Trichinella spiralis: vascular recirculation and organ retention of newborn larvae in rats

The recirculation of Trichinella spiralis newborn larvae was studied in inbred AO rats. Newborn larvae collected after in vitro incubation of adult T. spiralis worms for 2 or 24 hr were injected into rats through the tail vein or hepatic portal vein. Blood samples from the femoral vein, hepatic portal vein, and abdominal aorta were collected at intervals from 1 min to 24 hr after larval injection. Newborn larvae of both ages (24 hr or 2 hr old) persisted in femoral vein blood for less than or equal to 5 hr after injection, but they could be detected in portal vein blood by 24 hr after injection. The injection of larvae into a tail vein or the portal vein did not influence the pattern of larval circulation, although there was a 1-5 min delay in newborn larval appearance time after injection into the portal vein. Transcapillary migration through tissue and back to the circulation was evident in the appearance of newborn larvae in the thoracic duct lymph up to 24 (occasionally 48) hr after tail vein injection of newborn larvae. During the course of a natural primary infection, no evidence for trapping of larvae in the mesenteric lymph node could be found despite direct larval migration through this organ. Injected newborn larvae were retained in the lungs, and small numbers could be recovered 24 hr after intravenous injection. We conclude that a proportion of newborn larvae recirculates within the vasculature for several hours; a smaller population extravasates but can reenter the circulatory system via the lymphatics. Furthermore, some newborn larvae are found in organs rich in capillaries up to 24 hr after their entry into the blood.     

STUDIES ON THE REGULATION OF LYMPHOCYTE PRODUCTION IN THE MURINE THYMUS AND SOME EFFECTS OF A CRUDE THYMUS EXTRACT

Cell proliferation in the murine thymus was studied in vivo under normal conditions and from 0 to 24 hr after a single injection of a water-soluble extract from mouse thymus, mouse spleen, and mouse skin. The thymus extract reduced during the first 24 hr the mitotic activity 40%; the spleen extract had a weaker inhibitory effect. The skin extract had no such effect. The thymus extract and spleen extract inhibited the flux of cells into the S phase 0–8 hr after the injection of the extract. Initial labelling index was also reduced in this period. Eight hours after injection of the thymus or spleen extracts the inhibited cells initiated DNA synthesis. The rate of progression of blast cells through the cell cycle was normal 24 hr after the injection of the extracts. It was deduced from the analysis that the thymus extract inhibits processes triggering Go/Gi cells into DNA synthesis, the inhibition of G₂ efflux being of minor importance. Finally a model for the regulation of proliferating thymic blast cells and the emigration of small lymphocytes from the thymus is proposed.     

Sequential metabolic events and morphological changes during in vivo large granular lymphocyte activation and proliferation

Interferon (IFN) and IFN inducers are known to boost natural killer (NK) activity in vivo and in vitro. In vivo enhancement of NK activity results from activation of preexisting NK cells as well as from an increased number of large granular lymphocytes (LGL), with a portion of them undergoing cell division. Our study was addressed to analyze the sequence of metabolic events occurring within the LGL population of Fischer rats treated with poly(I:C), as an IFN inducer. The increase in cytotoxic activity and LGL number in the peripheral blood already reached maximal levels by 12 hr after poly(I:C) injection, remained on a plateau 24 to 48 hr later, then slightly decreased on Day 4, and returned to control levels by Day 6. A similar kinetics was observed for RNA synthesis. In contrast DNA synthesis first increased at 24 hr, peaked at 48 hr, then decreased on Day 4, and was not detectable on Day 6. Percoll fractionation resulted in 92-97% of LGL in fraction 1, and cells in this fraction accounted for the increase of cytotoxicity as well as for newly synthesized RNA and DNA. However, LGL recovered on Day 1 or 2 after poly(I:C) stimulation displayed quite heterogeneous morphology, and a number of mitotic configurations were seen on Day 2 within the LGL population. Our results indicate that the boosting of NK activity by poly(I:C) is always associated with an increase in LGL numbers, the enhanced lytic capacity is associated in vivo with new RNA synthesis by the NK cells, and only in a later phase NK cell proliferation may account for the increase in LGL numbers.     

