Internalization of cellular fragments in a human embryo: time-lapse recordings.

An observation is reported of internalization of a cellular fragment into a blastomere from a human embryo, as documented by time-lapse photography. The fragment, created during the first mitotic cleavage was reabsorbed into one of the mother blastomeres in less than 5 min. The time-lapse sequence, shown here as a series of still photographs, provides the first direct evidence that cellular fragments in human embryos can 'disappear' during the culture period, a phenomenon that is common in human IVF. The time-lapse sequence itself may be viewed on the internet at www.rbmonline.com/Article/633.

A method for longitudinal microscopic in vivo examinations of morphology, vascularity, and motility in the ovary and the oviduct of the rat.

OBJECTIVE: We developed an in vivo model to enable observation of dynamic changes in morphology, vascularity, and motility of the rat adnexa. METHODS: Immature Sprague-Dawley rats (n = 16) were primed with equine chorionic gonadotrophin (eCG;15 IU) followed by human chorionic gonadotrophin (hCG; 15 IU) 48 hours later to induce ovulation. The experiments were performed during prolonged (up to 12 hours) thiobarbiturate anesthesia. During laparotomy the periovarian bursa was retracted, whereafter the oviductal-ovarian complex was submerged into an organ chamber. Water immersion lenses (4x-40x; final magnification up to 810x) enabled detailed observations that were recorded on Beta-SP videotape. RESULTS: Capillary flow was monitored easily. At the level of the follicle, top blood flow velocity variations (8-10 per minute) were observed in the microvasculature. Ovulations were followed in detail, and oocyte-cumulus complexes were seen later in the oviductal ampulla. Regular contractions in the oviduct were synchronous with the oocyte-cumulus complexes moving back and forth in the oviductal lumen over a distance of about 900 microm. These contractions were more frequent (13-16 per minute) in the postovulatory phase compared with the time before ovulation (9-10 per minute). The oviductal contractions were initiated alternately from either end of the ampulla and were accompanied by a denudation of the oocytes, with a stream of cumulus cells seen moving in an abovarian direction in between contractions. CONCLUSION: High-magnification video recording in vivo was useful for capturing microcirculatory events as well as structural and functional changes of the ovary and the oviduct.

The allergenic yeast Malassezia furfur induces maturation of human dendritic cells.

BACKGROUND: The yeast Malassezia furfur (M. furfur), present in the normal microflora of human skin, can act as an allergen that incites specific IgE reactivity and T cell proliferation in atopic dermatitis (AD) patients. The role of antigen presenting dendritic cells (DCs) in the onset and maintenance of AD is not well established. OBJECTIVE: The objective of the present study was to assess whether the interaction of M. furfur with human DCs will result in DC maturation, cytokine production and lymphocyte proliferation. METHODS: Monocyte-derived dendritic cells (MDDCs) were generated from human peripheral blood. Immature MDDCs were cultured with or without M. furfur or plastic beads, and with or without CD40L stimulation. Interaction of yeast cells by MDDCs was studied by time-lapse photography and cytokines were detected in culture supernatants with ELISA. The ability of MDDCs pre-incubated with M. furfur to induce proliferation in autologous lymphocytes was measured by [(3)H]-thymidine incorporation. RESULTS: Time-lapse photography showed that the majority of immature MDDCs internalized whole M. furfur yeast cells within 1 h. The presence of M. furfur induced maturation (CD83 expression) of MDDCs, and up-regulation of the costimulatory molecules CD80 and CD86. Production of TNF-alpha, IL-1 beta and IL-18 by MDDCs increased significantly (P < 0.05 for TNF-alpha and IL-1 beta, and P < 0.01 for IL-18) after the addition of M. furfur, while IL-10 and IL-12p70 levels remained unaltered. The CD40L-stimulated IL12p70 production by MDDCs was decreased in the presence of M. furfur (P < 0.05). Finally, immature MDDCs pre-incubated with M. furfur induced a proliferative response in autologous CD14-depleted peripheral blood mononuclear cells, in a dose-dependent manner. CONCLUSION: The data indicate that immature MDDCs can internalize the opportunistic yeast M. furfur. This process was associated with MDDC maturation, production of pro-inflammatory and immunoregulatory cytokines, which might favour induction of a Th2-type immune response, and a capacity to stimulate lymphocyte proliferation. This chain of events most likely contributes to the inflammatory reaction in AD.

IgE-mediated mast cell degranulation and recovery monitored by time-lapse photography.

BACKGROUND: Mast cells are long-lived resident cells that are of great importance in an allergic reaction. It has previously been suggested that after IgE-mediated degranulation mast cells can undergo regranulation. Such a process is probably of great importance with respect to the severity and perpetuation of the allergic response. OBJECTIVE: Our purpose was to investigate whether mast cells recover from degranulation and whether they still have the potential to release a granule-associated mediator and upregulate certain cytokine genes. METHODS: Mouse mast cells were repeatedly activated by IgE and specific antigen with a 24-hour or 48-hour interval. During each of the 2 activation stages, release of beta-hexosaminidase was measured by means of enzymatic colorimetric analysis, and IL-13 and IL-6 mRNA was detected by ribonuclease protection assay. Both scanning electron microscopy and time-lapse photography were used to reveal the process of mast cell recovery. RESULTS: We found that re-activation of degranulated mast cells in response to high-affinity IgE-receptor cross-linkage triggers beta-hexosaminidase release and upregulation of IL-13 and IL-6 gene expression levels similar to what is seen in the initial activation. Scanning electron microscopy documented cells at various stages during the recovery process 30 minutes after the activation. With time-lapse photography, a single cell that had undergone degranulation could be visualized consecutively during its recovery process. CONCLUSION: Mast cells can recover after an IgE-mediated activation and can repeatedly release beta-hexosaminidase and express IL-6 and IL-13 mRNA after re-activation.

