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At least 1,549 records · Page 86Linked to original sources
PubMed1995

Cloning of Thai strain Giardia intestinalis.

Axenic cultures of Giardia intestinalis trophozoites were successfully established from human fecal specimens and rectal swabs from dogs using sucrose gradient centrifugation to separate the cysts from fecal material, the excystation method of Robert-Thompson et al. and culture of the preparation in TYI-S-33 medium supplemented with human serum, vitamin mixture and piper acillin and amikacin antibiotics, respectively. Fungal contamination could be controlled by amphotericin B at 10 micrograms per ml of medium. Clones of the parasites were obtained using a combination of dilution method and micromanipulation technique.

Animals↗
PubMed1994

Expression of interleukin-1 beta gene in candidate human hematopoietic stem cells.

Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect interleukin-1 beta (IL-1 beta) mRNA in candidate human hematopoietic stem cells. The cells, obtained from adult bone marrow (BM) or umbilical cord blood, had a CD34+ CD45RAlo CD71lo phenotype and were further fractionated into CD38+ and CD38- or Thy-1+ and Thy-1- subpopulations. The purity of these fractions was always more than 99%. IL-1 beta and CD34 mRNA were detected in pools of 30 BM-derived CD34+ CD45RAlo CD71lo cells. To further exclude any contribution by contaminating cells, individual cells were analyzed for CD34 and IL-1 beta mRNA. Positive results were obtained with 2 of 5 individual BM-derived CD34+ CD45RAlo CD71lo CD38+ cells isolated by micromanipulation after overnight culture in serum-free medium without any exogenous cytokines, and 1 of 10 individual CD34+ CD45RAlo CD71lo CD38- cells isolated immediately after sorting. Moreover, of 10 pools of three BM-derived CD34+ CD45RAlo CD71lo cells cultured overnight in the presence of a mixture of various cytokines (Steel factor, IL-3, IL-6, macrophage colony-stimulating factor [M-CSF], erythropoietin, and IL-3/granulocyte-macrophage colony-stimulating factor [GM-CSF] fusion protein), 5 were positive for IL-1 beta mRNA. This result was compatible with more than 20% (95% confidence limit 0.06-0.61) of the BM cells with the CD34+ CD45RAlo CD71lo phenotype expressing IL-1 beta mRNA. IL-1 beta expression was also consistently observed from day 0 to day 9 in liquid cultures of cord-blood-derived CD34+ CD45RAlo CD71lo Thy-1+ or Thy-1- cells. The cultures contained the same combination of cytokines and resulted in an expansion of cell numbers of up to 400-fold. GM-CSF mRNA was not detected in the equivalent of 75 cells at any day, even though it could be detected with high sensitivity in control stromal cells. Because IL-1 beta is a powerful and pleiotropic biomodulator of cytokines and adhesion molecules, our observations suggest that at least some primitive hematopoietic cells do not merely respond passively to signals from their environment, but may themselves regulate the paracrine production of cytokines from neighboring stromal cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Antigens, CD↗
PubMed1995

An optical pressure chamber designed for high numerical aperture studies on adherent living cells.

We have developed an optical pressure chamber designed for use with high numerical aperture oil immersion microscope objectives at working pressures up to 1,000 psi (67 atm abs). The chamber is optimized for studies of living, adherent, cultured mammalian cells using high resolution epifluorescence and phase contrast microscopy, and biophysical techniques such as fluorescence redistribution after photobleaching and optical trapping. The primary optical window assembly of the chamber can be removed and placed into a standard 35-mm tissue culture dish, allowing for culture, microinjection, and micromanipulation of adherent cells before they are loaded into the chamber. The chamber is designed to fit into a commercially available stage heater for temperature control, and we used a computer-controlled high pressure liquid chromatography pump for pressure control. A graphic software interface allows the user to program "dive" profiles and to link temperature and pressure data with digital image files of specimens under study. A minor modification of the present design will allow perfusion at high pressure.

Adhesives↗
PubMed1993

Microinjection of cells: basic considerations.

