Cytoplasmic vacuolization responses to cytopathic bovine viral diarrhoea virus

Bovine Viral Diarrhea Virus (BVDV) is a positive sense, single-stranded RNA virus which exhibits two biotypes in standard cell culture systems. The cytopathic strains of this virus (cpBVDV) induce dramatic cytoplasmic vacuolization in cell cultures, while infection with the non-cytopathic (NCP-BVDV) strains produces no overt changes in the host cells. Our results show that extensive cytoplasmic vacuolization is the earliest morphological change in response to cpBVDV infection in MDBK cells. Cells with extensive vacuolization showed no co-existing chromatin condensation, caspase activation, or loss of membrane integrity. In addition, the caspase inhibitor (zVAD-fmk), although improving cell viability of infected cells from 6.7+/-2.2% to 18.8+/-2.2%, did not prevent vacuolization. On the ultrastructural level, the virus-induced cytoplasmic vacuoles are single membrane structures containing organelles and cellular debris, which appear capable of fusing with other vacuoles and engulfing surrounding cytoplasmic materials. LysoTracker Red which marks lysosomes did not stain the virus-induced cytoplasmic vacuoles. In addition, this lysosomal dye could be observed in the cytoplasm of vacuolized cells, suggesting a lysosomal abnormality. Our data demonstrate that cpBVDV induced a novel cell death pathway in MDBK cells that is primarily associated with lysosomal dysfunction and the formation of phagocytic cytoplasmic vacuoles, and this mode of cell death is different from apoptosis and necrosis.     

内体和溶酶体来源的胞质空泡研究进展

细胞应对细菌或病毒感染及来自环境的各种化学药物时常表现为在细胞质内形成大量空泡,其中有些空泡来源于内体和溶酶体,称为内体和溶酶体的空泡化.空泡的形成过程及分子机制复杂,有些细胞形成空泡时还伴随着细胞死亡,但空泡化与细胞死亡的关系仍不明确.因而对内体和溶酶体空泡化进行研究,以便更好地理解细胞对外源刺激的应激反应及其分子机制.     

Ultrastructural studies on the replication of herpes simplex virus in PK and XTC-2 cells

Ultrastructural changes showed the following characteristics of restricted replication of herpes simplex virus 1 (HSV 1) strains MA and HSZP in PK and XTC-2 cells: 1) minimal cytopathic changes in PK cells as compared to more pronounced alterations in XTC-2 cells; 2) formation of single nucleocapsids or their absence in the nuclei of PK cells infected with the HSZP strain; 3) lack of budding and envelopment and absence of reduplication of the nuclear membrane; 4) persistence of partially uncoated virions within the endocytic vacuoles in the cytoplasm of PK cells; and 5) formation of dense inclusion bodies in addition to the presence of defective virions in the cytoplasm of XTC-2 cells and vacuolation of their cytoplasmic membranes. The replication of HSV 1 in PK and XTC-2 cells seemed to be blocked at both early and late stages of virus replication. At low multiplicity of infection, no virus particles were formed.     

Cytoplasmic vacuoles: Clue in the fine‐needle aspiration diagnosis of melanoma

For the fine‐needle aspiration (FNA) diagnosis of melanoma recognition of characteristic morphologic features is key. In our practice we noted that cytoplasmic vacuoles in Romanowsky‐stained FNA smears of melanoma appeared to be a frequent finding. To investigate this premise, we examined 36 consecutive melanoma FNA cases that had both adequate Papanicolaou‐stained and Romanowsky‐stained smears in an effort to determine the prevalence, and thereby, the potential diagnostic utility of cytoplasmic vacuoles in the diagnosis of melanoma compared to established cytomorphologic parameters of melanoma. We found pigment in 21 cases (58%) and plasmacytoid cells in 28 cases (78%), and in all 36 cases (100%) we found bi/multinucleation, intranuclear cytoplasmic inclusions and prominent nucleoli. Cytoplasmic vacuoles were present in 30 cases (83%) and were found in air‐dried Romanowsky‐stained smears only. These findings suggest that in FNA smears of melanoma cytoplasmic vacuoles in Romanowsky‐stained smears have prevalence and potential diagnostic utility that are comparable to widely recognized cytomorphologic features of melanoma. Numerous articles have focused on the cytomorphologic criteria for the FNA diagnosis of melanoma, but few have noted the presence of cytoplasmic vacuoles in FNA smears of melanoma, and rare reports suggest this finding to be a useful clue to the FNA diagnosis of melanoma. This report appears to be the first to focus on the prevalence and potential diagnostic utility of cytoplasmic vacuoles in FNA samples of melanoma. Diagn. Cytopathol. 2014;42: 143–146. © 2013 Wiley Periodicals, Inc.     