Structure and development of the larval visual system in embryos of Lytta viridana leconte (coleoptera,meloidae)

At hatching (252–264 hr. at 25 ± 0.5°C), the visual system in larvae of Lytta viridana consists of paired stemmata, stemmatal nerves, optic neuropiles, and inner and outer imaginal optic lobe anlagen. It originates between 64 and 72 hr. with invagination of an optic lobe primordium in the side of each protocephalic lobe. These primordia later differentiate into protocerebral ganglion cells and the imaginal optic lobe anlagen. Each stemma arises at 72 hr. from epidermis below and behind the optic lobe invagination and subsequently becomes cupshaped, closes over, and differentiates. At hatching, it consists of a planoconvex corneal lens, a corneagenous layer, and an everse retina of numerous, pigmented retinular cells, each with a terminal rhabdomere. Between 96 and 104 hr, proximal ends of the retinular cells grow posteromedially into a transverse, horizontal fold in the posterior wall of each optic lobe invagination and along its length to the protocerebral neuropile, which they contact by 112 hr. As the brain withdraws posteriorly within the head, these axons elongate correspondingly. Sheath cells of stemmata and stemmatal nerves descend either from protocerebral perineurium or the optic lobe primordia. Structure and development of the larval visual system in L. viridana are compared with those of other insects and its various components are shown to be homologous throughout the Insecta. However, the stemmata of this insect more closely resemble the atypical imaginal eyes of male scale insects than the photoreceptors of other holometabolous larvae–a similarity arising through convergence.     

Suppression of rabbit lymphocyte functions by antibodies specific for allotypic membrane determinants

Regulation of immunoglobulin synthesis and secretion was analyzed by exposing spleen cells of b4b4 rabbits to anti-b4 for 24 hr in culture. As noted previously, no lymphocytes with membrane-bound b4 were found immediately after pulse treatment, but substantial regeneration of membrane Ig (mIg) occurred on further culture in antibody-free medium. Splenocytes cultured either in the presence or absence of anti-b4 showed a marked loss of Ig-secreting cells (ISC) after 24 hr in culture but recovered and exhibited peak numbers of ISC on Day 2. However, ISC formation in cultures of antibody-treated cells was significantly suppressed and thereafter declined at a more rapid rate than in control cultures. Polyclonal B cell activators from Nocardia and from gram-negative bacteria stimulated ISC formation in cultures of normal spleen cells, but responsiveness to these activators was depressed following antibody treatment. Antibody-induced suppression of Ig synthesis was attributed to interference with differentiation of B lymphocytes to the secretory stage.     

In Vitro Studies on the Mechanism of Acquired Resistance to Tuberculous Infection

The relationship between lymphocytes and macrophages in cellular immunity against tuberculous infection was studied by means of an in vitro cell culture system without addition of streptomycin. The peritoneal macrophages were obtained from normal mice or mice immunized with heat-killed tubercle bacilli in paraffin oil, boosted with live BCG and infected with H37Rv cells in vitro. The infected monolayers of macrophages were cultivated for 48 hr with immune lymphoid cells obtained from immunized mice. The intracellular growth of H37Rv cells 3,5 and 7 days after infection was examined by counting tubercle bacilli within infected macrophages under a microscope. 1) The increase of bacilli within macrophages derived from immunized mice was slightly smaller than that in normal macrophages. 2) The addition of immune lymph node cells to the macrophage monolayers resulted in a marked decrease in the number of bacilli within both normal and “immune” macrophages. Conversely, normal lymph node cells exhibited an enhancing effect on the intracellular bacillary growth. 3) Immune lymph node cells showed a higher capacity to cause macrophages to suppress intracellular growth of bacilli than that of splenic lymphoid cells or thymocytes after addition to macrophage monolayers. 4) The treatment of lymphoid cells with inhibitors of protein synthesis, cycloheximide or streptovitacin A, resulted in a remarkable reduction of the ability of sensitized lymphocytes to cause macrophages to suppress multiplication of intracellular bacilli.     

