Measurement of S-phase duration of adult stem cells in the flatworm Macrostomum lignano by double replication labelling and quantitative colocalization analysis.

Platyhelminthes are highly attractive models for addressing fundamental aspects of stem cell biology in vivo. These organisms possess a unique stem cell system comprised of neoblasts that are the only proliferating cells during adulthood. We have investigated Ts (S-phase duration) of neoblasts during homoeostasis and regeneration in the flatworm, Macrostomum lignano. A double immunohistochemical technique was used, performing sequential pulses with the thymidine analogues CldU (chlorodeoxyuridine) and IdU (iododeoxyuridine), separated by variable chase times in the presence of colchicine. Owing to the localized nature of the fluorescent signals (cell nuclei) and variable levels of autofluorescence, standard intensity-based colocalization analyses could not be applied to accurately determine the colocalization. Therefore, an object-based colocalization approach was devised to score the relative number of double-positive cells. Using this approach, Ts (S-phase duration) in the main population of neoblasts was ∼13 h. During early regeneration, no significant change in Ts was observed.

Immunofluorescent detection of two thymidine analogues (CldU and IdU) in primary tissue.

Accurate measurement of cell division is a fundamental challenge in experimental biology that becomes increasingly complex when slowly dividing cells are analyzed. Established methods to detect cell division include direct visualization by continuous microscopy in cell culture, dilution of vital dyes such as carboxyfluorescein di-aetate succinimidyl ester (CFSE), immuno-detection of mitogenic antigens such as ki67 or PCNA, and thymidine analogues. Thymidine analogues can be detected by a variety of methods including radio-detection for tritiated thymidine, immuno-detection for bromo-deoxyuridine (BrdU), chloro-deoxyuridine (CldU) and iodo-deoxyuridine (IdU), and chemical detection for ethinyl-deoxyuridine (EdU). We have derived a strategy to detect sequential incorporation of different thymidine analogues (CldU and IdU) into tissues of adult mice. Our method allows investigators to accurately quantify two successive rounds of cell division. By optimizing immunostaining protocols our approach can detect very low dose thymidine analogues administered via the drinking water, safe to administer to mice for prolonged periods of time. Consequently, our technique can be used to detect cell turnover in very long-lived tissues. Optimal immunofluoresent staining results can be achieved in multiple tissue types, including pancreas, skin, gut, liver, adrenal, testis, ovary, thyroid, lymph node, and brain. We have also applied this technique to identify oncogenic transformation within tissues. We have further applied this technique to determine if transit-amplifying cells contribute to growth or renewal of tissues. In this sense, sequential administration of thymidine analogues represents a novel approach for studying the origins and survival of cells involved in tissue homeostasis.

Replication labeling with halogenated thymidine analogs.

In this unit, several basic protocols to identify sites of DNA replication utilizing incorporation of halogenated thymidine analogs into DNA, followed by immunofluorescent imaging are described. Antibodies specific for halogenated thymidine analogs such as bromodeoxyuridine (BrdU), chlorodeoxyuridine (CldU), and iododeoxyuridine (IdU) can provide a rapid, nonhazardous, and sensitive method for detecting DNA replication in single cells, in a manner analogous to the traditional use of tritiated thymidine. In combination with different techniques to prepare the DNA template, a variety of DNA replication-related events can be examined by conventional fluorescence-microscopic approaches. Because origin firing and the progression of replication forks are regulated in the context of subnuclear compartments through protein-protein interactions, chromatin modifications, and subnuclear localization of replication clusters, visualizing replication foci significantly facilitates understanding of nuclear dynamics during S-phase.

Thymidine analogues to assess microperfusion in human tumors.

