human thyroglobulin in relation to hormone release

Reduced and S-alkylated thyroglobulin (Tgb) from different species were shown by SDS-PAGE to contain small peptides (from 45-9 kDa) rich in thyroxine. Several hypotheses were proposed to explain their origin. The polypeptide composition of iodine-poor (Tgb A) and normally iodinated (Tgb B) human Tgb prepared by two different procedures (one minimizing and the other favoring post-mortem proteolysis) was compared in the native state and after in vitro iodination. Results show that one of the hormonogenic sites of human Tgb is part of a domain of the molecule most susceptible to proteolysis, especially when it is very iodinated.     

A simple filtration device for separating nonadherent microorganisms from mammalian cells in in vitro studies on microbial adherence

In adherence studies, the removal of nonadherent microorganisms is essential for the valid enumeration of microorganisms that adhere to host cells. Although filtration devices are available commercially for the removal of nonadherent microorganisms, these are expensive and not reusable. In this article, we describe a simple, inexpensive, and reusable filtration device composed of two chambers of nylon, a nylon membrane of desired pore size, a rubber washer, and supporting stainless steel mesh. The device was effective in in vitro adherence assays for removing nonadherent endospores of Rhinosporidium seeberi from human buccal epithelial cells, providing valid counts of adherent microorganisms.     

Direct and indirect effects of recombinant human granulocyte-colony stimulating factor on in vitro colony formation of human bladder cancer cells

Although the present experimental use of recombinant human granulocyte-colony-stimulating factor (rG-CSF) has been proven to alleviate the myelosuppression induced by antitumor chemotherapy, it is also believed to stimulate growth of some nonhematopoietic tumor cells. We investigated both the direct and indirect effects of rG-CSF on in vitro colony formation of human bladder cancer cell lines using a modified human tumor clonogenic assay. Peripheral blood mononuclear cells (PBMC) were used as feeder cells (a mixture of 5×10⁴ monocytes/dish and 5×10⁵ lymphocytes/dish obtained from healthy donors). Human bladder cancer cell lines KK-47, TCCSUP and T24, all derived from human transitional-cell carcinomas, were incubated continuously with various concentrations of rG-CSF ranging from 0.01 ng/ml to 10 ng/ml both with and without PBMC for 7–21 days. The concentrations of rG-CSF used were chosen as being in the range of achievable serum concentrations in patients treated with rG-CSF. At the end of incubation, colonies were counted under an inverted phase-contrast microscope, and an increase in the number of colonies in comparison with the control was used to evaluate the effects of rG-CSF. Results were expressed as a percentage of controls. rG-CSF in the upper layer at concentrations ranging from 0.1 ng/ml to 10 ng/ml stimulated the colony formation of all the cancer cell lines tested in the absence of PBMC in the feeder layer, whereas cells with PBMC in the feeder layer were significantly stimulated more than those without PBMC in the feeder layer (P<0.05) up to a certain concentration, which varied from cell line to cell line. At higher concentrations of rG-CSF, no further stimulation but, on the contrary, a decrease in colony formation was observed in cells with PBMC in the feeder layer in all the cell lines tested. Colony formation in KK-47 and T24 cell lines was significantly inhibited at 5 ng/ml and/or 10 ng/ml rG-CSF compared with cells without PBMC in the feeder layer. Our results suggest that rG-CSF may have both direct and indirect stimulatory effects on the growth of human bladder cancer cell lines in vitro. The results obtained also raise the possibility of adverse effects of rG-CSF in bladder cancer patients whose malignant cells may be directly and indirectly stimulated by this factor while it is being used clinically to alleviate the myelosuppression induced by antitumor chemotherapy.     