Effect of calcium-binding protein regucalcin on Ca2+ transport system in rat liver nuclei: stimulation of Ca2+ release

The basis for the hypersensitive response of glycogen phosphorylase to epinephrine stimulation was investigated in adult rat cardiomyocytes isolated from normal and alloxan-diabetic animals. To assess potential G-protein involvement in the response, normal and diabetic derived myocytes were incubated with either cholera or pertussis toxin prior to hormonal stimulation. Pretreatment of cardiomyocytes with cholera toxin resulted in a potentiated response to epinephrine stimulation whereas pertussis toxin did not affect the activation of this signaling pathway. To determine if the enhanced response of phosphorylase activation resulted from an alteration in adenylate cyclase activation, the cells were challenged with forskolin. After 3 hr in primary culture, diabetic cardiomyocytes exhibited a hypersensitive response to forskolin stimulation relative to normal cells. However, after 24 hr in culture, both normal and diabetic myocytes responded identically to forskolin challenge. The present data suggest that a cholera toxin sensitive G-protein mediates the hypersensitive response of glycogen phosphorylase to catecholamine stimulation in diabetic cardiomyocytes and this response which is present in alloxan-diabetic cells and is induced in vitro in normal cardiomyocytes is primarily due to a defect at a post-receptor site.     

A SIMPLE METHOD FOR PRODUCING DIFFERENT GROWTH FRACTIONS IN VITRO FOR USE IN ANTI-CANCER DRUG STUDIES

A simple technique has been used experimentally to produce in vitro Chinese hamster ovary cells with growth fractions ranging from 0 to 100%. Known numbers of exponentially growing and plateau-phase tissue culture cells were mixed in various proportions to yield the desired final growth fraction. Cells attach to the culture flask surface within 1 hr of mixing. Treatment at that time with the nitrosourea compounds, CCNU and MeCCNU, resulted in differential drug survival sensitivities that were dependent upon the growth fraction of the population treated.     

HORMONAL CONTROL OF PEROXIDASE ACTIVITY IN CULTURED PELARGONIUM PITH

Freshly excised Pelargonium pith tissue lacks peroxidase activity toward guaiacol or benzidine, but it develops such activity within 24–36 hr in aseptic culture. All the activity is manifested as a single enzyme moving toward the cathode during electrophoresis on starch gel at pH 9.0. This development of peroxidase activity is at first (up to ca. 50 hr in culture) inhibited and later (ca. 100–150 hr in culture) promoted by IAA. This dual effect of IAA resembles that previously reported for specific isoperoxidases in tobacco pith cells. Kinetin alone also inhibits peroxidase formation, but in the presence of IAA those concentrations which enhance growth enhance peroxidase formation as well.     

The participation of nitric oxide in peritoneal exudate cell cytotoxicity of mice by Fusobacterium nucleatum

Previously we reported that mice infected recurrently with live Fusobacterium nucleatum(Fn) synthesize a significant amount of NO between 12 hr and 24 hr after Fn injection. Fn is a gram-negative rod periodontal pathogen. NO could not be induced by heat-killed Fn or in untreated mice. This NO, derived from the iNOS after infection of live Fn, was not involved in the Fn reduction because Fn clearance occurs within 6 hr. We investigated in this study whether this NO was involved in cytotoxicity in peritoneal exudate cells (PEC) in vivo. The mice were divided into two groups: those treated with live Fn (immune) and those left untreated (normal). PEC number, NO production, detection of apoptosis or death cells, and lactate dehydrogenase (LDH) release activity after injection of live Fn were compared in these groups. In the immune group, the increase of the total cell numbers caused by an increase in neutrophils, a significant NO production only after injection of live Fn at 24 hr and identification of iNOS positive macrophages were confirmed. The apoptotic rate was very low and did not increase at 24 hr in vivo. Therefore, apoptosis was seldom relevant to the NO. In the immune group, LDH activity was remarkable high at 24 hr, and dead cells and macrophages phagocytizing cell fragments increased at the same time. Pretreatment of L NMMA, an inhibitor of iNOS, suppressed LDH activity and cell death. Therefore, the NO derived from the iNOS is involved in the cytotoxicity. These results suggest that NO may contribute to the inflammatory response during Fn infection in periodontitis.     