PURPOSE: To validate the use of the thymidine analogues as local perfusion markers in human tumors (no labeling indicates no perfusion) by comparison with the well-characterized perfusion marker Hoechst 33342. METHODS AND MATERIALS: Human tumor xenografts from gliomas and head-and-neck cancers were injected with iododeoxyuridine (IdUrd) or bromodeoxyuridine (BrdUrd) and the fluorescent dye Hoechst 33342. In frozen sections, each blood vessel was scored for the presence of IdUrd/BrdUrd labeling and Hoechst in surrounding cells. The percentage of analogue-negative vessels was compared with the fraction of Hoechst-negative vessels. Collocalization of the two markers was also scored. RESULTS: We found considerable intertumor variation in the fraction of perfused vessels, measured by analogue labeling, both in the human tumor xenografts and in a series of tumor biopsies from head-and-neck cancer patients. There was a significant correlation between the Hoechst-negative and IdUrd/BrdUrd-negative vessels in the xenografts (r = 85, p = 0.0004), despite some mismatches on a per-vessel basis. CONCLUSIONS: Thymidine analogues can be successfully used to rank tumors according to their fraction of perfused vessels. Whether this fraction correlates with the extent of acute hypoxia needs further confirmation.

Temporal changes in the distribution of thoracic duct lymphoblasts to synovium and other tissues of rats with adjuvant-induced arthritis.

The distribution of lymphoblasts(lymphocytes in cell cycle) obtained from the central lymph of donor rats and transferred adoptively to syngeneic recipients has been shown previously to be influenced by the presence of arthritis in either donor or recipient rats. The intent of the present study was to examine patterns of distribution of lymphoblasts in the early period after transfer, when extravasation of donor lymphoblasts was expected to occur. Thoracic duct lymphoblasts labelled in vitro with [125I]-iododeoxyuridine were detected in recipient rats by external radiometry and autoradiography. Irrespective of donor status, fewer donor lymphoblasts accumulated in the feet of normal recipients when compared to arthritic recipients at 15 min, 2 h and 24 h after cell transfer.When recipients of similar disease status were compared, the percentages of injected lymphoblasts from normal and arthritic donors recovered in the feet were similar at 15 min and 2 h after transfer. The proportions of lymphoblasts recovered in the feetat 24 h after injection declined in normal recipients and arthritic recipients of cells from normal donor rats. Importantly,this decline did not occur when both the donor and the recipient were arthritic. In the hindpaws, donor lymphoblasts were located predominantly in the bone marrow, except in transfers between arthriticrats, when at 24 h they were predominantly in the synovium. At 15 min, lymphoblasts were detected within the lumen of vessels within synovium, whereas by 2 h extravasation of these cells was evident. In conclusion, lymphoblasts accumulate more readily in hindfeet that are inflamed. In the early hours after injection, lymphoblasts from normal and arthritic donors are recruited equally, but these early levels are only maintained for 24 hin the combination of arthritic donor and arthritic recipient. Adramatic change in the proportion of lymphoblasts located in synoviumat this later time suggests that a dynamic process of relocation,retention and/or local cell division maintains the numbers of arthritic donor cells in the latter combination.

Purification and use of granzyme B.

Granzyme B (GrB) is the primary molecular mediator of apoptosis by cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. It is a unique mammalian aspartic acid-cleaving serine protease. On T cell receptor activation, GrB is released from the CTL cytoplasmic granules by exocytosis, enters the target cells and, in the presence of the granule pore-forming protein perforin, it initiates the processing of caspases and apoptosis. GrB apoptosis is also activated by adenovirus, which can effectively replace perforin. Methods for the purification and quantitation of GrB and perforin, and the preparation and titration of adenovirus, are described. In addition, methods for application of these reagents to the initiation of apoptosis in tumor target cells, with several assays for detecting GrB apoptotic activity, are detailed.

Preparation and in vitro studies of [125I]IUDR-T101 antibody conjugate.

Idoxuridine labeled with 125I was conjugated to polylysine. This conjugate was then coupled to the carbohydrate side chains of T101 monoclonal antibody (anti-CD5). The immunoreactivity, cell retention, cytotoxicity, and intracellular localization of this conjugate was tested in CCRF-CEM cells (CD5 positive). The conjugate had 68% immunoreactivity. The retention of 125I by CCRF-CEM cells was higher for the conjugate than for T101 directly labeled with 125I and more of it localized in the nucleus than did the 125I-labeled T101. The 125I IUDR-polylysine-T101 conjugate was more cytotoxic than the 125I-labeled T101. In conclusion, the conjugation of [125I]IUDR to T101 is feasible, and preferential targeting of the 125I to the nucleus is obtained.