At least four types of technological advances have made cell microinjection procedures precise and practical, while minimizing negative impacts on cell viability: 1) hydraulic and electromechanical (stepper) micromanipulators, which provide operators with precise spatial control; 2) high-precision syringe and automated pneumatic microinjectors, with high reproducibility in the injected volume; 3) regulated pipet-pulling devices that yield highly reproducible tip diameters, down to 0.1 microns; and 4) specially adapted microscopes in both upright and inverted configurations, which ensure high-quality observation of cellular features in brightfield and all modern contrasting techniques, while reducing movement-induced vibrations at the contact point with the cell.

Cells↗
PubMedDate not supplied

[Response of neurons of the sensomotor region of the cerebral cortex to low-intensity pulsed ultra-high frequency irradiation].

A peculiarity of the work is an analysis of bioelectrical activity of single neurones under irradiation with low-level pulsed microwaves (6 GHz, 50 Hz, 0.2 mW/cm2). A specially designed micromanipulator made of organic glass was used. The results are presented in comparison with appropriate reactions of neurones to other physical factors considered like microwaves as weak stimuli for central nervous system.

Animals↗
PubMed1995

The influence of cerebrospinal fluid pressure on the lamina cribrosa tissue pressure gradient.

PURPOSE: To measure the tissue pressure gradient through the optic disk and to determine the relationship between intraocular, cerebrospinal fluid, and retrolaminar tissue pressures. The relationship of optic nerve subarachnoid space pressure to intracranial cerebrospinal fluid pressure also was explored. METHODS: Micropipettes coupled to a pressure transducer were passed through pars plana and vitreous to enter the optic disk in the anesthetized dog. Using a micromanipulator, pipettes penetrated the optic disk in steps while pressure measurements were taken. In some animals, pipettes also were passed into the optic nerve subarachnoid space. Lateral ventricle cerebrospinal fluid pressure, intraocular pressure, and arterial blood pressure were measured concurrently, and the effect of raising CSF pressure was explored. RESULTS: Retrolaminar tissue pressure was largely dependent on the surrounding cerebrospinal fluid pressure, which was on average 8.6 +/- 3.5 mm Hg (SD, n = 8) higher, and was independent of intraocular pressure. Most (85% +/- 15% [SD, n = 8]) of the pressure drop between intraocular pressure and retrolaminar pressure occurred across the anterior 400 microns of disk tissue. When the intraocular pressure was 21 mm Hg and the cerebrospinal fluid pressure was zero, retrolaminar tissue pressure averaged 7 mm Hg and the translaminar pressure gradient was 3.08 +/- 0.29 mm Hg/100 microns tissue (SD, n = 3). Optic nerve subarachnoid space pressure was equivalent to lateral ventricular pressure. CONCLUSIONS: These results show that cerebrospinal fluid pressure largely determines retrolaminar tissue pressure; hence, along with intraocular pressure, it is of major importance in setting the translaminar tissue pressure gradient. Results also demonstrate hydrostatic continuity between the optic nerve subarachnoid space and the lateral ventricle. That the translaminar pressure gradient can vary independently of intraocular pressure may be of importance in understanding the pathophysiology of glaucoma.

Animals↗
PubMed1995

Lymphohematopoietic progenitors of normal mice.

We have identified and characterized the lymphohematopoietic progenitors in the bone marrow of normal mice using a single-step methylcellulose culture assay. Lineage-negative Ly-6A/E (Sca-1)+ progenitors isolated from normal mice were plated in methylcellulose culture containing steel factor (SF), interleukin-7 (IL-7), erythropoietin (Ep), and IL-11. After 16 to 17 days of culture, pre-B-cell-containing multilineage myeloid colonies can be microscopically identified; however, flow-cytometric analysis of individual colonies for B220-positive cells proved superior to in situ microscopic identification of lymphomyeloid colonies. Approximately 10% (6/66) of the mixed colonies without a conspicuous B-cell component had B220-positive cells. The single cell origin of the lymphomyeloid colonies was confirmed by micromanipulation. Although the combination of SF, IL-7, and Ep was sufficient to support formation of lymphomyeloid colonies, addition of IL-11, granulocyte colony-stimulating factor or IL-12 to the combination of SF, IL-7, and Ep increased the number of lymphomyeloid colonies. IL-1 alpha and IL-3 independently inhibited the expression of the B-lymphoid lineage when added to the combination of SF, IL-7, Ep, and IL-11. Approximately four times more lymphohematopoietic progenitors are present in normal mice than in mice treated with 5-fluorouracil.