In vitro culture of Cryptosporidium muris in a human stomach adenocarcinoma cell line

We investigated the optimal culture conditions for Cryptosporidium muris in a human stomach adenocarcinoma (AGS) cell line by determining the effects of medium pH and of selected supplements on the development of C. muris. The optimum pH of the culture medium required for the development of C. muris was determined to be 6.6. The number of parasites significantly increased during cultivation for 72 hr (p < 0.05) at this level. On the other hand, numbers decreased linearly after 24 hr of incubation at pH 7.5. When cultured in different concentrations of serum, C. muris in media containing 5% FBS induced 4-7 times more parasites than in 1% or 10% serum. Of the six medium supplements examined, only 1 mM pyruvate enhanced the number of C. muris in vitro. Transmission electron microscopic observation showed the developmental stages of C. muris in the cytoplasm of the cells, not in an extracytoplasmic location. The growth of C. muris in AGS cells provides a means of investigating its biological characteristics and of testing its response to therapeutic agents. However, a more optimized culture system is needed for the recovery of oocysts on a large scale in vitro.     

用于蛋白质体外分子进化研究的DNA随机突变技术

蛋白质体外分子进化是模拟自然的进化过程,利用基因随机突变和定向筛选（选择）技术,以获得具有预期新功能的突变体分子。虽然体外进化近几年才产生,但已成为医药和工业领域中筛选具有特殊催化性质的酶的最重要的方法之一。ＤＮＡ随机突变技术是蛋白质体外分子进化研究的基础,本文将对几种最重要的突变方法：倾向错误的ＰＣＲ、ＤＮＡ重排、模板交错延伸反应和随机延伸突变的原理和应用等加以介绍。     

Establishing in vitro in vivo correlations to screen monoclonal antibodies for physicochemical properties related to favorable human pharmacokinetics

Implementation of in vitro assays that correlate with in vivo human pharmacokinetics (PK) would provide desirable preclinical tools for the early selection of therapeutic monoclonal antibody (mAb) candidates with minimal non-target-related PK risk. Use of these tools minimizes the likelihood that mAbs with unfavorable PK would be advanced into costly preclinical and clinical development. In total, 42 mAbs varying in isotype and soluble versus membrane targets were tested in in vitro and in vivo studies. MAb physicochemical properties were assessed by measuring non-specific interactions (DNA- and insulin-binding ELISA), self-association (affinity-capture self-interaction nanoparticle spectroscopy) and binding to matrix-immobilized human FcRn (surface plasmon resonance and column chromatography). The range of scores obtained from each in vitro assay trended well with in vivo clearance (CL) using both human FcRn transgenic (Tg32) mouse allometrically projected human CL and observed human CL, where mAbs with high in vitro scores resulted in rapid CL in vivo. Establishing a threshold value for mAb CL in human of 0.32 mL/hr/kg enabled refinement of thresholds for each in vitro assay parameter, and using a combinatorial triage approach enabled the successful differentiation of mAbs at high risk for rapid CL (unfavorable PK) from those with low risk (favorable PK), which allowed mAbs requiring further characterization to be identified. Correlating in vitro parameters with in vivo human CL resulted in a set of in vitro tools for use in early testing that would enable selection of mAbs with the greatest likelihood of success in the clinic, allowing costly late-stage failures related to an inadequate exposure profile, toxicity or lack of efficacy to be avoided.     

Regulatory perspective on in vitro potency assays for human T cells used in anti-tumor immunotherapy

The adaptive immune system is known to play an important role in anti-neoplastic responses via induction of several effector pathways, resulting in tumor cell death. Because of their ability to specifically recognize and kill tumor cells, the potential use of autologous tumor-derived and genetically engineered T cells as adoptive immunotherapy for cancer is currently being explored. Because of the variety of potential T cell-based medicinal products at the level of starting material and manufacturing process, product-specific functionality assays are needed to ensure quality for individual products. In this review, we provide an overview of in vitro potency assays suggested for characterization and release of different T cell-based anti-tumor products. We discuss functional assays, as presented in scientific advices and literature, highlighting specific advantages and limitations of the various assays. Because the anticipated in vivo mechanism of action for anti-tumor T cells involves tumor recognition and cell death, in vitro potency assays based on the cytotoxic potential of antigen-specific T cells are most evident. However, assays based on other T cell properties may be appropriate as surrogates for cytotoxicity. For all proposed assays, biological relevance of the tests and correlation of the read-outs with in vivo functionality need to be substantiated with sufficient product-specific (non-)clinical data. Moreover, further unraveling the complex interaction of immune cells with and within the tumor environment is expected to lead to further improvement of the T cell-based products. Consequently, increased knowledge will allow further optimized guidance for potency assay development.     