Cytoplasmic vacuoles of Rous virus transformed cells are organelles involved in cation uptake.

Cytoplasmic vacuoles induced during transformation of cells by Bryan strain Rous sarcoma virus (RSV-BH) have been studied using the cationic dye, neutral red(NR). Both the rate of uptake and the accumulation of NR are greater in RSV-BH transformed cells than non-transformed cells however, uptake was greater in vacuolated than in non-vacuolated cells, whether or not they were transformed. The NR was incorporated into pre-existing vacuoles in the absence of cytoplasmic staining, suggesting the existence of direct channels from the cell surface to the vacuoles. Other low mol. wt. cationic dyes could also be incorporated into vacuoles, although those with branched structures or cationic weights greater than 330 were excluded. No anionic dyes were incorporated. Infection of cells with a virus mutant, RSV-BH-Ta, induces temperature-dependent vacuolization. After a shift to the vacuole-permissive temperature, vacuoles developed at different rates and with morphological variations with different cations. Vacuoles which had formed in the presence of several cations, (K+, Rb+, tris+, choline+) failed to disappear when cells were incubated at a temperature sufficient to revert vacuoles formed in Na+-containing medium. No short-term effects of Cl-replacements (Br-, I-, or SO2-4) on vacuolization or reversal were observed. The results suggest that these vacuoles are organelles involved in cation uptake. A possible function for these organelles in RSV-BH induced malignancy is discussed.     

Nucleolar and cytoplasmic RNA density-concentration in leukemia granulocytic progenitors in human bone marrow biopsies: A short cytochemical note

The present study was undertaken to provide more information on the differentiation and maturation of human granulocytes using computer-assisted image RNA densitometry at single-cell level. The bone marrow of patients suffering from chronic phase of chronic myeloid leukemia represents a very convenient model for such measurements because of the satisfactory number of early stages, as well as advanced stages, of the granulocytic cell lineage represented by neutrophils. In contrast to the erythroid cell lineage, similar nucleolar and cytoplasmic RNA density-concentration values were found only in early granulocytic progenitors such as myeloblasts and promyelocytes. In advanced stages of the granulocytic development starting with myelocytes, these cells were characterized by a larger decrease in the cytoplasmic RNA concentration in comparison with that of the nucleoli. Thus, the nucleolar to cytoplasmic RNA concentration ratio in these cells was above 1. On the other hand, it should be pointed out that late differentiation stages of granulocytes, starting with myelocytes, possessed nucleolar bodies (nucleoli without surrounding perinucleolar chromatin) of a markedly reduced size.     

Cytoplasmic sequestration phenomena in smooth muscle cells of kidney resistance vessels and epithelioid cells of the juxtaglomerular apparatus