Animals↗
PubMed1995

Hodgkin and Reed-Sternberg cells do not carry T-cell receptor gamma gene rearrangements: evidence from single-cell polymerase chain reaction examination.

Hodgkin and Reed-Sternberg (H&RS) cells are generally accepted to be the neoplastic cells of Hodgkin's disease (HD), even though they represent only a minority of the cellular infiltrate in affected tissues. Recent immunologic studies and Southern blot analyses of DNA extracted from whole lymph node tissue favored, but did not convincingly prove a lymphoid origin of H&RS cells. To detect rearrangements of the T-cell receptor gamma chain (TCR gamma) genes at the single-cell level as an indication of early T-cell lymphoid differentiation, we isolated H&RS cells by micromanipulation from cytospin preparations of fresh biopsy material. TCR gamma chain rearrangement was detected by polymerase chain reaction using four "forward primers" that were constructed corresponding to all four V families and two "reverse primers" corresponding to consensus sequences of J segments. Rearrangements of all V families in combination with the different J segments were detected in human peripheral blood and tonsillar T cells. Although rearrangements of TCR gamma chain genes were shown in single cells of 10 of 10 T-cell leukemias, no rearrangement of these genes was found in single H&RS cells from 13 consecutive patients with HD. Our results indicate that H&RS cells from the vast majority of cases are not derived from T cells. This finding may have implications for the pathogenesis of HD and the development of more effective treatment regimens.

Base Sequence↗
PubMed1994

Amplification of beta-globin gene from single blastomeres.

Prenatal diagnosis of beta-thalassemia can be performed by chorionic villus sampling as early as the ninth gestational week, and early amniocentesis within 12 weeks of gestation. Selective abortion or termination should be suggested if the fetus is affected. With polymerase chain reaction (PCR) and micromanipulation techniques, the preimplantation diagnosis of beta-thalassemia has become possible. A total of 60 single blastomeres were collected and equally divided at random into two groups. Eight DNA primers (A to H) were designed for 15 mutations of beta-thalassemia in Chinese people. Two beta-globin gene fragments (602 bp and 423 bp) were amplified by PCR through two sequential reactions with two sets of primers (A+B and C+D, E+F and G+H), respectively. The amplified products were confirmed by Southern blotting with DNA probes hybridization. The amplification rates were 54% (16/30) and 60% (18/30), respectively. All of the positive and negative controls showed the presence or absence of amplification, respectively. Our study provides a possible approach for the preimplantation diagnosis of beta-thalassemia.

Base Sequence↗
PubMed1994

Cerebrospinal fluid collection in rats: modification of a previous technique.

Modification of a previously published technique for cerebrospinal fluid collection in rats is described. This technique uses general anesthesia, a supporting platform to flex the head-neck junction, a surgical approach to the dorsal atlanto-occipital region, and a micromanipulator to hold and control the approach of the collection needle. Critical steps for success of the procedure are correct positioning, avoiding premature incision of the subarachnoid space, and alignment of the collection needle with the midline and longitudinal axis of the head. The authors had a 95% success rate in obtaining > 0.1 ml of cerebrospinal fluid, using this technique.

Anesthesia, General↗
PubMed1994

Qualitative and quantitative analysis of subpopulations of cytotoxic effector cells by flow cytometry.

The heterogeneous nature of cytotoxic effector cells presents a difficult obstacle to the study of developmental, biochemical and molecular events governing cytotoxic T lymphocytes (CTL) and natural killer (NK) cell biology. In previous studies, there were no available methods, other than the single cell assay or micromanipulation techniques that could distinguish between lytic and non-lytic cells present in purified populations or clones. Further, there were no available means to isolate these subsets for further investigation. The studies presented here describe a novel approach to identify and isolate cells based on their functional characteristics. Thus, non-lytic free and conjugate forming cells as well as killer cells could be identified, quantified, and further purified by flow cytometric cell sorting for further investigations. Studies of several properties of isolated NK subpopulations (free, non-lytic binders, and killers) are presented.

