Coronavirus-associated mucormycosis: different from sinonasal mucormycosis.

OBJECTIVE: To study unusual presentations of coronavirus-associated mucormycosis that are rarely seen in sinonasal mucormycosis cases. METHOD: The data of 400 rhino-orbito-cerebral mucormycosis patients admitted to Sawai Man Singh Hospital, Jaipur, from May 2021 to June 2021, were retrospectively collected. The diagnosis of mucormycosis was made by histological examination of biopsy samples. RESULTS: Out of 400 patients, 62 had symptoms other than common symptoms of rhino-orbito-cerebral mucormycosis. Thirty-four patients had facial palsy, 19 complained of gum ulcers, 6 developed a cheek abscess, 2 complained of maggots in the nose along with common rhino-orbito-cerebral mucormycosis symptoms, and 1 had a cerebellar infarct. CONCLUSION: Mucormycosis is a disease with various presentations, and coronavirus-associated mucormycosis has added unusual presentations to the existing list of manifestations of rhino-orbito-cerebral mucormycosis. In this coronavirus disease era, mucormycosis should always be considered as a diagnosis in patients with these unusual presentations.

Activation of nuclear factor-kappa B signalling promotes cellular senescence.

Cellular senescence is a programme of irreversible cell cycle arrest that normal cells undergo in response to progressive shortening of telomeres, changes in telomeric structure, oncogene activation or oxidative stress. The underlying signalling pathways, of major clinicopathological relevance, are unknown. We combined genome-wide expression profiling with genetic complementation to identify genes that are differentially expressed when conditionally immortalised human fibroblasts undergo senescence upon activation of the p16-pRB and p53-p21 tumour suppressor pathways. This identified 816 up and 961 downregulated genes whose expression was reversed when senescence was bypassed. Overlay of this data set with the meta-signatures of genes upregulated in cancer showed that nearly 50% of them were downregulated upon senescence showing that even though overcoming senescence may only be one of the events required for malignant transformation, nearly half of the genes upregulated in cancer are related to it. Moreover 65 of the up and 26 of the downregulated genes are known downstream targets of nuclear factor (NF)-κB suggesting that senescence was associated with activation of the NF-κB pathway. Direct perturbation of this pathway bypasses growth arrest indicating that activation of NF-κB signalling has a causal role in promoting senescence.

Transcriptional networks and cellular senescence in human mammary fibroblasts.

Senescence, the molecular program that limits the finite proliferative potential of a cell, acts as an important barrier to protect the body from cancer. Techniques for measuring transcriptome changes and for modulating their expression suggest that it may be possible to dissect the transcriptional networks underlying complex cellular processes. HMF3A cells are conditionally immortalized human mammary fibroblasts that can be induced to undergo coordinated senescence. Here, we used these cells in conjunction with microarrays, RNA interference, and in silico promoter analysis to promote the dissection of the transcriptional networks responsible for regulating cellular senescence. We first identified changes in the transcriptome when HMF3A cells undergo senescence and then compared them with those observed upon replicative senescence in primary human mammary fibroblasts. In addition to DUSP1 and known p53 and E2F targets, a number of genes such as PHLDA1, NR4A3, and a novel splice variant of STAC were implicated in senescence. Their role in senescence was then analyzed by RNA silencing followed by microarray analysis. In silico promoter analysis of all differential genes predicted that nuclear factor-kappaB and C/EBP transcription factors are activated upon senescence, and we confirmed this by electrophoretic mobility shift assay. The results suggest a putative signaling network for cellular senescence.

Conditional immortalization of freshly isolated human mammary fibroblasts and endothelial cells.

