Large Amounts Of Cellular Debris Research Articles

Avian mycobacteriosis in a naturally infected captive nocturnal curassow (Nothocrax urumutum)

A senile female nocturnal curassow (Nothocrax urumutum) kept under human care was found dead. Grossly, the lungs were congested and had a nodule with 2.0 cm in diameter adhered to the left lung. Histologically, there were multifocal to coalescent areas of necrosis characterized by deposition of amorphous and eosinophilic material with large amount of cellular debris, surrounded by numerous epithelioid macrophages, multinucleated Langhans giant cells, occasional lymphocytes and plasma cells. Ziehl-Neelsen stained sections demonstrated myriad of intralesional alcohol-acid resistant bacilli (BAAR). DNA extracted from lung samples were evaluated by nested polymerase chain reaction resulting in mycobacterial DNA identification. This is the first reported case of mycobacteriosis in a nocturnal curassow.

AUTOMATED AND MANUAL SEMEN ANALYSIS: THE COMPARATIVE CHARACTERISTICS

Currently, spermogram parameters are routine criteria for assessing male fertility. There are two methods of semen analysis: traditional manual and automated. The concentration of spermatozoa of various motility categories is important in predicting physiological conception, as well as in choosing fertilization methods in assisted reproductive technology programs. The introduction of automatic semen analyzers has shown that their use can be considered as an alternative to the routine manual analysis method, which can contribute to laboratory standardization. Initially, these devices demonstrated difficulty in accurately indicating sperm concentration due to the presence of sperm aggregation and large amounts of cellular debris. In the present study, the main focus was on the analysis of sperm concentration by manual and automated methods. A total of 50 sperm samples were analyzed from patients participating in assisted reproductive technology programs. Manual analysis was performed in a Makler's chamber according to the standard method in 10 small squares. The motility of each spermatozoon was classified into categories. Automated analysis was carried out using the CASA sperm analyzer computer analysis system (MICROPTIC, Spain), which uses the principle of microscopic imaging and processing to detect motile and immobile spermatozoa through fast and consistent images. Statistical methods for independent variables were chosen to evaluate the obtained data. As part of the study, by the method of determining confidence intervals, a statistically significant difference was found between automated and standard or manual methods of analysis when evaluating spermatozoa with the highest speed of 0,025 mm/sec and rectilinear and translational movement, as well as with a lower speed, either aging or with broken morphology. Most likely, this is due to the objective difficulty of visual assessment of spermatozoa of such motility categories. The data obtained suggest that automated analysis has a higher degree of objectivity in assessing mobile biological objects, in particular male germ cells.

Necrotizing Tracheobronchitis Caused by Aspergillus fumigatus in a Cow

This report describes a case of tracheobronchitis caused by Aspergillus fumigatus in a cow. A 4-year-old Jersey cow was submitted for necropsy examination. Grossly, the mucosa of the trachea and bronchi was diffusely reddened and was covered by a moderate amount of yellow, caseous material, sometimes with small foci of grey 'cotton-like' appearance, suggestive of filamentous fungus. Microscopical analysis of the trachea and bronchi showed marked diffuse mucosal and submucosal necrosis with a large amount of cellular debris, fibrinous exudation and inflammatory infiltration dominated by macrophages and neutrophils. There were large numbers of septate fungal hyphae with acute bifurcation angles and conidia and vesicles typical of Aspergillus spp. that were stained by the Grocott method. A. fumigatus growth in mycological culture from the trachea and bronchi was confirmed by molecular identification. Tracheobronchitis caused by A. fumigatus infection in cattle is poorly described and requires further research to improve the therapeutic management of the disorder.

Radiofrequency ablation of liver metastasis: potential impact on immune checkpoint inhibitor therapy.

