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PubMed1996

Mapping the location of functional nicotinic and gamma-aminobutyric acidA receptors on hippocampal neurons.

To assess the density and distribution of functional nicotinic acetylcholine receptors (nAChRs) and gamma-aminobutyric acid (GABA) receptors on hippocampal neurons, we have combined infrared videomicroscopy with a nanorobotic micromanipulator system and studied the receptor-mediated currents by using the whole-cell patch-clamp technique. Acetylcholine or GABA was applied by pressure ejection onto a small segment of either cell soma or dendrite, and the resulting current was measured in cultured hippocampal neurons by using a whole-cell pipette positioned at the cell soma. Type IA nicotinic currents, sensitive to blockade by methyllycaconitine (1 nM) and alpha-bungarotoxin and associated with alpha 7-subunit-containing nAChRs, type II currents, sensitive to blockade by dihydro-beta-erythroidine (100 nM) subserved by alpha 4 beta 2 nAChRs, and GABA-mediated currents were evoked when the agonists were applied to either cell soma or the dendrites. Analysis of the current amplitude with respect to the membrane area covered by the applied agonist provided an estimation of the receptor density along the somato-dendritic axis of the hippocampal neurons. Such analysis revealed: 1) a nonuniform distribution for the receptor types studied; 2) a higher density of nAChR and GABA receptors at the dendrites than at the soma; and 3) an increasing density for both nAChR subtypes with distance from the center of the cell soma, but increasing and then decreasing density for the GABA receptor. Exposure of cultured hippocampal neurons to colchicine (100 nM for 3 days) produced a dramatic reduction in the dendritic branching, and this morphological feature was associated with a significant decrease in the receptor density, such an effect being more prominent for nAChRs than for GABA receptors. Given the high Ca+2 permeability of nAChRs, the dendritic localization of nAChRs suggest that they are involved in modulating the synaptic efficacy at the level of the dendrites.

Aconitine↗
PubMed1996

Single-cell mutation analysis of tumors from stained histologic slides.

Formalin-fixed and paraffin-embedded tissues are a valuable resource for diagnosis and research. PCR is one of the most powerful methods of retrospective analysis of the DNA present in fixed tissues. One major problem with the molecular analysis of tissue samples, however, is cellular heterogeneity, ie, the large variety of cell types usually present in these specimens can mask cell-specific genetic alterations associated with disease. Herein we describe a procedure for obtaining and analyzing single cells recovered from stained histologic tissue sections without risking contamination from neighboring cells. An ultraviolet laser microbeam was used to physically destroy the tissue surrounding the single cells of interest. These cells, now freed from adjacent cells, were then easily retrieved with a motorized, computer-controlled micromanipulator and molecularly characterized through the use of PCR-based microanalysis. This accurate microdissection technique, followed by DNA amplification and direct sequencing, revealed a novel mutation in the gene coding for the cell adhesion molecule E-cadherin in single tumor cells that was absent in the adjacent single epithelial cells of a patient with early gastric cancer of the diffuse type. In this form of malignancy, tumor cells lose homophilic cell-to-cell interactions and invade the connective tissue as single cells. E-cadherin gene mutations have previously been detected in advanced diffuse-type gastric cancer and gastric carcinoma cell lines. The present study suggests that E-cadherin gene mutations may be an early event in gastric tumorigenesis. The laser-based isolation and subsequent molecular characterization of individual cells, as described herein, allows for micrometer-sized precision and should prove useful in detecting the nucleic acid abnormalities that underlie cancer, infection, and genetic disease.

Base Sequence↗
PubMed1997

Testis biopsies frequently demonstrate sperm in men with azoospermia and significantly elevated follicle-stimulating hormone levels.

PURPOSE: Men with azoospermia, markedly elevated serum follicle-stimulating hormone levels and testicular atrophy were previously considered irreversibly infertile. Thus, testicular biopsy in this patient population was considered unnecessary. However, presently men with even the most severe infertility disorders are potentially able to initiate a pregnancy with intracytoplasmic sperm injection provided sperm can be recovered in even relatively few numbers directly from the testicular tissue. For these reasons we sought to reevaluate the findings from testicular biopsies in these men in the era of advanced micromanipulation techniques. MATERIALS AND METHODS: Chart review identified men with azoospermia, confirmed on a pelleted specimen, and a serum follicle-stimulating hormone level of 3 or more times normal. Mature sperm in the touch preparation cytology and testis biopsy specimen were confirmed. RESULTS: A total of 57 men, most with testicular atrophy, underwent a testicular biopsy and in 17 (30%) mature sperm were identified. The most common diagnosis in these men was severe hypospermatogenesis. Men without sperm most commonly had a pure Sertoli-cell-only pattern. CONCLUSIONS: Men with azoospermia and testicular atrophy with significantly elevated follicle-stimulating hormone levels should undergo testicular biopsy if in vitro fertilization with intracytoplasmic sperm injection is an acceptable approach for the couple.