Animals↗
PubMed1994

Assisted reproductive techniques and male infertility.

For couples in whom improvement in spermatogenesis is not possible or is unsuccessful in producing conception, ARTs offer much promise. For mild to moderate male factor couples, IUI combined with superovulation is often the initial procedure employed. If unsuccessful, IVF or one of its variations should be considered. With severe male factor cases, couples should proceed directly to IVF or one of its variations in which specific techniques may be used to improve the chances of fertilization. Currently both specialized semen-processing techniques and micromanipulation are used either singly or in combination. More experience is needed to determine which couples will benefit from which techniques. Although the urologist may not actively participate in all the actual ART procedures, it is important that the urologist have a thorough understanding of the procedures and their indications. This allows for accurate counseling of the male factor couple.

Humans↗
PubMed1975

Transvitreal chorioretinal biopsy.

A technique for transvitreal chorioretinal biopsy has been developed which employs a stereotaxic intraocular micromanipulator and a newly designed biopsy instrument. Biopsy is performed by trephinating and removing from the eye a 1.5-mm diameter specimen of posterior eye wall. Hemostasis and chorioretinal adhesion are achieved by mechanical compression of the tissues surrounding the eye wall defect. The eye wall defect is sealed by a preplaced silicone explant and repaired by fibrovascular ingrowth from the surrounding choroid and episclera. Normal histological features of the biopsy specimen are preserved except for edema of the tissues, separation of the neurosensory retina from the pigmented epithelium, and occasional hemorrhagic detachment of the pigmented epithelium.

Animals↗
PubMed1993

Analysis of human in vitro fertilization failure.

Fertilization failure is a serious problem in human in vitro fertilization (IVF) programs and deserves further investigation and management. Two hundred and ninety-four IVF cycles performed at the National Taiwan University Hospital from July 1989 to June 1991 were retrospectively analyzed. Thirty-seven (13%) of the 294 cycles were observed to have fertilization failure. The incidence of fertilization failure in male factor patients was significantly higher (p < 0.05) than in others. Patients with oligoasthenospermia tended to have a higher rate of fertilization failure than patients with oligospermia or asthenospermia alone. In non-male factor patients, a smaller number of oocytes and mature oocytes was found in patients with fertilization failure than in patients achieving fertilization. Sperm penetration assay (SPA) using zona-free hamster eggs was performed for 15 patients with fertilization failure; no correlation was found between SPA and the fertilizing ability of the sperm in vitro. Four patients with severe oligoasthenospermia had repeated fertilization failure in subsequent IVF cycles. The other five patients, including four non-male factor patients and one male factor patient, achieved fertilization in their second IVF trial. Our results suggest that severe oligoasthenospermic patients with repeated fertilization failure should be candidates for micromanipulation of gametes in subsequent IVF trials.

Female↗
PubMed1994

Aberration of genomic DNA in association with human hepatocellular carcinomas detected by 2-dimensional gel analysis.

Alterations of genomic DNAs in primary hepatocellular carcinomas (HCCs) were examined by restriction landmark genomic scanning (I. Hatada et al., Proc. Natl. Acad. Sci. USA, 88: 9523-9527, 1991) which is a 2-dimensional gel analysis that allows detection of deletion, amplification, or other rearrangements of genomic DNA. Sixteen HCC samples together with their normal counterparts were tested in this manner. Each HCC sample was micromanipulated to minimize possible carryover from non-malignant cells. DNAs from HCCs and their normal counterparts were cleaved with the restriction enzyme NotI, end labeled with 32P, and size fractionated by 2-dimensional electrophoresis using HinfI as the second cleavage enzyme. The resulting spots (about 2000) in HCC samples were compared with their normal counterparts. Five spots were more intense in 10-14 of the 16 HCCs (63-88%). The intensity of several spots was reduced to about half, suggesting the loss of one of two alleles. Some of these decreases were observed frequently in different HCC samples, whereas others were sporadic. Sixty of these spots reproducibly decreased in > 2 cases, with 27 showing a decrease in > 50% of the informative cases. The highest incidence was observed in 14 of 16 samples (88%). No significant correlations were observed between these changes in spots and hepatitis B virus or hepatitis B virus infection. The use of landmarks that show a reproducible increase or decrease in intensity is discussed in conjunction with future studies of genomic alterations inherent in HCC.