Percutaneous radiofrequency ablation (RFA), a generally accepted alternative therapy for patients with liver metastases, is a minimally invasive approach with a favorable safety profile and a lower rate of major complications. The use of RFA or combined RFA plus resection can produce total tumor clearance in patients with unresectable liver metastases. However, the relatively high rate of local tumor progression has prevented the widespread use of RFA. Furthermore, its efficacy is controversial because there have been no comparisons for its effect on overall survival compared with standard options such as systemic chemotherapy. Meanwhile, immunotherapy has become a major research focus for oncology based on the recent successes reported for immune checkpoint inhibitors for melanoma, non-small cell lung cancer, gastric cancer, and other cancers. Immune checkpoints negatively regulate T cell function, and inhibition prevents the blockade of the immune system by cancer cells to prevent their destruction. Unfortunately, only some patients (< 25%) respond to immuno-oncology drugs, whereas other patients acquire resistance. However, RFA can induce massive necrotic cell death which might activate immunity and the presentation of cryptic antigens to induce tumor-specific T cell response. Because RFA can induce the rapid release of large amounts of tumor antigens, it can potentially stimulate transient immune responses to much tumor antigens. Combination therapies have induced synergistic enhancement of anticancer immune response in preclinical studies, indicating great promise for the future of oncologic treatment.Key Points • Only some patients respond to immuno-oncology drugs. • RFA causes the release of large amounts of cellular debris, a source of tumor antigens that elicit immune responses against tumors. • Combination RFA for liver metastases and immune checkpoint inhibitor therapies might synergistically enhance antitumor immunity.

Automated enumeration and viability measurement of canine stromal vascular fraction cells using fluorescence-based image cytometry method.

In recent years, the lipoaspirate collected from adipose tissue has been seen as a valuable source of adipose-derived mesenchymal stem cells for autologous cellular therapy. For multiple applications, adipose-derived mesenchymal stem cells are isolated from the stromal vascular fraction (SVF) of adipose tissue. Because the fresh stromal vascular fraction typically contains a heterogeneous mixture of cells, determining cell concentration and viability is a crucial step in preparing fraction samples for downstream processing. Due to a large amount of cellular debris contained in the SVF sample, as well as counting irregularities standard manual counting can lead to inconsistent results. Advancements in imaging and optics technologies have significantly improved the image-based cytometric analysis method. In this work, we validated the use of fluorescence-based image cytometry for SVF concentration and viability measurement, by comparing to standard flow cytometry and manual hemocytometer. The concentration and viability of freshly collected canine SVF samples are analyzed, and the results highly correlated between all three methods, which validated the image cytometry method for canine SVF analysis, and potentially for SVF from other species.

Neurotoxicity of coral snake phospholipases A2 in cultured rat hippocampal neurons

The neurotoxicity of two secreted Phospholipases A2 from Brazilian coral snake venom in rat primary hippocampal cell culture was investigated. Following exposure to Mlx-8 or Mlx-9 toxins, an increase in free cytosolic Ca2+ and a reduction in mitochondrial transmembrane potential (ΔΨm) became evident and occurred prior to the morphological changes and cytotoxicity. Exposure of hippocampal neurons to Mlx-8 or Mlx-9 caused a decrease in the cell viability as assessed by MTT and LDH assays. Inspection using fluorescent images and ultrastructural analysis by scanning and transmission electron microscopy showed that multiphase injury is characterized by overlapping cell death phenotypes. Shrinkage, membrane blebbing, chromatin condensation, nucleosomal DNA fragmentation and the formation of apoptotic bodies were observed. The most striking alteration observed in the electron microscopy was the fragmentation and rarefaction of the neuron processes network. Degenerated terminal synapses, cell debris and apoptotic bodies were observed among the fragmented fibers. Numerous large vacuoles as well as swollen mitochondria and dilated Golgi were noted. Necrotic signs such as a large amount of cellular debris and membrane fragmentation were observed mainly when the cells were exposed to highest concentration of the PLA2-neurotoxins. PLA2s exposed cultures showed cytoplasmic vacuoles filled with cell debris, clusters of mitochondria presented mitophagy-like structures that are in accordance to patterns of programmed cell death by autophagy. Finally, we demonstrated that the sPLA2s, Mlx-8 and Mlx-9, isolated from the Micrurus lemniscatus snake venom induce a hybrid cell death with apoptotic, autophagic and necrotic features. Furthermore, this study suggests that the augment in free cytosolic Ca2+ and mitochondrial dysfunction are involved in the neurotoxicity of Elapid coral snake venom sPLA2s.