Adult↗
PubMed1996

Andrological work-up of patients undergoing microsurgical epididymal sperm aspiration or testicular sperm extraction.

Microsurgical epididymal sperm aspiration (MESA) and testicular sperm extraction (TESE) require close cooperation between andrologists, urologists and gynaecologists. Intracytoplasmic sperm injections were established in Giessen in March 1994 and embryo transfer (ET) was performed in 342 of 375 patients (91.2%). The percentage of pregnancies and ongoing pregnancies are 35.4% per ET (32.3% per cycle) and 25.1% per ET (22.9% per cycle), respectively. Microsurgical procedures such as epididymovasostomy or vasovasostomy and cryopreservation of human semen are also established methods. The purpose of the present study was to describe the andrological work-up for patients before MESA and TESE. Experiments demonstrate that incubation of testicular tissue samples in IVF medium and treatment with 1 mg ml-1 pentoxifylline increase the number of extracted motile spermatozoa. Centrifugation of the medium results in a further concentration of sperm cells. If no motile spermatozoa can be found in the supernatant medium, they may be extracted directly from the testicular tissue samples by means of a micromanipulator.

Cell Separation↗
PubMedDate not supplied

[Experimental data on reaction of neurons of the brain to low-intensity package-pulsing microwave irradiation].

In experiments on non-movement rabbits with help specially made from organic glass of micromanipulator and non-artefactions electrodes and wires study the bioelectrical activity separate neurons on moment of package-pulsing microwaves irradiation (1.5 GHz, duration of pulse-0.4 ms, frequency their recurrences 1000 Hz, duration of packs-16 ms, frequency their recurrences-0.12 Hz, energy-0.3 mW/sm2). The results of experiments are compared with control series, where is carried out false irradiation.

Animals↗
PubMed1997

Improving the cost-to-benefit ratio of in-vitro fertilization.

In-vitro fertilization (IVF) has been criticized for being too costly to permit ready access to needy patients, either through their own funds or through those of third party providers. European groups have managed to offer these procedures at a fraction of the cost incurred by their United States counterparts by streamlining their protocols. Accordingly, we present our methods for reducing the cost of IVF. The main modification was made by performing the IVF procedures in the clinic under i.m. analgesia, avoiding the costs of a surgery suite and anaesthesia. In addition, donor, oocyte, micromanipulation, and cryopreservation services were not offered, reducing overall personnel and equipment costs. Overall costs were reduced from customary levels of $7,000--$11,000 in the United States to $3,409 per cycle initiated, while maintaining good ongoing/delivered pregnancy rates (30.0% versus 18.6% nationally) per cycle reaching aspiration. We conclude that, through the elimination of less necessary and/or utilized procedures, IVF may be performed in a more cost-effective manner while maintaining good success rates. For the patients who desire IVF services not offered under such a system, referral to a more specialized IVF center is appropriate.

Clinical Protocols↗
PubMed1997

[Physiology of human gamete interaction].

Subzonal insemination was the first micromanipulation technique used in the treatment of male factor sterility. Analysis of the sperm pathology prior to patient inclusion in the program allowed us to study gamete interaction in the human and the early embryogenesis. A series of 523 cycles and 3,027 micro-injected oocytes is reported.

Animals↗
PubMed1997

Clonal proliferation and cytokine requirement of murine progenitors for natural killer cells.

We have established a clonal cell culture system that supports the proliferation of committed natural killer (NK) cell progenitors of mice to investigate the pathway and cytokine regulation of NK cell development. Day 14 fetal thymocytes cultured in methylcellulose with interleukin-7 (IL-7), IL-15, and steel factor (SF) formed diffuse colonies that could not be classified to known colony types. Single-cell origin of the colonies was established by micromanipulation of the colony-forming cells. Cells in the colonies are very blastic, showing no cytoplasmic differentiation, and express Ly5, Thy-1, and CD25 but not myeloid, B, mature T, or NK cell markers. The cells lack T, B, and myeloid potentials but can differentiate to mature NK cells in fetal thymus organ culture, suggesting that the colonies consist of NK committed progenitors. Examination of the minimal cytokine requirement for the NK colony formation showed that IL-7 and SF are indispensable for the formation of immature NK cell colonies. Both IL-2 and IL-15 increased the frequency of colonies. In contrast to IL-2, IL-7, and IL-15, IL-4 strongly inhibited the formation of the colonies. This quantitative clonal culture will provide a useful means to examine the mechanism of NK cell development.