Summary The occurrence of vacuoles in cells of contractile tissues and especially in media cells of resistance vessels has been known for quite some time. Recently, it has been widely accepted that these vacuoles, characteristically lined by a double membrane, result from herniation of one vascular smooth muscle cell into the other as a result of vasoconstriction. In our electronmicroscopic investigations we found double membrane-bounded vacuoles not only in kidney resistance vessels of rats and mice under conditions of vasoconstriction, but also in control animals and animals with maximal renal vasodilation. Part of our observations are compatible with the assumption that such vacuoles arise from a damage of club-shaped, musculo-muscular contacts due to shape changes of media cells during maximal vasoconstriction or vasodilation. However, serial thin sectioning revealed that some of the cytoplasmic vacuoles have no connections with neighbouring cells. This finding and various parallels to the generation of autophagic vacuoles indicate that the so-called herniations may also represent demarcations of large cytoplasmic areas within an individual cell. Irrespective of the origin of these vacuoles, their contents show different stages of deterioration. At later stages, the vacuoles appear to be adjacent, with only one membrane, to the extracellular space, into which they are believed to discharge finally. Cytoplasmic vacuolization has not only been observed in smooth muscle cells, but also in juxtaglomerular epithelioid cells of the afferent arteriole. Here the vacuoles - besides other organelles - also contain secretory granules; it is therefore proposed that autophagic phenomena with final extrusion of cytoplasmic material may be involved in the programmed down-regulation of the granular renin store following inhibition of renin synthesis and secretion.These studies were supported by the German Research Foundation within the Forschergruppe Niere, Heidelberg     

Light and electron microscopic analysis of human testicular spermatozoa and spermatids from frozen and thawed testicular biopsies.

The morphological changes caused by freezing and thawing human testicular spermatozoa have been assessed here. Retrieval of testicular biopsies was carried out on six patients with obstructive azoospermia preparatory to intracytoplasmic sperm injection (ICSI). Light microscope analysis was carried out on testicular cells and ultrastructural analysis was carried out on spermatozoa and different spermatid stages before and after the freezing procedure. Upon examination under light microscopy, all germ cells presented increased vacuolization in their cytoplasm and shrinkage or swelling of the nuclei and cytoplasmic membranes. These altered structures were accentuated in the spermatocyte I cell which often presented disrupted membranes. The ultrastructural findings under transmission electron microscopy demonstrated that after freezing and thawing the major types of cryoinjury were the swelling and rupture of inner and outer acrosomal and plasma membranes. The acrosome material often appeared as dispersed material or as condensed spots or was even lost. Such damage was observed mainly at the spermatozoa and late spermatid stages. We conclude that the freezing and thawing of testicular biopsies causes similar morphological damage to testicular spermatozoa and frozen-thawed ejaculated spermatozoa. It is still unclear whether these changes in testicular spermatozoa after freezing and thawing may compromise its use in the ICSI procedure.     

Ultrastructural alterations induced by the neolignan dihydrobenzofuranic eupomatenoid-5 on epimastigote and amastigote forms of Trypanosoma cruzi

Eupomatenoid-5, a compound isolated from leaves of Piper regnellii var. pallescens, showed antiprotozoal activity against the epimastigote proliferative stages and intracellular amastigote forms of Trypanosoma cruzi Y strain. Eupomatenoid-5, at 7.0 μg/ml (50% growth inhibition concentration) produced morphological changes in epimastigote forms of the parasite, such as intense cytoplasmic vacuolization, mitochondrial swelling, kinetoplast alteration, presence of myelin-like figures, and mitochondrial damage, as observed by transmission electron microscopy. In amastigote forms in LLCMK₂ cells, at 7.0-μg/ml concentration, the compound induced a decrease in the number of cells with internalized parasites and in the number of internalized parasites per LLCMK₂ cell, compared with untreated cells. Furthermore, intense cytoplasmic vacuolization and autophagic vacuoles were observed in T. cruzi intracellular amastigotes after 72 h of incubation. Scanning electron microscopy confirmed the alterations in the shape of the parasites.     

Vacuolar degeneration of the cochlea in young rats

In the course of investigations of the post-natal development of the ear in Sprague-Dawley rats, a hitherto unrecorded degenerative process was seen in two animals (7-and-8-days old). The process was characterized by vacuolization of almost all the cellular elements of the cochlear tissues. The overall shape and size of the cochlear structures remained well preserved. The organ of Corti did not atrophy but its components were vacuolized, as well as the cells of other inner ear tissues, except for nuclei in the lower part of the modiolus.     