Nonessential Role of β3 and β5 Integrin Subunits for Efficient Clearance of Cellular Debris after Light-Induced Photoreceptor Degeneration

During light-induced photoreceptor degeneration, large amounts of cellular debris are formed that must be cleared from the subretinal space. The integrins alphavbeta5 and alphavbeta3 are involved in the normal physiological process of phagocytosis in the retina. This study was conducted to investigate the question of whether the lack of beta5 and/or beta3 integrin subunits might influence the course of retinal degeneration and/or clearance of photoreceptor debris induced by acute exposure to light. Wild-type, beta5(-/-) and beta3(-/-) single-knockout, and beta3(-/-)/beta5(-/-) Ccl2(-/-)/beta5(-/-) double-knockout mice were exposed to 13,000 lux of white light for 2 hours to induce severe photoreceptor degeneration. Real-time PCR and Western blot analysis were used to analyze gene and protein expression, light- and electron microscopy to judge retinal morphology, and immunofluorescence to study retinal distribution of proteins. Individual or combined deletion of beta3 and beta5 integrin subunits did not affect the pattern of photoreceptor cell loss or the clearance of photoreceptor debris in mice compared with that in wild-type mice. Invading macrophages may contribute to efficient phagocytosis. However, ablation of the MCP-1 gene did not prevent macrophage recruitment. Several chemokines in addition to MCP-1 were induced after light-induced damage that may have compensated for the deletion of MCP-1. Acute clearance of a large amount of cellular debris from the subretinal space involves invading macrophages and does not depend on beta3 and beta5 integrins.

Intrastriatal grafts of rat colonic smooth muscle lacking myenteric ganglia stimulate axonal sprouting and regeneration.

Grafts of living or freeze-killed freshly dissected colonic smooth muscle from young inbred Fischer rats were implanted into the corpus striatum of adult Fischer rats. Sections of brain were examined electron microscopically 3 and 6 wk after implantation. At both times, living grafts were vascularised and contained healthy differentiated smooth muscle cells, fibroblasts, interstitial cells of Cajal and some macrophages. Large bundles of small nonmyelinated axons, identified as CNS axonal sprouts, could be observed in the brain at and near the interface between the living smooth muscle and the CNS tissue. Bundles of regenerating CNS axons, often associated with astrocyte processes, had grown into the grafts. Some axons within the grafts had matured, enlarged and become myelinated by oligodendrocyte processes or Schwann cells. In some cases, smooth muscle cells were observed in close and intricate association with axons. In contrast to the living grafts, grafts of freeze-killed smooth muscle, examined 3 and 6 wk after implantation, contained macrophages, fibroblasts, collagen and large amounts of cellular debris, but no living muscle cells, astrocytes or Schwann cells. The striatal neuropil around freeze-killed grafts did not contain large bundles of CNS axonal sprouts and bundles of axons were not observed within the freeze-killed graft. This study demonstrates that cells from the smooth muscle layers of the colon, in the absence of myenteric ganglia, can stimulate a vigorous regenerative response from CNS axons when implanted into the corpus striatum of adult rats.

Cellular evaluation of bovine nuclear transfer embryos developed in vitro.