Animals↗
PubMed1997

CO2 laser surgery of hypertrophied inferior turbinates.

The inferior turbinates are responsible for nasal obstruction in patients with allergic and vasomotor rhinitis. Until today there is no satisfactory means of treating hypertrophied turbinates. One hundred and eighty-four patients with nasal obstruction due to hypertrophied inferior turbinates were treated with the CO2 laser using the microscope and a micromanipulator. A few laser spots (1 W, 1 s, laser power density: 2,038 W/cm2) were applied to the head of the turbinate. After a few days a positive effect was present. One hundred and twelve patients were followed for over 2 years. Six months after laser surgery, 87.5% had excellent or good results. After one year, 82.1% of the patients were satisfied, and after 2 years 80.4% were satisfied. The procedure involves little bleeding, no pain and can be done under local anaesthesia in an outpatient setting. Less post-operative wound care is necessary. Therefore, the CO2 laser surgical technique can be considered as an effective method in the treatment of hyperthrophied turbinates.

Adult↗
PubMed1997

Laser beam microdissection in the diagnosis of cutaneous B-cell lymphoma.

Interpretation of molecular analyses of cutaneous lymphoid infiltrates may be difficult because a heterogeneous group of cells in usually present within the neoplasms. Extraction of DNA from tissue sections does not provide exact information about which cell population has been analysed. We present a laser microscope system that allows selective molecular analysis of single cells or small groups of cells in cases of cutaneous lymphoma. An ultraviolet (UV)-laser microscope system (PALM, Wolfratshausen, Germany) was used to isolate particular populations of cells from a routinely processed specimen of a cutaneous follicular lymphoid proliferation. Using the UV-laser beam, a circle was cut around a target germinal centre in order to separate it from neighbouring tissues and to isolate a pure population of germinal centre cells. Isolated cells were scraped off with a micromanipulator and placed in a proteinase-K solution. DNA was extracted and amplified by the polymerase chain reaction (PCR) technique. Analysis of immunoglobulin JH gene rearrangement showed a distinct monoclonal band. In a second phase, using the same procedure in the same specimen, mantle zone cells around a germinal centre and single interfollicular B lymphocytes were isolated for PCR analysis of immunoglobulin JH gene rearrangement. In this population of cells, no clonality could be detected. This new technique allows the selective elimination of undesired cells and tissue from cutaneous neoplasms. By destruction of unwanted tissues with laser-beam energy a contamination-free sample is obtained. Analysis of isolated cells in our case demonstrated a clonal rearrangement derived from germinal centre cells and not from other B lymphocytes in the specimen, confirming the diagnosis of cutaneous follicle centre lymphoma. The method described has exciting implications for dermatology and dermatopathology, allowing precise correlation of morphological features with findings by molecular genetics.

Cell Separation↗
PubMed1997

[Modification of in vitro insemination techniques in the treatment of severe male factors in assisted reproduction].

The aim of this study is to detect qualified in vitro insemination techniques in the treatment of the severe oligoasthenotheratospermia which is defined as total motile count in the pretreatment samples (< 5 x 10(6) with > 50% of abnormal morphology). These patients have taken part in the in vitro insemination program of the Assisted Reproduction Unit of the 2nd Department of Obstetrics and Gynecology at Rome University "La Sapienza" during a period between June and December 1995. Several modifications of the standard in vitro techniques have been developed such as: mechanical decumulation of the oocytes, reduction of the volume of culture medium, increase of spermatozoa and oocyte concentration at the moment of insemination. A good fertilization rate was achieved (33%) as regard to the semen sample and procedures utilized. Twelve Ets were performed and 4 clinical pregnancies (25% per patients and 33% per transfer) were achieved. These data demonstrate that by the modification of standard laboratory methods for in vitro insemination, a good fertilization rate and a high clinical pregnancy rate can be achieved in cases of severe male factor infertility without having to resort to micromanipulation techniques.

Adult↗
PubMed1997

Normal and malignant B-cell development with special reference to Hodgkin's disease.