Carcinoma, Hepatocellular↗
PubMed1994

Successful implementation of a regional in vitro fertilization program.

In vitro fertilization and other assisted reproductive technologies are becoming widely accepted modalities of treatment for infertile couples. Like other modern technologies, in vitro fertilization requires specially trained physicians and scientists who need specially dedicated equipment and space. In addition, patients having the procedure must undergo timely laboratory tests and sonographic evaluations for monitoring follicular maturation. To make in vitro fertilization available to patients in north central Florida, the Division of Reproductive Endocrinology and Infertility of the Department of Obstetrics and Gynecology at the University of Florida College of Medicine in Gainesville established a system that utilizes local resources in three distant locations. Patients in an assisted reproductive technologies cycle are monitored at satellite locations but coordinated through the University of Florida in vitro fertilization team. Oocyte retrieval, laboratory handling of gametes and embryo transfer are performed at the Shands Teaching Hospital in Gainesville. Micromanipulation for severe male factor infertility and oocyte donation are available when needed. Preimplantation diagnosis for genetic conditions is under development. The ongoing pregnancy and delivery rate is 21% per cycle initiated from July 1, 1992, to July 3, 1993, 62% above the reported national pregnancy rate for 1991. This program may be a useful model for the development of collaborative efforts in delivering highly complex medical services to large populations.

Academic Medical Centers↗
PubMed1994

[Studies on partial zona dissection in in vitro fertilization for severe male infertility].

The effects of partial zona dissection (PZD) were investigated in mouse in vitro fertilization (IVF) with an oligospermic model and then PZD was applied to patients with severe male infertility. Micromanipulated (PZD-treated) and zona-intact (control) mouse oocytes were inseminated with 1 x 10(4) (low sperm concentration) and 1 x 10(5), 1 x 10(6) (normal sperm concentration) motile sperm/ml. In mouse IVF with 1 x 10(4) sperm/ml, the fertilization rate in those PZD-treated were significantly higher than that in the control (21.3% vs 8.1%, p < 0.01). No polyspermy was observed. No significant difference in the rate of development to the blastocyst stage was seen between the PZD-treated and the control (71.0% vs 65.0%). The incorporation rate of 3H-leucine in embryos at the 2-cell and blastocyst stages also did not differ between the two groups of mice. Sixteen patients with severe male infertility who had failed to fertilize for the previous three consecutive IVF cycles were enrolled in the PZD protocol. The fertilization rate in PZD-treated cycles was significantly improved (19.6% in PZD-treated vs 0% in control cycles, p < 0.001). In ten patients who underwent replacement with PZD-treated embryos, one biochemical pregnancy occurred. The present study suggests that although PZD is useful for severe male infertility, further investigation is needed to evaluate PZD in assisted fertilization.

Animals↗
PubMed1993

Use of perfluoroperhydrophenanthrene in the management of posteriorly dislocated crystalline and intraocular lenses.

We describe a series of 28 patients in whom a posteriorly dislocated crystalline or intraocular lens was successfully removed with the intraoperative use of perfluoroperhydrophenanthrene (Vitreon), a heavy liquid perfluorocarbon. The Vitreon was used to float the lens into the midvitreous cavity, thereby avoiding hazardous micromanipulation with intraocular instruments in the macular area. In addition, the Vitreon served to protect the posterior retina by keeping the posteriorly dislocated lens fragments from falling backward onto the posterior pole as the lens was being removed from the eye. Postoperative visual acuity improved in 73% of the patients, remained the same in 15%, and worsened in 12%.

Aged↗