Cloned blastocysts developed in vitro for 7 d had a mean number of cells (82.86 +/- 5.35) as evaluated by nuclei counting in serial optical sections using confocal microscopy, after staining with propidium iodide. This number was not significantly different from that of control IVF embryos cultured under the same conditions during the same period (mean = 88.89 +/- 7.53). Semi-thin sections revealed that most of the blastocysts had an inner cell mass (10/12) and a blastocoele. Under transmission electron microscopy, the trophectoderm appeared well differentiated as a polarized epithelium with apical microvilli and lateral junctions including desmosomes with bound intermediate filaments. The cytoplasm sometimes contained immature mitochondria or a large number of residual bodies. About half of the blastocysts examined had a large amount of cellular debris in the perivitelline space or inside the blastocoele cavity. The cloned blastocysts were also able to hatch in vitro by day 8 and SEM indicated a normal morphology of the trophectoderm cells with numerous apical microvilli. The high number of excluded or degenerating cells found in some embryos may partially explain early embryonic mortality that follows transfer. However, these observations do not give a clear explanation for the high incidence of fetal losses.

Flow cytometric study of nodular fasciitis, proliferative fasciitis, and proliferative myositis

DNA ploidy was assessed retrospectively, using flow cytometry, in 13 nodular fasciitis (NF) lesions, three proliferative myositis (PM) lesions, one proliferative fasciitis lesion, and 12 other benign fibrous lesions (BFLs). All were diploid. In view of the large amounts of cellular debris, cell cycle analysis was possible in only seven NF lesions, three PM lesions, and six BFLs. The mean percentage of S phase did not differ markedly between the combination of NF and PM lesions (6.6%) and BFLs (7.1%); the mean percentage of G2+M phase (5.4%) of the NF PM lesion combination was twice as large as that of the BFLs (2.5%). No correlation was detected between the cell cycle analysis and the mitotic count, the predominant histologic type in NF lesions, or the predominant stroma in PM lesions.

Integrated study of the structural and metabolic degeneration of skin during 4 °C storage in nutrient medium

Integrated studies of the structural and metabolic degradation of skin during 4 °C refrigerated storage in Eagle's minimal essential medium were undertaken. Skin degeneration occurred in three phases during the first 7 weeks of refrigeration. Phase I (Days 1–10) was characterized by the release of cellular debris and leakage of intracellular enzymes into the storage medium, shrinkage of the epithelium, and vacuolization and cell loss in the vascular bundles of the papillary dermis. The release of debris was accelerated by a 2-hr incubation of the skin at 25 and 37 °C prior to 4 °C storage. The ability of dermal cells to metabolize glucose, aspartic acid, glutamic acid, ornithine, and orotic acid to carbon dioxide dropped to low levels during Phase I. Phase II (Days 11–30) was characterized by a period of less intense physical decay, but with continued cellular and vascular degeneration. By the third week, there was nearly total loss of recognizable vascular bundles in the papillary dermis. Phase III (Days 31–58) was characterized by the exfoliation of large amounts of cellular debris and significant loss of structural integrity. By the fifth week, nearly all of the nuclei in the epidermis became pyknotic, and the vascular bundles of the reticular dermis were lost. Previous reports of the maximum allowable duration of skin storage at 0–8 °C have ranged from 0.3 to 185 days; however, our findings suggest that the useful limit of refrigerated skin storage in nutrient medium is 1 week if physiological function and structural integrity are desired for optimum postgraft performance.

Regeneration by dissociated adult Hydra cells: a histologic study.

Adult Hydra attenuata with vitally stained gastrodermal cells were dissociated into their component cells which were then randomly reaggregated into pellets by low-speed centrifugation. Representative examples of these preparations, which develop into normal adult hydra if left undisturbed, were examined fresh at low magnification and at higher magnification in fixed, stained, and sectioned specimens. The actual pellet stage lasts less than 1 hour because the adult ectodermal and gastrodermal cells rapidly sort themselves into an inner and outer layer and seem to secrete a new mesoglea immediately thereafter. The "embryo" becomes trilaminar and attains a central cavity by extruding a large amount of cellular debris at the end of the first day. At about this same time, new tentacles begin to differentiate from rapidly dividing and undifferentiated interstitial cells. Regulation of tentacle number and position occurs at the end of two days, and the body form is essentially reestablished within 60 hours by further differentiation of the hypostomes and body wall. Complete separation of the preparation into individual polyps does not occur until about 190 hours of development.