Site-directed mutagenesis studies of human serum albumin define tryptophan at amino acid position 214 as the principal site for nitrosation

The patterns of nitric oxide (NO) release from nitrosated bovine serum albumin (BSA), human serum albumin (HSA) and a number of recombinant HSA mutants were compared. All albumin species were nitrosated by incubation with acidified NO(2)(-). The pattern of NO release from BSA nitrosated with acidified NO(2)(-) was in agreement with previous reports which indicated that Cys-34 is the primary target for nitrosation in BSA. In contrast, the pattern of NO release from HSA nitrosated with acidified NO(2)(-) indicated that the primary nitrosation target was an amino acid residue other than Cys-34. Based on our initial findings and a previous report that tryptophan is a potential target for nitrosation by acidified NO(2)(-), several recombinant HSA mutants were synthesized in the yeast species Pichia pastoris. The following recombinant HSA species were produced: wild-type, C34S, W214L, W214E and W214L/Y411W HSA. Nitrosation of these mutants using acidified NO(2)(-) showed that Trp-214 is the primary nitrosation target in HSA. Mutation of Trp-214 led to an increase in Cys-34 nitrosation, indicating possible competition between these two residues for reaction with N(2)O(3), the reactive nitrosating species formed in aqueous acidified NO(2)(-) solutions.     

In vitro biofilm model for studying tongue flora and malodour

AIMS: To develop a perfusion biofilm system to model tongue biofilm microflora and their physiological response to sulfur-containing substrates (S-substrates) in terms of volatile sulfide compound (VSC) production. METHODS AND RESULTS: Tongue-scrape inocula were used to establish in vitro perfusion biofilms which were examined in terms of ecological composition using culture-dependent and independent (PCR-DGGE) approaches. VSC-specific activity of cells was measured by a cell suspension assay, using a portable industrial sulfide monitor which was also used to monitor VSC production from biofilms in situ. Quasi steady states were achieved by 48 h and continued to 96 h. The mean (+/-SEM) growth rate for 72-h biofilms (n=4) was micro=0.014 h(-1) (+/-0.005 h(-1)). Comparison of biofilms, perfusate and original inoculum showed their ecological composition to be similar (Pearson coefficient>0.64). Perfusate and biofilm cells derived from the same condition (co-sampled) were equivalent with regard to VSC-specific activities which were up-regulated in the presence of S-substrates. CONCLUSIONS: The model maintained a stable tongue microcosm suitable for studying VSC production; biofilm growth in the presence of S-substrates up-regulated VSC activity. SIGNIFICANCE AND IMPACT OF THE STUDY: The method is apt for studying ecological and physiological aspects of oral biofilms and could be useful for screening inhibitory agents.     

In vitro rooting of Psoralea corylifolia Linn: Peroxidase activity as a marker

Induction of rooting in microshoots of Psoraleacorylifolia was achieved within 6–8 days of cultureon half-strength basal Murashige and Skoog's(1962) medium supplemented with 0.005–0.01 mg/lindole-3-acetic acid (IAA) and 2% (w/v) sucrose. Rooting was drastically reduced and friable callusformed at the cut end of the microshoots when themedium was supplemented with a higher concentration ofauxin. Rooting was totally inhibited when themicroshoots were cultured in vitro undercontinuous light. However, the maximum percentage ofmicroshoots rooted when incubated in continuous lightfor 4 weeks before transfer to the rooting media.Peroxidase activity increased considerably duringroot induction indicating a key role of peroxidase inrooting of microshoots of Psoralea corylifolia invitro.     