Helicobacter pylori entry into human gastric epithelial cells: A potential determinant of virulence, persistence, and treatment failures.

BACKGROUND AND OBJECTIVES: Intracellular location of Helicobacter pylori in human gastric epithelial cells has been observed in biopsies. Whether this reflects an ability to invade host cells and establish an intracellular niche remains to be determined. METHODS: The interactions between a clinical isolate of H. pylori and primary cell cultures from human gastric epithelium or the human epithelial cell line HEp-2 were monitored using time-lapse photography. This technique allows studies of the dynamics of host-microbial interactions. RESULTS: H. pylori cells readily approached and established close contacts with epithelial cells followed by uptake of the bacteria into the cellular cytoplasm. Entry into epithelial cells was achieved through an active process of bacterial motility and penetration of the cell membranes. In conventional invasion assays using HEp-2 cells, an increased internalization in a strain producing the vacuolating cytotoxin was observed, compared to the isogenic VacA knockout mutant. CONCLUSION: Invasion of gastric epithelium represents a hitherto unappreciated trait of H. pylori that could contribute to the bacterium's ability to establish persistent infection that evades the mucosal immune defense and sometimes also antimicrobial therapy. A small number of bacterial cells with a transient intracellular habitat could serve as a seeder population, providing a backup for a constantly challenged and fluctuating luminal population.

Control of gill filament blood flow by serotonin in the rainbow trout, Oncorhynchus mykiss.

The effects of exogenously applied serotonin [5-hydroxytryptamine (5-HT)] on the distal arterial vasculature of gill filaments were observed using an epi-illumination microscope equipped with a water-immersion objective and connected to a video camera. In addition, ventral aortic flow (Q) and celiac artery pressure (PCA) were measured. Intra-arterial injection of serotonin (100 nmol/kg) completely stopped the blood flow in the distal part of the filaments and caused a rapid decrease of PCA. Repeatedly, the flow reduction was found to coincide with a constriction of the distal portion of the efferent filamental vasculature. Because there was no concomitant reduction in Q, it is concluded that a redistribution of blood to more proximal parts of the filaments occurred. After treatment with the serotonergic receptor antagonist methysergide, the vasoconstrictor effect of serotonin on the filamental vasculature was eliminated, while a decrease in PCA was still observed. The results demonstrate a specific site(s) for the serotonergic vasoconstriction in the distal portion of the filament.

Anoxia and adenosine induce increased cerebral blood flow rate in crucian carp.

The crucian carp (Carassius carassius) has the rare ability to survive prolonged anoxia, indicating an extraordinary capacity for glycolytic ATP production, especially in a highly energy-consuming organ like the brain. For the brain to be able to increase its glycolytic flux during anoxia and profit from the large liver glycogen store, an increased glucose delivery from the blood would be expected. Nevertheless, the effect of anoxia on brain blood flow in crucian carp has never been studied previously. We have used epireflection microscopy to directly observe and measure blood flow rate on the brain surface (optic lobes) during normoxia and anoxia in crucian carp. We have also examined the possibility that adenosine participates in the regulation of brain blood flow rate in crucian carp. The results showed a 2.16-fold increase in brain blood flow rate during anoxia. A similar increase was seen after topical application of adenosine during normoxia, while adenosine was without effect during anoxia. Moreover, superfusing the brain with the adenosine receptor blocker aminophylline inhibited the effect of anoxia on brain blood flow rate, clearly suggesting a mediatory role of adenosine in the anoxia-induced increase in brain blood flow rate.

Ovulation in the isolated perfused rat ovary as documented by intravital microscopy.

Surface cell changes at the apices of preovulatory follicles and ovulations were documented in isolated perfused ovaries from immature rats treated with pregnant mare serum gonadotropin (20 IU) and 48 h later with human chorionic gonadotropin (hCG) (10 IU). A video camera coupled to an inverted microscope and a video recorder captured the preovulatory and ovulatory events at a cellular level. At around 8 h post-hCG, the follicular apex changed from a smooth and optically homogeneous appearance into a rough surface with bleb formation and extrusions of single cells through minute perforations (early stigma formation). At approximately 10 h, a sticky material formed a basketlike structure with trapped cells (late stigma formation). At 12 to 15 h, ovulation took place at a constant speed and with no contractions of the follicular wall. This indicates that ovulation can occur with no visible circumfollicular muscular activity. Furthermore, the observations of a leakage of cells over an extended period of time indicates that the follicular wall is partly digested several hours before ovulation occurs.

The study of ovulation in the isolated perfused rabbit ovary. II. Photographic and cinematographic observation.

Using the model of in vitro perfusion of the isolated rabbit ovary, in which follicular ruptures can be observed, in this paper we report on the photographic and cinematographic recording of the gross anatomy of ovulation. The observed sequence of events and their timing were very similar to that which has been reported for ovulation observed in vivo. In addition, the use of infrared film made it possible to visualize intrafollicular events prior to rupture.