The expression of the gene asebia in the laboratory mouse. I. Epidermis and dermis.

SUMMARYMice homozygous for the asebia mutation (ab/ab), which have defective sebaceous glands, display abnormalities in several other aspects of the integument. Histological sections showed that hyperplasia of the cellular layers and the stratum corneum of the epidermis is apparent at birth and increases markedly with age. Enlarged intercellular spaces are also noted in the epidermis. The thicker dermal layer of the asebic mice is characterized by increased vascularity, increased cellularity and the abnormal morphology of a large proportion of the ‘fibroblast’ population. Electron microscopy demonstrated the many abnormalities in the dermal fibroblasts as well as large amounts of cellular debris in the surrounding matrix. Collagen and elastin show alterations at the light microscopic and ultrastructural levels. Many features of the asebic dermis resemble those found with mild inflammation and with the lysosomal storage diseases. Changes in the dermis of asebic foetuses were noted prior to epidermal alterations and may mediate the latter.

Regulation of the bacterial microflora of the vagina in cyclic female rats.

The bacterial microflora was examined in the vagina of cyclic female rats kept under normal laboratory conditions. Large variations occurred during the cycle with high numbers of bacteria (10(5)-10(8) per vagina) during proestrus and estrus and low numbers (10(1)-10(4) per vagina) during the diestrus period. Histological analysis of in situ vaginal tissue and transplanted vaginal tissue revealed an association of high bacterial numbers with the presence of large amounts of cellular debris in the vaginal lumen during the period of epithelial keratinization. Absence of phagocytosis in leucocytes at mestestrus suggested that leucocytes did not play an active role in reduction of bacterial numbers between estrus and metestrus. Accurate measurement of the pH in the vaginal lumen failed to reveal differences which could explain the reduction in bacterial numbers between estrus and metestrus. The cyclic changes in the bacterial population-consisting of species which are normally present in the intestinal flora-- seem to be controlled by cyclic changes in the amounts of cellular debris in the vaginal lumen.

Changes in Small Blood Vessels During Cyclical Luteal Regression in Sheep

Changes in small blood vessels of corpora lutea (CL) undergoing cyclical regression were studied by light and electron microscopy and the progesterone content of some CL was assayed. Vascular changes were 1st identified at Day 14 of the estrous cycle were consistently present by Day 16 and were observed in all CL in which progesterone content was less than 10 mcg/gm luteal tissue. Vascular changes involved both the luminal contents and walls of capillaries and small venules. Many capillaries contained degenerating cellular fragments incorporating nuclear and cytoplasmic remnants together with blood cells. The walls of many vessels containing cellular fragments appeared normal. In others abnormalities observed included folding of the basal lamina increase in the size and number of gaps in the endothelium increased endothelial cell projection into the lumen the inclusion of hetero- and/or autophagic vacuoles in the cytoplasm of many endothelial cells and pericytes and endothelial cell degeneration leading to ultimate disintegration of affected vessels. By Day 1 of the following cycle the number of capillaries was much reduced. It is concluded that at a relatively early stage of luteolysis many luteal capillaries show changes involving obstruction of the lumina by large amounts of cellular debris at least a part of which is of endothelial origin. The relationship of these changes to other phenomena associated with luteolysis is discussed.(Authors modified)

Cell degeneration and recovery of the fetal mammalian brain after a chemical insult.