In vitro pollination as a model for studying fertilization in maize (Zea mays L.)

Summary In vitro pollination was conducted using excised segments of maize female spikelets to determine the effects of age and silk length on fertilization efficiency and developmental pattern. Ovary development after 15 days resulted in: (1) normal kernels, (2) abnormal kernels and (3) enlarged ovaries; the percentages of each class varied with age. Evidence of double fertilization was observed in both normal and abnormal kernels. In vitro fertilization was traced using silk excision and autoradiography with ³²P-radiolabelled pollen and occurred between 4 and 7 h after the pollination of 4.5-cm-long silks. This study supports the validity of the in vitro pollination method for studying fertilization and emphasizes the importance of using a developmentally sensitive index (silk length) for establishing female developmental stage.     

In vitro genotoxicity of polycyclic musk fragrances in the micronucleus test

The synthetic polycyclic musk fragrance compounds galaxolide (1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethylcyclopenta-(g)-2-benzopyrane), tonalide (7-acetyl-1,1,3,4,4,6-hexamerthyltetraline), celestolide (4-acetyl-1,1-dimethyl-6-tert-butylindane), phantolide (6-acetyl-1,1,2,3,3,5-hexamethylindane), cashmeran (6,7-dihydro-1,1,2,3,3-pentamethyl-4-(5H) indanone) and traseolide (5-acetyl-1,1,2,6-tetramethyl-3-isopropylindane) were examined for their genotoxicity in the micronucleus test (MNT) with human lymphocytes in vitro in the presence and absence of an exogenous metabolizing system containing rat liver S9 and the metabolically competent human hepatoma cell line Hep G2. Compound concentrations were employed up to cytotoxic doses. Galaxolide, tonalide, celestolide, phantolide, cashmeran and traseolide revealed no genotoxicity in the micronucleus test with human lymphocytes and with the human hepatoma cell line Hep G2.     

甲真菌病体外模型研究

目的 构建一个简单、经济的新的甲真菌病体外模型.方法 制备猪甲板,在其腹侧面中央黏贴一个“O”型圈,将受试菌液加入“O”型圈内,将甲板放置培养皿中培养,用大体观察及组织病理的方法动态观察不同真菌侵袭甲板的情况.结果 用猪甲板制备甲真菌病体外模型,通过该模型观察了红色毛癣菌、须癣毛癣菌以及白念珠菌穿透甲板的能力.通过组织病理学方法观察到了真菌动态穿透甲板的过程.结论 该模型适用于评估真菌对甲板的致病作用.     

In vitro toxicity of various classes of test agents using the neutral red assay on a human three-dimensional physiologic skin model

Summary A new three-dimensional human skin model consisting of several layers of actively dividing and metabolically active human neonatal foreskin-derived fibroblasts and epidermal keratinocytes grown on nylon mesh has been used to assess the in vitro toxicity of test agents from various classes. Utilizing a slight modification of the published neutral red viability assay for endpoint determination, we have assayed and obtained dose-dependent toxicity curves for test agents from the following classes: detergents (n=15), alcohols (n=5), metal chlorides (n=10), perfumes and colognes (n=5), shampoos (n=4), conditioners (n=3), moisturizers (n=3), pesticides (n=3), and antimicrobial preservatives (n=4). Limited comparisons to in vivo ocular irritancy data with alcohols and detergents are encouraging. We have demonstrated the utility of this metabolically active dermal substrate containing naturally secreted collagen and other extracellular matrix proteins along with the neutral red viability assay for assessing the toxicity of a number of test agents from a variety of different classes with broad industrial applications.     

In vitro adaptation of a ligase ribozyme for activity under a low-pH condition.