Lipid-rich carcinoid tumor of the thymus gland: diagnosis by fine-needle aspiration biopsy

In this report we describe the cytologic features of an unusual thymic carcinoid tumor containing prominent cytoplasmic lipid vacuoles that was diagnosed by fine-needle aspiration biopsy. The tumor presented as a presternal subcutaneous mass in an 81-yr-old female, with a contiguous mediastinal mass on computed tomography. The cytomorphologic features included numerous discohesive cells with eccentric, round to oval nuclei, granular chromatin, and scant cytoplasm containing numerous intracytoplasmic, clear vacuoles. The neoplastic cells were reactive for neuroendocrine markers by immunocytochemistry and showed reactivity of the intracytoplasmic vacuoles with an oil red-O stain for neutral lipid. Ultrastructural examination confirmed the presence of intracytoplasmic lipid vacuoles and neurosecretory granules. Subsequent surgical excision confirmed the diagnosis. We believe this to be the first report describing these features in a primary thymic carcinoid tumor. The differential diagnosis of mediastinal tumors with clear-cell features and cytoplasmic vacuolization is also discussed.     

Retroperitoneal PEcoma (angiomyolipoma) composed exclusively of epithelioid lipoblastic-like cells: a diagnostic conundrum on needle biopsy

Angiomyolipomas are one of several distinct entities that belong to the “PEComa” (perivascular epithelioid cell tumour) family that comprise varying degrees of fatty, smooth muscle and vascular components. Angiomyolipomas occurring in the retroperitoneum are relatively uncommon; however, when all of the three components are represented, the diagnosis can be readily made. Herein, we report the first case of a retroperitoneal angiomyolipoma composed exclusively of epithelioid and lipoblast-like cells. An extra-renal retroperitoneal mass was biopsied and revealed a relatively uniform population of epithelioid cells arranged in sheets separated by both a delicate vascular network and vessels of larger calibre. The tumour cells were small to medium in size with mild nuclear pleomorphism and cytoplasmic vacuolization ranging from multiple large vacuoles to larger vacuoles, resembling lipoblasts. Occasional nuclei were seen to have prominent clear nuclear pseudo-inclusions. Immunohistochemistry revealed the cells to be strongly positive for SMA and focally positive for HMB45, MiTF and S100. The tumour cells were negative for AE1/AE3, CAM5.2, CD45, Pax8, SF-1 and TFE3. Angiomyolipomas in the retroperitoneum share morphological similarities with a wide variety of tumours, chiefly liposarcoma. Awareness of angiomyolipomas composed purely of epithelioid and lipoblast-like cells is essential to avoid misdiagnoses.     

Studies on the progestational endometrium of the rabbit. II. Electron microscopy,day 0 to day 13 of gonadotrophininduced pseudopregnancy

The fine structure of the endometrial epithelium of the pseudopregnant rabbit from the day of induced ovulation (day 0) to the 13th day is here correlated with previously defined light microscopic phases. In Phase 1 (0–1 day), in which there is a presumed “priming” of the endometrium by ovarian steroidal hormones, no changes were observed. In Phase 2 (1–3 days), in addition to mitotic activity, the epithelium showed a disappearance of the mucification and lymphocytic migration typical of Phase 1 and also of the nonpregnant or “estrous” phase, and showed other nuclear and cytoplasmic changes which probably reflect endogenous growth and protein synthesis. In Phase 3 (4–6 days), two distinct populations of reacting cells were present: (1) surface and cryptal cells investing the now folded mucosal surface, and (2) glandular cells. The first group showed characteristic dome-like protrusions of the cytoplasm into the lumen, and also showed distinct cytoplasmic and nuclear changes which appear to be a prelude to the succeeding phase of fusion but are not necessarily secretory in type. The glandular cells, in contrast, showed cytoplasmic changes which appear to reflect active secretory activity (hypertrophy of the Golgi area, cytoplasmic vacuoles containing electron-opaque material, etc.). This phase coincides with the maximal secretion of uterine-specific proteins, and electron-opaque material is abundant within the endometrial lumen. In Phase 4 (6–8 days), the surface and cryptal epithelium undergoes a transformation into multinucleated cells, the result of a process of lysis of intervening plasma membranes, the precise mechanism of which (i.e., with or without initial membrane fusion) was not determined. Cell fusion proceeded earlier and more actively mesometrially than antimesometrially. The glandular cells showed evidence of reduced secretory activity, but did not at any stage undergo multinucleate-cell transformation. In Phase 5 (8–13 days) there was progressive fusion, and the number of nuclei per cytoplasmic sac appeared increased, presumably due to the continued action of progesterone which is maximal during this phase. Glandular cells showed further reduced secretory activity but remained columnar. Ciliation of the epithelium was sporadic in the pre-secretory phases and rare or absent in the secretory and fusion phases; it became widespread during the phase of decline after day 14, a period which will not be included in this study. The fine structure of the ciliated cells was the same at all stages; there was no evidence for their origin from a reserve population; it is possible that they arise by modification of the multinucleated cells. Cytoplasmic crystals and intramitochondrial densities or lamellae were observed during the secretory and fusion stages, the former only in the glands, the latter in the surface and cryptal epithelium. They appear to be associated with rising or maximal progesterone secretion.     