HYDROXYUREA EFFECTS IN THE C3H MOUSE: I. DUODENAL CRYPT CELL KINETICS

Duodenal crypt cell kinetics in C3H mice have been studied before and after the injection of a single dose (3 mg/g body weight) of hydroxyurea (HU). This was done by autoradiographic analysis of crypt cells which had been labeled with tritiated 5-iodo-2'-deoxyuridine. This dose of HU kills the cells which are synthesizing DNA at the time of injection, inhibits DNA synthesis completely for 4–5 hr, and causes a partial synchronization of the cells when they recover from the inhibitory effects of HU. Duodenal crypt recovery is manifested by a decrease in the mean cell cycle time, an increase in the proliferating fraction, and a lengthening of the crypts. The acute cellular responses are apparently complete within 24–48 hr, but the length of the crypt has not returned to normal by 48 hr after HU administration.     

Kinetics of long-term (72 hr) calcium content during mitogen activation of cultured human T lymphocytes

In vitro stimulation of human blood lymphocytes with mitogen resulted in an increased intracellular content of Ca2+ per unit cell volume. This increase in Ca2+ content of lectin-activated cells reached a maximum after 24 hr of culture and thereafter slowly declined. Brief treatment of cells at 24 hr of culture with the Ca2+ ionophore A23187 in combination with EGTA resulted in a larger release of Ca2+ from cells in mitogen-stimulated cultures than from cells in control cultures. This indicates that the Ca2+ is accumulated intracellularly but is readily exchangeable. At 24 hr of culture the increase in cellular Ca2+ correlated well with the proliferative response as measured by 3H-thymidine incorporation. Ca2+ influx at 24 and 48 hr of culture was markedly enhanced in the mitogenically stimulated cells as compared either to cells cultured for 1 and 72 hr or cells cultured without mitogen.     

The ability to achieve meiotic maturation in the dog oocyte is linked to glycolysis and glutamine oxidation

We tested the hypothesis that meiotic competence of dog oocytes is tightly linked with donor follicle size and energy metabolism. Oocytes were recovered from small (<1 mm diameter, n = 327), medium (1–<2 mm, n = 292) or large (≥2 mm, n = 102) follicles, cultured for 0, 24, or 48 hr, and then assessed for glycolysis, glucose oxidation, pyruvate uptake, glutamine oxidation, and nuclear status. More oocytes (P < 0.05) from large follicles (37%) reached the metaphase‐II (MII) stage than from the small group (11%), with the medium‐sized class being intermediate (18%; P > 0.05). Glycolytic rate increased (P < 0.05) as oocytes progressed from the germinal vesicle (GV) to MII stage. After 48 hr of culture, oocytes completing nuclear maturation had higher (P < 0.05) glycolytic rates than those arrested at earlier stages. GV oocytes recovered from large follicle oocytes had higher (P < 0.05) metabolism than those from smaller counterparts at culture onset. MII oocytes from large follicles oxidized more (P < 0.05) glutamine than the same stage gametes recovered from smaller counterparts. In summary, larger‐sized dog follicles contain a more metabolically active oocyte with a greater chance of achieving nuclear maturation in vitro. These findings demonstrate a significant role for energy metabolism in promoting dog oocyte maturation, information that will be useful for improving culture systems for rescuing intraovarian genetic material. Mol. Reprod. Dev. 79: 186–196, 2012. Published 2011. This article is a U.S. Government work and is in the public domain in the USA.     

Characterization of the Melanoplus sanguinipes hemolymph after infection with Beauveria bassiana or wounding

• 1.1. The phenoloxidase activity, protein and carbohydrate levels were studied for 24 hr in the hemolymph of the migratory grasshopper, Melanoplus sanguinipes after artificial wounding of the insect cuticle or the injection of Beauveria bassiana conidia.
• 2.2. Injection or wounding induced a primary response and phenoloxidase activity was found to increase within 10–60 min. The values for phenoloxidase activity in viable B. bassiana-injected insects exhibited a secondary response, i.e., an increase 24 hr after injection.
• 3.3. In wounded insects and those injected with inactivated conidia, the phenoloxidase activity receded after the initial increase and remained at low levels.
• 4.4. Protein concentrations in the hemolymph increased immediately after infection and wounding and returned to basal levels during the course of the experiment.
• 5.5. Injection of viable B. bassiana resulted in a gradual increase in the protein concentrations between 12 and 24 hr.
• 6.6. There was no apparent change in the carbohydrate levels in either B. bassiana-infected or wounded insects.
• 7.7. These results are discussed in relation to their possible role(s) and interrelationships in the immune response to infection or wounding. Furthermore, we suggest that a “factor” is released after mechanical injury of the integument.