Reports differ as to whether reconstitution of telomerase activity alone is sufficient for immortalization of different types of human somatic cells or whether additional activities encoded by other "immortalizing" genes are also required. Here we show that ectopic expression of either the catalytic subunit of human telomerase (hTERT) or a temperature-sensitive mutant (U19tsA58) of simian virus 40 large-tumor antigen alone was not sufficient for immortalization of freshly isolated normal adult human mammary fibroblasts and endothelial cells. However, a combination of both genes resulted in the efficient generation of immortal cell lines irrespective of the order in which they were introduced or whether they were introduced early or late in the normal proliferative lifespan of the cultures. The order and timing of transduction, however, did influence genomic stability. Karyotype analysis indicated that introduction of both transgenes at early passage, with hTERT first, yielded diploid cell lines. Temperature-shift experiments revealed that maintenance of the immortalized state depended on continued expression of functional U19tsA58 large-tumor antigen, with hTERT alone unable to maintain growth at nonpermissive temperatures for U19tsA58 large-tumor antigen. Such conditional diploid lines may provide a useful resource for both cell engineering and for studies on immortalization and in vitro transformation.

Establishment and characterization of equine autonomic ganglion cell lines to enable direct testing of candidate toxins involved in equine dysautonomia (grass sickness).

To enable direct testing of a range of potential toxins or pathogens that might be involved in grass sickness, equine thoracic sympathetic chain ganglion cell lines were established from primary cell cultures by retroviral-mediated transduction of the temperature-sensitive mutant of the establishment oncogene encoding SV40 large T antigen. Morphological and behavioral features, temperature dependence, and immunocytochemical characteristics of the cell lines were investigated. The majority of cells were noradrenergic neurons in which dopamine-beta-hydroxylase, the enzyme that catalyzes norepinephrine synthesis, and neuropeptide Y coexisted. Cells treated with plasma from grass sickness cases that had previously been shown to induce autonomic nervous system damage when injected into normal horses showed significantly decreased mitochondrial function after 1 day. After 3 days exposure most cells showed severe degeneration in contrast to those treated with normal plasma. Liver and lung cell lines were also susceptible to plasma, suggesting that the toxin is not specifically neurotoxic.

BAP37 and Prohibitin are specifically recognized by an SV40 T antigen antibody.

We have identified two cellular proteins that are specifically immunoprecipitated by an anti-SV40 T antigen monoclonal antibody. This antibody, PAb419, recognizes an epitope contained within a region of T antigen which we have recently demonstrated is required for the initiation of immortalization by SV40 T antigen, but is not essential for maintenance of the immortal state. The two proteins were identified as BAP37 and Prohibitin. Recent results suggest Prohibitin may enhance the transcriptional inactivation of E2F by the retinoblastoma family of pocket proteins (pRb, p107, p130). BAP37 and Prohibitin are specifically recognized by PAb419 and PAb210, another anti-SV40 T antigen monoclonal antibody, which has an overlapping epitope, but not by other anti-SV40 T antigen monoclonal antibodies, demonstrating the specificity of the interaction.

Different functions are required for initiation and maintenance of immortalization of rat embryo fibroblasts by SV40 large T antigen.

We have used two different, but complementary assays to characterize functions of SV40 T antigen that are necessary for its ability to immortalize rat embryo fibroblasts. In accordance with previous work, we found that several functions were required. These include activities that map to the p53 binding domain and the amino terminal 176 amino acids which contain the J domain as well as the CR1 and CR2 domain required for binding and sequestering the RB family of pocket proteins. Moreover, we found that even though activities dependent only upon the amino terminus were sufficient for immortalization they were unable to maintain it. This suggests that immortalization by these amino terminal functions requires either additional events or immortalization of a subset of cells within the heterogeneous rat embryo fibroblast population. We further found that an activity dependent upon amino acids 17 - 27 which remove a portion of the CR1 domain and the predicted alpha-1 helix of the J domain was not necessary to maintain growth but was required for direct immortalization suggesting that at least one of the functions required initially was not required to maintain the immortal state. This represents the first demonstration that some of the functions required for maintenance of the immortal state differ from those required for initiation of immortalization.

Cloning and identification of genes that associate with mammalian replicative senescence.