Incorporation of 5-iodo-2'-deoxyuridine and 5-bromo-2'-deoxyuridine into rodent DNA as determined by neutron activation analysis.

Using 5-iodo-2'-deoxyuridine (IdU) and 5-bromo-2'-deoxyuridine (BrdU) as DNA precursors, neutron activation analysis (NAA) of iodine and Br was developed as a quantitative method for determining DNA synthesis. Endogenous rodent tissue concentrations of bromine (Br) and iodine ranged 100-fold from a low of 0.06 microgram of iodine/g of rat gastrointestinal tract (GIT) to a high of 5.99 micrograms of Br/g of rat kidney. All 10 rodent tissues had concentrations of Br 4 to 76 times higher than those of iodine. Rat hepatic Br concentrations could be reduced 17-fold by dietary and pharmacological methods. Female Fischer 344 rats and male C57BL/6 mice were given 4-8 intraperitoneal injections of either IdU or BrdU as a DNA precursor. Tissue clearance of iodine in IdU-treated rodents was both faster and more complete (in mice 4 and in rats 17 h or less) than Br clearance from BrdU-treated rodents (at 162 h nonincorporated Br label still remains). In rat liver, lung, and kidney, the iodine label incorporated from IdU into DNA was stable for at least 162 h. The incorporation ratio is defined as the microgram halogen/tissue for either IdU- or BrdU-treated rodents divided by the microgram halogen/g tissue of untreated rodents. NAA-based studies of DNA synthesis gave high incorporation ratios in rat liver (5.3), rat lung (6.7), rat GIT (19.0), rat spleen (24.0), mouse GIT (10.1), and mouse spleen (25.8).

Specific staining of iododeoxyuridine and bromodeoxyuridine in tumors double labelled in vivo: a cell kinetic analysis.

The simultaneous and specific staining of iododeoxyuridine (IdUrd) and chlorodeoxyuridine (CldUrd) allows for more accurate estimates of potential doubling time (Tpot). Because CldUrd is not approved for human use, the procedure was adapted for the staining of IdUrd and bromodeoxy-uridine (BrdUrd). The fluorescein isothiocyanate-conjugated B44 antibody (B44-FITC) stained both IdUrd and BrdUrd in tumor nuclei labelled singly with one or the other pyrimidine analogue. However, when MCaK tumors in exponential growth in vivo were pulse labelled with both IdUrd and BrdUrd, the staining of BrdUrd was not seen, and the labelling pattern reflected specificity to IdUrd. These observations were confirmed using tumors pulse labelled with IdUrd and/or BrdUrd at 6 h and/or 0.3 h prior to tumor removal in all possible combinations. Simultaneous specific staining of BrdUrd by Br3 and of IdUrd by B44-FITC was documented by quantification of labelling indices (LIs) from double-labelled tumors. The specificity of B44-FITC for IdUrd in double-labelled tumors was due to a greater affinity of this antibody for IdUrd than for BrdUrd. This technique allowed for two independent estimates of LI and Tpot when tumors were double labelled for 3.0 and 5.5 h. Both IdUrd and BrdUrd are approved for clinical use, and this double-labelling technique should prove to be valuable for measuring the cell kinetics of solid tumors in vivo.

Idoxuridina jalea: parte II: estabilidad / Idoxuridine gel: part II: stability

Se realizó el estudio de estabilidad de una jalea de idoxuridina para el tratamiento de la gingivo estomatitis herpética aguda, mediante una técnica de análisis específica por cromatrografía líquida de alta resolución, con la que se puede cuantificar el contenido de idoxuridina en presencia de sus productos de degradación. Las muestras se almacenaron a diferentes temperaturas y se analizaron de acuerdo con el diseño previamente establecido. se demostró que la formación es estable químicamente y que la reacción de degradación hidrolitíca de la idoxuridina en la jalea es de primer orden; se plantea para este producto una fecha de vencimiento de 5 años, si se almacena a temperatura de refrigeración