     

Cumulus cell function during bovine oocyte maturation,fertilization, and embryo development in vitro

Several contemporary micromanipulation techniques, such as sperm microinjection, nuclear transfer, and gene transfer by pronuclear injection, require removal of cumulus cells from oocytes or zygotes at various stages. In humans, the cumulus cells are often removed after 15–18 hr of sperm-oocyte coincubation to assist the identification of the fertilization status. This study was designed to evaluate the function of cumulus cells during oocyte maturation, fertilization, and in vitro development in cattle. Cumulus cells were removed before and after maturation and after fertilization for 0,7,20, and 48 hr. The cumulus-free oocytes or embryos were cultured either alone or on cumulus cell monolayers prepared on the day of maturation culture. Percentages of oocyte maturation, fertilization, and development to cleavage, morula, and blastocyst stages and to expanding or hatched blastocysts were recorded for statistical analysis by categorical data modeling (CATMOD) procedures. Cumulus cells removed before maturation significantly reduced the rate of oocyte maturation (4–26% vs. 93–96%), fertilization (0–9% vs. 91–92%), and in vitro development at all stages evaluated. Cumulus cells removed immediately prior to in vitro fertilization (IVF) or 7 hr after IVF reduced the rates of fertilization (58–60% and 71%, respectively, vs. 91–92% for controls), cleavage development (40–47% and 53–54% vs. 74–78% for controls), and morula plus blastocyst development (15% and 24% vs. 45%, P < 0.05). Cumulus cell co-culture started at various stages had no effect on fertilization and cleavage development but significantly improved rates of embryo development to morula or blastocyst stages (P < 0.05). Cumulus cell removal at 20 hr after IVF resulted in similar development to controls (P > 0.05) at all stages tested in this study. The intact state of surrounding cumulus cells of oocytes or embryos appears to be beneficial before or shortly after insemination (at or before 7 hr of IVF) but not essential at 20 hr after IVF. © 1995 Wiley-Liss, Inc.     

Estrogen action in the mouse uterus: An additional nuclear event

An $\text{in vivo}$ injection of ³H-estradiol to an ovariectomized mouse resulted in the appearance of two increases of nuclear receptor: the first at 1 hr and the second occurring at approximately 7–8 hrs post-injection. These findings were substantiated by quantifying cytoplasmic and nuclear receptor levels with the exchange assay after injections of unlabeled estradiol. Cytosol receptor levels were depleted at 0.5–1 hr and replenished from 2–10 hr with values at 15–24 hr significantly greater than those at zero time. Nuclear receptors decreased precipitously to near control values after the initial translocation event. At 7–8 hr a second smaller nuclear increase occurred which then declined to near zero levels. Both of these nuclear events appear to be dose related. Similar experiments using cycloheximide did not abolish either of these events, but did significantly diminish the cytosol receptor replenishment.     

Regeneration of appendages in the large milkweed bug, Oncopeltus fasciatus

An analysis was made of the regeneration of legs and antennae of Oncopeltus. Amputations were performed on first instar larvae within 24 hr after hatching, and on later instars within 24 hr after ecdysis. The resulting regenerates were then measured at each instar. When amputations were performed soon after hatching, there was no significant effect on the duration of any instar. The regenerate was usually visible after the second post-operative ecdysis, and was smaller than a normal appendage (hypomorphic). Removal of the three distal segments of the antenna usually resulted in regeneration of only one segment which was abnormally long and showed a combination of the bristle patterns characteristic of the two most distal segments of the control. In a few such cases a partial intersegmental membrane was present in the regenerated segment. Removal of the tarsus resulted in a structurally complete regenerate which was smaller than the control tarsus. The largest leg regenerates were obtained when amputation was performed through the tibia. With amputation through the femur, a decrease in length of the remainder of this segment was observed after the first ecdysis. This type of amputation and amputation through the trochanter in some cases resulted in the formation of a globular stump containing tarsal claws. The results indicate that amputation of part of an appendage in Oncopeltus does not stimulate an increased growth rate in the stump, but merely causes reorganization of the stump material which subsequently grows at the normal rate. Since even the most hypomorphic regenerates contained well-formed claws, even though proximal parts were missing, it appears that the reorganization process must begin at the most distal point and proceed proximally.     