During their development, B lymphocytes are repeatedly selected for the expression of an appropriate surface receptor: the pre-B-cell receptor at the pre-B-cell stage and surface immunoglobulin (Ig) at the transition from a pre-B cell to a mature B cell. Furthermore, stringent selection for B cells expressing high affinity antibodies operates when antigen-activated B cells proliferate within germinal centers (GC). Here, somatic point mutations are introduced into rearranged V region genes at a high rate, and B cells acquiring favorable mutations are selected to differentiate into memory B cells or plasma cells. In the frame of this developmental scheme, extending a recent analysis, we investigated 10 primary cases of Hodgkin's disease (HD) for B-lineage origin and clonality [1]. Single Hodgkin and Reed-Sternberg (H-RS) cells were micromanipulated from frozen tissue sections and analyzed by PCR for rearranged V genes. Clonal VH and/or V kappa/ V delta gene rearrangements were obtained from 9 of the cases. This shows that H-RS cells represent a clonal, B-lineage-derived population of tumor cells. Somatic mutations were found in all clonal VH gene rearrangements. Interestingly, mutations leading to stop codons in in-frame V gene rearrangements were detected in four cases. Since GC B cells acquiring such crippling mutations are usually efficiently eliminated within the GC, the finding of those mutations indicates that H-RS cells are derived from precursors within the GC that escaped apoptosis by a transforming event.

Animals↗
PubMed1997

Development of natural killer cells, B lymphocytes, macrophages, and mast cells from single hematopoietic progenitors in culture of murine fetal liver cells.

We have recently established a clonal culture system that supports the growth of immature natural killer (NK) cells from murine fetal thymocytes. We now describe a culture system for mixed NK cell colony formation from single lymphohematopoietic progenitors. When Sca-1+c-kit+ fetal liver cells were cultured in methylcellulose media with interleukin (IL)-2, IL-7, IL-11, and steel factor (SF), we found mixed colonies consisting of diffuse small round cells characteristic of immature NK cells and other types of cells. The single cell origin of the mixed colonies was established by micromanipulation. Individual mixed colonies derived from single cells were characterized by flow cytometric analysis and May-Grünwald Giemsa staining. All mixed colonies contained Thy-1+B220- cells, which can differentiate to mature NK cells in fetal thymus organ culture. Most of the colonies contained B220+ B-lineage cells and macrophages, and some contained mast cells. IL-1alpha and IL-3, which have previously been shown to inhibit the T- and B-cell potentials of blast colonies, suppressed the formation of mixed NK cell colonies. The clonal culture assay presented here may be useful in analysis of the developmental pathway and commitment of NK cells from multipotential progenitors.

Animals↗
PubMed1997

[Prenatal diagnosis of Duchenne muscular dystrophy using single fetal cell in maternal blood].

We developed a method that allows prenatal diagnosis of Duchenne muscular dystrophy using a single nucleated erythrocyte (NRBC) isolated from maternal blood. Maternal blood was obtained at 8 to 20 weeks of gestation. NRBCs were separated with Percoll using a discontinuous density gradient method and then collected by micromanipulator under microscopic observation. The entire genome of a single cell amplified by primer extension preamplification (PEP). Sex was determined from a small aliquot of the PEP reaction. After an NRBC was determined to be male and confirmed to be of fetal origin, dystrophin exons 4, 8, 12, 45, 48, 50, and 51 were determined from the same PEP reaction. This diagnostic method using maternal blood is safer than amniocentesis or cordocentesis and can be applied to other X-linked disease.

Dystrophin↗
PubMed1976

Is Arthroderma simii the perfect state of Trichophyton Quinckeanum?

Gymnothecia with asci and ascospores developed repeatedly and in abundance whenever Trichophyton quinckeanum isolates X-392 and IMI 140691 were paired with Arthroderma simii 678 A. However, repeatable fertile crosses did not occur when all of our 19 isolates of T. quinckeanum were paired with 10 other isolates of A. simii comprising A and a mating types. Additional crosses between these 19 isolates of T. quinckeanum with A. simii and with A. benhamiae revealed that 11 of the 19 isolates of T. quinckeanum produced fertile gymnothecia only when crossed with A. simii 678 A but not when crossed with another isolate of A. simii of the A mating type nor when crossed with mating type a. All 19 isolates mated with A. benhamiae mating type A; therefore, all of our T. quinckeanum isolates were of the a mating type. Study of asci and random ascospores, selected with a micromanipulator, from crosses between several isolates of T. quinckeanum and A. simii 678 A revealed irregularities in the maturation of ascospores, poor ascospore germination, and a predominance of the A. simii phenotype and mating type for those ascospores that germinated and produced mature colonies. All these reactions are characteristic of interspecific crosses. In contrast, ascus analysis of the F1 progeny of crosses between these same isolates of T. quinckeanum with A. benhamiae revealed regular maturation of ascospores, better germination, and essentially a 1:1 segregation of mating types and parental phenotypes. This study confirms the concept that T. quinckeanum is not an independent species and that it is best considered to be a variety of T. mentagrophytes, as reported by Ajello, Bostick & Cheng (1968). Furthermore, our data indicate that T. mentagrophytes var. quinckeanum appears to be more closely related to A. simii than the granular variety of T. mentagrophytes, since fertile gymnothecia resulted from crosses between T. mentagrophytes var. quinckeanum and A. simii but not between T. mentagrophytes var. mentagrophytes and A. simii. Further implications of this study suggest the importance of extensive ascospore analysis when investigating the perfect states of fungi in order to avoid creating unwarranted new species.