AbstractIn this experiment 12‐day pregnant mice were treated with 5‐fluorodeoxyuridine, an inhibitor of DNA‐synthesis, to examine the influence of the drug on the central nervous system of the embryos. More specifically the sites of cell degeneration, the type of cells affected, and the elimination of cellular debris were investigated.Approximately two hours after treatment with FUdR, mitotic activity in the neocortex ceased and the first degenerating cells became visible in the outer zone of the neuroepithelial layer, e.g., the DNA‐synthetic zone. During the following 8 to 11 hours degenerating cells increased in number and were seen throughout the neuroepithelial layer, suggesting either that other than DNA‐synthesizing cells were affected or that degenerating cells moved from the outer zone towards the inner zone. Since all dying cells were labeled when tritiated thymidine was given shortly before FUdR treatment, it is concluded that DNA synthesizing cells have moved to the lumen despite blockage of DNA synthesis and degeneration of the nucleus. At 24 hours after treatment all degenerating cells were found in the outer region of the neuroepithelial zone, indicating that they had moved from the inner zone (near the lumen) to the outer region. Hence, the degenerating cells did not remain in the DNA‐synthetic zone, but apparently moved or were pushed from the outer to the inner zone, and vice versa.When a search was made for cells cleaning up the debris, relatively few macrophages were seen. In addition only a few normal neuroepithelial cells could be identified as containing phagocytosed material. Many degenerating cells apparently were not phagocytosed but underwent lysis, leaving temporarily large holes in the tissue. It is thus suggested that in the embryo the large amount of cellular debris is eliminated mainly by autolysis, partially by macrophages, and also to some extent by neighboring neuroepithelial cells.Despite the presence of many degenerating cells, a number of mitotic cells were seen 11 hours after treatment, indicating that the drug had ceased to act. During the following 24 hours the neuroepithelial zone is reconstituted, but few if any neuroblasts are formed. Hence, despite the extensive repair which follows the action of a chemical compound, a permanent shortage of neurons results.

CHANGES IN THE SHAPE AND SIZE OF BACTERIUM COLI AND BACILLUS MEGATHERIUM UNDER THE INFLUENCE OF BACTERIOPHAGE—A MOTION PHOTOMICROGRAPHIC ANALYSIS OF THE MECHANISM OF LYSIS

This paper contains the records of a motion photomicrographic investigation of the lysis of Bact. coli and B. megatherium by bacteriophage. The bacteria mixed with bacteriophage were grown on moist nutrient agar in small culture chambers on the stage of a microscope in an incubator maintained at 37 degrees C. The apparatus used permitted continuous inspection of the preparations. Photographs were made at the rates of 2 and 30 per minute and at the rate of 8 per second during the terminal stage of lysis of Bact. coli. The accurately timed films were studied by rapid projection and by the projection of single frames. Measurements of dimensions of cells, calculations of volumes, information on generations, generation times and duration spans are presented in the tables. Similar information on normal cultures grown and photographed in the same way is furnished for comparison. Groups of serial photographs are reproduced in the plates to illustrate the special features observed. These observations seem to us to warrant the following conclusions: 1. Enlargement or swelling of the cells of Bact. coli usually, but not always, precedes lysis. Some of the enlargement is an expression of increase of cell substance and is not altogether due to imbibition of water. Cells of early generations of Bact. coli enlarge to greater absolute and relative proportions than cells of later generations. Enlargement does not occur before lysis in B. megatherium. 2. The terminal stage of lysis of Bact. coli is explosive, occupying (1/2) to (7/8) second. The terminal stage of lysis of B. megatherium is a slow disintegrative process, extending over 2-10 minutes. 3. Bacteriophage inhibits fission of some cells, but does not stop the reproduction of other cells in contact with it. The genealogical records of six generations of cells of Bact. coli and of two generations of cells of B. megatherium indicate that bacteriophage may be transmitted through parents to the offspring which ultimately undergo lysis. 4. Bacteriophage spreads by contact through a group of cells and also along paths determined by genetical relationships. 5. A large amount of cellular debris remains after the lysis of the cells in both of these species of bacteria. This residue of material is in the form of irregularly shaped masses and granules. This material is not in solution at the time of lysis and appears not to be digested or hydrolized. 6. Theories of the mechanism of lysis are discussed. It is suggested that reduction of surface tension of the cells may be an important factor in the mechanism of lysis.