Idoxuridina jalea. parte II: estabilidad / Idoxuridine gel: part II: stability

Se realizó el estudio de estabilidad de una jalea de idoxuridina para el tratamiento de la gingivo estomatitis herpética aguda, mediante una técnica de análisis específica por cromatrografía líquida de alta resolución, con la que se puede cuantificar el contenido de idoxuridina en presencia de sus productos de degradación. Las muestras se almacenaron a diferentes temperaturas y se analizaron de acuerdo con el diseño previamente establecido. se demostró que la formación es estable químicamente y que la reacción de degradación hidrolitíca de la idoxuridina en la jalea es de primer orden; se plantea para este producto una fecha de vencimiento de 5 años, si se almacena a temperatura de refrigeración (AU)

Calculating potential doubling time using monoclonal antibodies specific for two halogenated thymidine analogues.

PURPOSE: A new flow cytometric technique that allows for two-incorporated thymidine analogues to be measured simultaneously and independently has been used to improve the accuracy of in vivo cell kinetic estimates, i.e., the length of S-phase (TS) and potential doubling time (Tpot). METHODS AND MATERIALS: The analogues chlorodeoxyuridine and iododeoxyuridine were injected at different times into mice bearing the mouse mammary tumor MCaK. At different times after labeling, the tumors were harvested and prepared for three color flow cytometric analysis of DNA, chlorodeoxyuridine, and iododeoxyuridine. Control experiments showed that similar estimates of Tpot were obtained from each label when administered singly, or as staggered pulses. Comparisons were made between TS and Tpot calculated from a single label (single point), from the averaged result of the two labels from the same tumor (two point-ave), and from the simultaneous nonlinear fitting of the measured parameters from the two labels, from the same tumor (two point-fit). These estimates of TS and Tpot were then compared to reference values obtained by fitting the pooled measured parameters from all the tumors, that were labeled for different periods of time. RESULTS: While all of the methods resulted in similar mean estimates of TS and Tpot that were close to the reference values, the fewest assumptions, and the least variability in the results, were obtained using the two point-fit data. CONCLUSION: The estimation of Tpot using two thymidine analogues is more accurate than that obtained from a single label.

Idoxuridina jalea: determinación cuantitativa / Idoxuridine jelly: quantitative determination

Se desarrolloó un método para la valoración de idoxuridina en una jalea, mediante la cromatrografía líquida de alta resolución, en el que se utilizó fase reversa de C-8 como fase estacionaria, metanol-agua como fase móvil, sulfatiazol como stándar interno y detección ultravioleta a Landa=295 nm. Previamente se extraen los parabenos y se precipita la hodroxipropilmetilcelulosa que interfiere en la determinación de idoxuridina por dicho método. Los resultados obtenidos fueron reproducibles y el recobrado se encontró dentro de los límites establecidos

Antivirais / Antivirals

Doenças causadas por vírus representam dificuldades terapêuticas. O conjunto de medicamentos antivirais disponíveis é ainda relativamente pequeno, mas tem crescido nos últimos anos em razäo de enormes investimentos materiais e de talentos: a síndrome da imunodeficiência adquirida é, certamente, o motivo principal dessa procura, mas näo o único. Com a presente atualizaçäo, dedicada a oftalmologistas, o modo de açäo, as aplicaçöes, os efeitos adversos, as apresentaçöes e a posologia dos principais quimioterápicos desse grupo, assim como novidades e perspectivas, säo aqui resumidas

Idoxuridina jalea. determinación cuantitativa / Idoxuridine jelly: quantitative deterination

Se desarrolloó un método para la valoración de idoxuridina en una jalea, mediante la cromatrografía líquida de alta resolución, en el que se utilizó fase reversa de C-8 como fase estacionaria, metanol-agua como fase móvil, sulfatiazol como stándar interno y detección ultravioleta a Landa=295 nm. Previamente se extraen los parabenos y se precipita la hodroxipropilmetilcelulosa que interfiere en la determinación de idoxuridina por dicho método. Los resultados obtenidos fueron reproducibles y el recobrado se encontró dentro de los límites establecidos (AU)

Liquid chromatographic study of the stability of 5-halogeno-2'-deoxyuridines.