Ultrastructural studies of the envelopment and release of guinea pig herpes-like virus in cultured cells

The process of envelopment and release of guinea pig herpes-like virus was examined in both infected guinea pig kidney and thymus tissue culture cells by electron microscopy. The majority of the nucleocapsids were enveloped by budding into nuclear vacuoles; some were enveloped by budding from the inner nuclear membrane. Budding into cytoplasmic vacuoles was also seen. Many enveloped virus particles inside the nuclear vacuoles were pear shaped with a tail-like structure. Approximately 23% of pear-shaped virus particles were seen in the infected thymus fibroblastic cells, but only 6% were found in the infected epithelial cells. The envelopes of all nuclear enveloped virus particles appeared as smooth membranes, while the majority of particles exhibiting fuzzy and thick dense envelopes were seen in the cytoplasm or extracellular space. The average diameter of the cytoplasmic or extracellular enveloped virus particles was approximately 167 nm, and the average diameter of the nuclear enveloped virus particles was about 146 nm.Data also showed that mature nuclear virus particles were first released into perinuclear cisterna and then traveled through cytoplasmic channels to the extracellular space.     

Elektronenmikroskopische Untersuchungen der Leber von Maus und Ratte bei akuter Phalloidin-Vergiftung

Zusammenfassung Die Struktur der Leberzellen wurde an der Maus nach subcutaner Injektion von 10 Phalloidin/g Körpergewicht und an der isolierten, durchströmten Leber von Ratten nach Zusatz von 20 mg Phalloidin zur Perfusionslösung zu Zeitpunkten zwischen 10 und 100 min nach der Vergiftung elektronenmikroskopisch untersucht.Die Strukturveränderungen sind in beiden Versuchsanordnungen gleich. Die Vergiftung führt über eine zunehmende Erweiterung der Zisternen des endoplasmatischen Reticulums zum Auftreten großer, fettfreier, fibrinhaltiger Vacuolen, die schließlich alle Leberzellen durchsetzen. Etwas später finden sich autolytische Vacuolen, in denen Cytoplasmabestandteile eingeschlossen werden. In der Umgebung autolytischer Vacuolen treten herdförmige Cytoplasmanekrosen auf, die teilweise durch eine Membran abgegrenzt werden. Die Kernund Mitochondrienstruktur bleibt intakt.Die Strukturveränderungen sind nicht spezifisch für die Phalloidinwirkung. Die peribiliären Lysosomen sind an der Vacuolisierung des endoplasmatischen Reticulums nicht beteiligt; umschriebene Cytoplasmanekrosen in der Umgebung autolytischer Vacuolen könnten jedoch durch einen Austritt von Hydrolasen nach Schädigung der Membran der autolytischen Vacuolen verursacht sein.