Cellular senescence and limited proliferative capacity of normal diploid cells has a dominant phenotype over immortality of cancerous cells, suggesting its regulation by the expression of a set of genes. In order to isolate the genes that associate with senescence, we have employed a clonal system of conditional SV40 T antigen rat embryo fibroblast cell lines which undergo senescence upon T antigen inactivation. Construction of cDNA libraries from two conditional cell lines and application of differential screening and subtractive hybridization techniques have resulted in the cloning of eight senescence-induced genes (SGP-2/Apo J, alpha 1-procollagen, osteonectin, fibronectin, SM22, cytochrome C oxidase, GTP-alpha, and a novel gene) and a senescence-repressed gene (FRS-2). Three of these genes encode for extracellular matrix proteins, others are involved in the calcium-dependent signal transduction pathways, while the SGP-2/Apo J gene may have a cellular protective function. RNA analysis has shown that the senescence-associated genes are overexpressed in both normal rat embryonic fibroblasts and human osteoblasts cell cultures undergoing aging in vitro. In comparison, the expression of these genes in a rat fibroblast immortalized cell line (208F cells) was down-regulated after both its partial and its full transformation by ras oncogenes. Thus, cloning of senescence-associated genes opens up new ways to elucidate and/or to modulate aging and cancer.

Immortalization of rat embryo fibroblasts by a 3'-untranslated region.

We have exploited a cross-species expression screen to search for cellular immortalizing activities. A newt blastemal cDNA expression library was transfected into rat embryo fibroblasts and immortal cell lines were selected. This identified a 1-kb cDNA fragment which has a low representation in the cDNA library and is derived from the 3'-UTR of an alpha-glucosidase-related mRNA. Expression of this sequence in rat embryo fibroblasts has shown that it is active in promoting colony formation and immortalization. It is also able to cooperate with an immortalization-defective deletion mutant of SV40 T antigen, indicating that it can exert its growth-stimulatory activity in the pathway activated by a viral immortalizing oncogene. This is the first example of an immortalizing activity mediated by an RNA sequence, and further analysis of its mechanism should provide new insights into senescence and immortalization.

Recovery of spatial learning by grafts of a conditionally immortalized hippocampal neuroepithelial cell line into the ischaemia-lesioned hippocampus.

Transient global cerebral ischaemia in rats causes relatively circumscribed and specific damage to the CA1 pyramidal cells of the dorsal hippocampus, along with a cognitive deficit manifest as difficulties in the performance of a range of spatial learning and memory tasks. Our previous studies have shown that restoration of behavioural performance in ischaemic rats by neural grafts taken relatively late in fetal development occurs only after local replacement of cells homotypic to those lost through the ischaemic insult. This lesion-plus-behaviour model therefore offers a powerful means for establishing whether multipotent embryonic neuroepithelial cells will engraft the damaged CA1, develop into appropriate neuronal phenotypes and produce behavioural recovery. Here we report that, in rats subjected to 15 min of global cerebral ischaemia, intrahippocampal implants of a conditionally immortal, multipotent cell line, directly derived from the embryonic day 14 hippocampal neuroepithelium of the H-2Kb-tsA58 transgenic mouse, selectively repopulated the lesioned CA1 pyramidal layer and restored ischaemia-induced deficits in acquisition of a hidden platform location in the Morris water maze.

Expression of p24, a novel p21Waf1/Cip1/Sdi1-related protein, correlates with measurement of the finite proliferative potential of rodent embryo fibroblasts.

Normal mammalian fibroblasts undergo a limited number of divisions when cultured in vitro before entering a state of replicative senescence. The molecular basis for the determination of the finite mitotic potential is not known. Nevertheless, simian virus 40 T antigen, among other oncogenes, is able to prevent senescence in rodent embryo fibroblasts. T antigen immortalized cells are dependent upon this protein for maintaining growth once their normal mitotic life span has elapsed. Even though the mechanism that measures the finite mitotic potential of rodent fibroblasts is not known, it has been shown that it continues to function normally in the presence of this immortalizing gene. Accumulation of cyclin-dependent kinase inhibitors such as p21Waf1/Cip1/Sdi1 could potentially be a component of the mechanism that determines the finite life span. Here we show that accumulation of p21Waf1/Cip1/Sdi1 does not correlate with this biological counting mechanism, but we have identified p24, a p21Waf1/Cip1/Sdi1-related protein, whose accumulation does correlate with the measurement of the finite proliferative potential of rodent embryo fibroblasts and suggest that sequestration might be a mechanism by which its activity is regulated.