The stability of a series of 5-halogeno-2'-deoxyuridines was investigated using liquid chromatography as the analytical technique. Characteristics and profiles of the acidic, neutral and alkaline degradation are described, together with Arrhenius relationships and activation parameters for weakly acidic media.

An indirect immunofluorescence double staining procedure for the simultaneous flow cytometric measurement of iodo- and chlorodeoxyuridine incorporated into DNA.

In this paper we describe an indirect fluorescence double staining procedure for the simultaneous detection of IdUrd and CldUrd in the same cell nucleus. Two commercially available antibodies were selected for this purpose. A rat anti-BrdUrd monoclonal antibody from Sera-lab was found to bind specifically to CldUrd and BrdUrd. A mouse monoclonal anti-BrdUrd antibody from Becton Dickinson used in a 1:2 dilution binds to all halogenated deoxyuridines but, when the cells were extensively washed with Tris buffer with a high salt concentration, almost no binding to CldUrd was observed. An immunofluorescence procedure was developed, based on these primary antibodies, raised in different species (rat and mouse), in combination with highly purified second antibodies: FITC conjugated goat antirat and Texas-Red conjugated goat antimouse.

A method for determination of iododeoxyuridine substitution of thymidine using reversed-phase high-performance liquid chromatography.

An assay for thymidine substitution by iododeoxyuridine (IdUrd) using reversed-phase high-performance liquid chromatography (HPLC) has been developed. Three principal steps in this procedure are: extraction of DNA from cell or tissues, hydrolysis of DNA into deoxynucleosides and separation using HPLC. Approximately 1 microgram of DNA was recovered from 10(5) cells by phenol extraction, and subjected to hydrolysis into deoxynucleosides which required a three-stage DNA digestion using enzymes DNAse I. phosphodiesterase I and alkaline phosphatase. The deoxynucleosides were separated on the Microsorb C18 column with isocratic elution; 90-100% of the DNA was recovered as deoxynucleosides on the column. The method was used to determine quantitatively the percent IdUrd substitution of thymidine in Chinese hamster lung cells in vitro and BA1112 rhabdomyosarcoma in WAG/Rij rats perfused with IdUrd. It was possible to determine the thymidine substitution by IdUrd as small as 1% using a few micrograms of DNA. The close correspondence between the percent substitutions determined by HPLC and those determined by radioactive assay using [125I]-labelled IdUrd, confirmed the accuracy of our HPLC method. The HPLC analysis is especially suitable for the determination of percent IdUrd substitution of thymidine in tissue biopsies from animals used in in vivo experiments or humans undergoing radiation treatment.

5-iodo-2'-deoxyuridine-protein conjugates: synthesis and enzymatic degradation.

Several halogenated analogs of thymidine and cytidine possess antineoplastic and antiviral activity. They are also powerful sensitizers of bacterial and mammalian cells to lethal effects of x-irradiation. An important factor limiting the effectiveness of these agents in therapy is their extremely short half-life in circulation due to rapid hepatic dehalogenation. An approach to this problem is to deliver the drug directly to its target using monoclonal antibodies. This study evaluates the lysosomotropic delivery systems of halogenated pyrimidines using 5-iodo-2'-deoxyuridine [IUdR] as a model. IUdR, derivatized and activated at either the 3'- or the 5'-position forms covalent adducts with the epsilon-amino groups of the lysine residues in proteins (bovine serum albumin [BSA], and immunoglobulins [IgG]). Two methods suitable for conjugation of IUdR to proteins involving either the formation of acyl-imidazoles in the reaction of IUdR succinates with N,N'-carbonyldiimidazole or the preparation of N-succinimidyl esters of IUdR succinates were established. Both derivatives express comparable reactivity toward proteins. The degree of IUdR incorporation is easily controlled by the ratio of reagents. The succinate "arm" linking IUdR to protein is susceptible to lysosomal hydrolysis in vitro releasing intact IUdR. The half-life of the IUdR-IgG conjugate in the presence of the lysosomal enzymes was shown to be approximately twice that of the IUdR-BSA conjugate.