Effect of in vivo administration of different adjuvants on the in vitro candidacidal activity of mouse peritoneal cells

The candidacidal activity (CA) of peritoneal cells (PC) in vitro was used as a measure of nonspecific microbicidal activity of phagocytes after intraperitoneal injection of mice with different adjuvants. Dilutions of PC were incubated with constant numbers of C. parapsilosis in a 96-well culture plate. The PC number causing 50% reduction of yeast colonies formed after 48 hr at 37 degrees C was called 1 CA50 unit. CA was expressed in CA50 units per 10(6) PC. Optimal reduction of the number of viable candida cells in vitro was established within 1.5 hr while 50% reduction was reached after 0.5 hr. In this test CA was, within limits, independent of the number of viable candida cells added per well (22 to 152 yeast cells), of the concentration of fetal calf serum (1-20%) and of the presence of heat-labile serum components. The CA of PC of individual mice was measured 6, 24, and 96 hr after injection of an adjuvant. In most instances optimal CA was observed 6 hr after administration of adjuvant and varied from 3.7 (methylamine) to 50 (Corynebacterium parvum strain 4982) units. With respect to the titer and duration of CA, the adjuvants were arranged in the following order of increasing efficacy: methylamine, heparin, polyol L 121, suramin, dextran sulfate, polyol L 101, dimethyldioctadecylammonium bromide, Liquoid, heat-killed Listeria monocytogenes, formalin-killed C. parvum strain 10387, and strain 4982. The CA induced by the latter strain persisted at least till 96 hr after injection. The induction of CA was accompanied by recruitment of polymorphonuclear cells. The contribution of distinct phagocytic effector cells to CA and the correlation between modulation of the specific and nonspecific immunity are discussed.     

The Growth of the Emt6 Tumour In the Lungs of Balb C Mice Following Intravenous Inoculation of Tumour Cells From Culture

The growth of the EMT6 tumour in the lungs of Balb C mice has been studied following intravenous inoculation of different numbers of tumour cells taken from culture. At various times after injection of cells into mice, cell suspensions have been prepared from pairs of lungs and the number of in vitro colony forming cells assayed by plating into petri dishes. Following intravenous injection of 10⁵ cells, the time required for doubling of the number of clonogenic tumour cells appearing in the cell suspension is around 17 hr until such time that the total tumour cell population per set of lungs reaches 10⁸ cells (at 10–12 days). This doubling time has to be corrected for changes in ability to extract cells from the lungs into the cell suspension at various times and also for possible changes in plating efficiency in vitro. When these correction factors are applied, the most likely value for the doubling time of clonogenic tumour cells in the lungs is in the range 20–24 hr. This is a similar figure to that previously deduced for the EMT6 flank tumour during its microscopic period of growth. After reaching a total size of 10⁸ tumour cells, the time for doubling of the number of clonogenic tumour cells in the lung increases. During the later stages of tumour growth a good correlation is seen between total lung tumour weight and the number of clonogenic cells present. For the final 3–4 days of the initial period of rapid tumour growth, it is possible to carry out a haemocytometer count of tumour cells in the lung suspension and hence surviving fraction experiments may be carried out after various forms of treatment. In this way the response to treatment of microscopic tumour foci may be determined.     

The Effect of Some Factors on the Cell Volume of Rumen Ciliates

The effect of diluting rumen samples with water and that of formol fixation, on the volume of ophryoscolecid rumen ciliates, was studied. The dilution with water caused within 2 hrs. the ciliates to swell to an average of 60 % (15.4–107.3). During 15 days of formol fixation, the shrinkage was on an average 10 % (0.0–19.8) of the original volume. When the thickness of the cells was conceived equal to the width, volumes 35–77 % too large were obtained. The influence of these sources of error on calculations of rumen fauna volumes is discussed.