A ligase ribozyme accelerating a ligation reaction with oligonucleotide under a low-pH condition was selected by in vitro adaptation. A ribozyme active at pH 7 was randomly mutated, and the resultant RNA library was subjected to in vitro adaptation under a low-pH reaction condition. At pH 4, the adapted RNAs reacted with the oligonucleotide substrates about 200 times faster than the original ribozyme. When the ribozyme was cloned and sequenced, 10 of the 30 clones sequenced had identical sequences. The differences in sequence from the original ribozyme were found at four positions in the middle region and at the 3' end. A few sequential differences dominated the activity of the ribozyme under the extreme condition. The adapted ribozyme had one repeating sequence that was critical for the activity.     

Validation of HB-EGF and amphiregulin as targets for human cancer therapy

Aberrant expression levels of epidermal growth factor receptor (EGFR) and its cognate ligands have been recognized as one of the causes of cancer progression. To investigate the validity of EGFR ligands as targets for cancer therapy, we examined the expression of EGFR ligands and in vitro anti-tumor effects of small interference RNA (siRNA) for EGFR ligands in various cancer cells. HB-EGF expression was dominantly elevated in ovarian, gastric, and breast cancer, melanoma and glioblastoma cells, whereas amphiregulin was primarily expressed in pancreatic, colon, and prostate cancer, renal cell carcinoma and cholangiocarcinoma cells. Transfection of siRNAs for HB-EGF or amphiregulin into these cells significantly increased the numbers of apoptotic cells with attenuation of EGFR and ERK activation. In lung cancer cells, any EGFR ligand was not recognized as a validated target for cancer therapy. These results suggest that HB-EGF and amphiregulin are promising targets for cancer therapy.     

In vitro activity of immunoconjugates between cisplatin and an anti-CA125 monoclonal antibody on ovarian cancer cell lines

Cis-diammine dichloro platinum (II) (CDDP), is a highly potent antineoplastic agent that is used in the treatment of ovarian cancer. However, the clinical use of CDDP is restricted by its severe side effects. In order to reduce these side effects and to enhance its therapeutic efficacy, we developed specific immunoconjugates consisting of the murine monoclonal antibody OC125 and CDDP, using diethylene triamine pentaacetic acid (DTPA) as a linker. The coupling efficiencies of the different preparations synthesized, varied between 1.10±0.42 and 2.65±1.60 mol of CDDP per mol of antibody protein. Despite the chemical modification of the antibody molecule, specific binding activity of the OC125-CDDP conjugates toward the CA125 antigen was maintained as was demonstrated by means of immunohisto-/cytochemical staining of frozen sections of ovarian cancer tissue, amniotic epithelium, and the CA125 positive ovarian cancer cell line NIH:OVCAR 3. The antiproliferative activity of the immunoconjugates was tested against the human ovarian cancer cell lines NIH:OVCAR3 and SKOV 3, applying a kinetic crystal violet microassay. Despite the promising results obtained with the specific immuno-staining of the target cells, no significant antiproliferative activity of our immunoconjugates against the cell lines tested was observed. One possible explanation for the lack of antitumor activity could be the fact that CA125 is released in large amounts by the NIH:OVCAR 3 cells. This may have prevented an efficient immunotargeting of the cancer cells by the formation of soluble immune complexes.     

Etiolation as a pretreatment for in vitro establishment and multiplication of mature chestnut

Localized etiolation of branches in the crown of a 30-year-old chestnut tree produced plant material that responded much better to establishment and multiplication in vitro than unetiolated material, whose cultures were very difficult to maintain (response being measured in terms of the percentage of cultures established, the mean number of shoots formed per explant, the number of 8-mm segments per new shoot, the length of longest shoot in each culture, and the coefficient of multiplication). Only 22% of the initial explants from unetiolated material were successfully established, as against 79% for etiolated material, with similar differences between the coefficients of multiplication of the two lines in successive cultures. Accordingly, partial etiolation of branches is proposed as a suitable pretreatment for in vitro propagation of selected mature trees, when physiologically juvenile materials such as stump sprouts, epicormic shoots or root suckers are not available.