Ascomycota↗
PubMedDate not supplied

[The role of fertilization in vitro in the treatment of infertility].

Artificial insemination presents of one broadly accepted methods of assisted reproduction. Indicative spheres for the healing of sterility by this method are permanently outspreading. Its primary application has been induced due to irreparable damages of oviducts but the indications for its use have been expanded in both women and men. One third of the total number of sterile married couples has the need for some of existing methods for assisted reproduction. Micromanipulative techniques of artificial insemination have practically solved all until recently unbridgeable difficulties in the treatment of marital sterility caused by man. Artificial insemination presents a treatment methodology tightly connected with a series of ethical, social and legal problems which are of great interest for the society also.

Female↗
PubMed1998

Origin of the Hodgkin/Reed-Sternberg cells in chronic lymphocytic leukemia with "Hodgkin's transformation".

A lymphoma with the characteristic features of Hodgkin's disease (HD) occasionally develops in patients with B-cell chronic lymphocytic leukemia (CLL), and has been called Richter's syndrome with HD features. In such cases, large tumor cells have the morphological and immunophenotypic features of classical Hodgkin and Reed-Sternberg (H-RS) cells. However, it is not known whether the H-RS cells arise from transformation of the underlying CLL cells or from a different pathological process. We report herein a study of the clonal relationship between the CLL cells and the H-RS cells in three cases of Richter's syndrome with HD features by using a single cell assay. We isolated single CLL cells and H-RS cells from immunostained tissue sections by micromanipulation. The immunoglobulin heavy chain gene (IgH) complementarity determining region (CDR) III of each cell was amplified by the polymerase chain reaction (PCR). The products were then compared by gel electrophoresis and nucleotide sequencing. The IgH CDRIII sequences from the H-RS cells were identical to those from the CLL cells in two cases. In one case, the clonal relationship between the two types of cells could not be determined because PCR products could not be obtained from any of the H-RS cells. This study shows that the H-RS cells and the CLL cells belong to the same clonal population in some cases of Richter's syndrome with HD features. Furthermore, our findings indicate that mature B cells can undergo transformation to cells with the features of H-RS cells, in association with a cellular background typical of HD. This study also supports recent findings suggesting that the H-RS cells in classical HD are derived from transformed B cells.

Cell Transformation, Neoplastic↗
PubMed1998

Fetal mouse NK cell clones are deficient in Ly49 expression, share a common broad lytic specificity, and undergo continuous and extensive diversification in vitro.

NK cells obtained by exposing mouse fetal thymocytes to appropriate combinations of IL-4, IL-2, and PMA are phenotypically indistinguishable from cultured adult splenic NK cells with the exception that they generally lack measurable expression of all of the inhibitory Ly49 molecules that can currently be detected with Abs (Ly49A, -C, -G, and -I) and of the activating molecule Ly49D. Despite this deficiency, fetal NK cells have a similar specificity to Ly49-expressing adult splenic NK cells. Individual fetal NK cell clones display an essentially invariant and broad specificity similar to that of polyclonal populations of fetal or adult NK cells, although significant differences in the fine specificity of clones can occasionally be detected. Most remarkably, cloned fetal NK cell lines display heterogeneous expression of a restricted set of surface molecules that includes 10A7, Ly6C, 3C2, CD8, certain isoforms of CD45, and also, occasionally, Ly49 molecules. This heterogeneity is not related to the cell cycle or activation status of the cells, and micromanipulation recloning demonstrates unambiguously that it is not due to a lack of a single cell origin. Diversity is generated rapidly and the capacity for diversification appears to persist indefinitely in vitro. The expression of individual variable Ags is independent and stochastic, resulting in fetal NK "clones" being potentially composed of hundreds of phenotypically distinct cells. We hypothesize that fetal NK cells behave as progenitor cells that are undergoing a process of rapid, extensive, and continuous diversification and that are individually capable of generating and regenerating a complex NK cell repertoire.

Animals↗