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Electron microscopic studies of mouse and rat hepatic cells after acute phalloidin poisoning

Summary The fine structure of liver cells was investigated (a) in mice at time points between 10 and 90 mins after subcutaneous injection of 10 phalloidin/g body weight, and (b) in the isolated perfused livers of rats at time points between 10 and 100 mins after addition of 20 mg phalloidin to the perfusate. The structural alterations were identical in both experiments. The cisternae of the endoplasmic reticulum were dilated in the early stages of intoxication and later turned into large vacuoles which were free of fat but contained fibrin; in the later stages all cells became vacuolated. Autolytic vacuoles sequestering cytoplasmic constituents appeared later than the changes of the reticulum. Foci of cytoplasmic necrosis, partially surrounded by a membrane, were found in the vicinity of autolytic vacuoles. The structure of nuclei and mitochondria remained unaltered. The structural changes were not specific for phalloidin poisoning. Peribiliary lysosomes were not involved in the vacuolization of the endoplasmic reticulum. Foci of cytoplasmic necrosis in the vicinity of autolytic vacuoles could have been caused by leakage of hydrolases through the damaged membrane of the autolytic vacuoles.

     

Development of an Equine Herpesvirus in Two Cell Culture Systems: Light and Electron Microscopy

Development of equine herpesvirus strain 82A was studied in cells from primary horse kidney (HOK) cultures and an equine dermis (ED) cell strain. HOK and ED cells are equally susceptible to the 82A virus infection and yield about the same amount of infectious virus. Intranuclear inclusions were present in both cell systems, but a ring-shaped syncytial formation was observed only in infected ED cells. Ultrastructural studies revealed the presence of dense granules 30 nm in diameter and characteristic star-like clusters of granules in the infected HOK cells, but these granules were rarely seen in the infected ED cells. Viral nucleocapsids were associated with homogenous nuclear matrices, with moderate electron density in both cell systems. Viral nucleocapsids acquired envelopes by budding into the nuclear vacuoles in both HOK and ED cells. Budding from inner nuclear membranes into perinuclear cisterna or into cytoplasmic vacuoles also was observed frequently in HOK cells but was not seen often in infected ED cells. Multiple, membrane-bound intranuclear inclusions of fibrillar material which may be associated with virus envelopes were seen only in infected ED cells. Enveloped virus particles seen in nuclear vacuoles or perinuclear cisterna were more regular in shape and had a 130-nm diameter, whereas the enveloped virus particles seen in the cytoplasm and extracellular space were more irregular in shape and had a 130- to 160-nm diameter.     

The cytopathology and development of a human polyoma virus (B.K.)

Summary Light and electron microscopic studies on foetal human fibroblast and glial cells infected with B. K. virus show a cytopathogenic effect similar to but not identical with that caused by polyoma and SV 40 viruses in murine and monkey cells, respectively. The first cytopathogenic effects appear at approximately 14 days and are complete at about 30 to 40 days. Light microscopy of unstained monolayers demonstrates roughening of the cell surface, liberation of exudate, cytoplasmic vacuolization and rounding of cells. Stained preparations show the same features and a series of nuclear changes consisting of light eosinophilic patches in the nucleoplasm, chromatin clumping and the development of a typical inclusion body. The nucleolus is not involved.Electron microscopy of the cells reveals virus crystals in intact nuclei followed by breakdown of the nuclear membrane, clumping of chromatin into dense masses and liberation of the viruses which often become associated with cytoplasmic membranes. Mass liberation of virus crystals occurs by disruption of the cell membrane. Liberation of virus particles from cells with intact nuclei occurs occasionally. Budding from the nucleus or cell membrane was not observed.     

VACUOLATED MENINGIOMA

Two unusual cases of meningioma with extensive vacuolization have been studied by light and electron microscopy. There were two kinds of vacuoles in the tumor cells which had the characteristic ultrastructure of meningioma as well. The smaller intracytoplasmic vacuoles were lipid droplets, while the larger, more prominent vacuoles were found to be extracellular spaces probably containing plasmatic fluid. The tumor cells were very much stellate with extremely thin and long cytoplasmic processes having desmosome junctions and forming cavernous intercellular spaces, some of which contained collagen fibers and fibrils. Although the xanthomatous change has been well known, the latter features provide a reasonable interpretation for the histology of the present tumors and fat-negative vacuoles in the ordinary meningiomas. The picture may be recapitulation of the subarachnoid structure. Furthermore, recognition of this type of meningioma is practically important especially in frozen section diagnosis not to misinterpret the tumor as liposarcoma, chordoma or metastatic adenocarci-noma. ACTA PATH. JAP. 27: 557–566, 1977.