Rat embryo fibroblasts immortalized with simian virus 40 large T antigen undergo senescence upon its inactivation.

Introduction of simian virus 40 T antigen into rodent fibroblasts gives rise to cells that can proliferate indefinitely but are dependent upon it for maintenance of their growth once the normal mitotic life span has elapsed. Inactivation of T antigen in these immortalized cells causes rapid and irreversible cessation of growth. To determine whether this growth arrest is associated with entry into senescence, we have undertaken a genetic and biological analysis of conditionally immortal (tsa) cell lines derived by immortalizing rat embryo fibroblasts with the thermolabile tsA58 T antigen. This analysis has identified the following parallels between the tsa cells after inactivation of T antigen and senescent rat embryo fibroblasts: (i) growth arrest is irreversible; (ii) it occurs in G1 as well as G2; (iii) the G1 block can be partially overcome by stimulation with 20% fetal calf serum, but the G2 block cannot be overcome; (iv) 20% fetal calf serum induces c-fos, but c-myc is unaltered; and (v) fibronectin and p21(Waf1/Cip1/Sdi1) are upregulated upon growth arrest. These results suggest that T-antigen-immortalized fibroblasts are committed to undergo senescence but are prevented from undergoing this process by T antigen. Inactivation of T antigen removes this block and results in senescence of the cells. Thus, these cell lines may represent a powerful system for study of the molecular basis of entry into senescence.

Immortalization of epithelial cells.

The methodologies for isolating cell lines have become very powerful, particularly in terms of retaining differentiated features of the parent cells. Cell lines can be developed from primary or early passage cells as well as from transgenic animals that carry an immortalizing gene. Cell lines from epithelia have been selected for their polar orientation, tight junction formation, and expression of differentiated markers or functions. These cell lines provide useful models for studying cell biology of specific tissues, tumorigenicity, genetic abnormalities, or to help screen for effective methods of gene therapy.

Vascular smooth muscle cells of H-2Kb-tsA58 transgenic mice. Characterization of cell lines with distinct properties.

BACKGROUND: The vascular wall is composed of at least two different populations of smooth muscle cells that are distinct in their structure and protein composition. According to the developmental stage of tissue taken for culture, the ratio between cells of epithelioid phenotype and spindle-shaped cells is variable. In particular, the epithelioid cells display characteristic features associated with immaturity. Because their increased appearance can be observed in endothelial denudation, the represent a dedifferentiated, proliferative smooth muscle cell type with a repair function in vascular injury. METHODS AND RESULTS: To investigate this cellular heterogeneity, we established vascular smooth muscle cell lines from H-2Kb-tsA58 transgenic mice. Due to temperature-sensitive expression of the SV 40 large T-antigen in cells derived from this mouse strain, our smooth muscle lines were conditionally immortalized from the onset of their life in culture. Thus, we were able to clone cell lines representing the two different phenotypes described so far. Epithelioid cells derived from newborn animals are characterized by their expression of cytokeratins and the development of tight junctional complexes. Spindle-shaped cells, which could be isolated from newborn or adult animals, corresponded in phenotype and protein expression to smooth muscle cell lines established previously. CONCLUSIONS: The special properties of vascular smooth muscle cells of the epithelioid phenotype suggest an endothelial replacement function in the course of injury to the vascular wall.

The H-2KbtsA58 transgenic mouse: a new tool for the rapid generation of novel cell lines.

The ability to generate expanded populations of individual cell types able to undergo normal differentiation in vitro and in vivo is of critical importance in the investigation of the mechanisms that underly differentiation and in studies on the use of cell transplantation to repair damaged tissues. This review discusses the development of a strain of transgenic mice that allows the direct derivation of conditionally immortal cell lines from a variety of tissues, simply by dissociation of the tissue of interest and growth of cells in appropriate conditions. In these mice the tsA58 mutant of SV40 large T antigen is controlled by the interferon-inducible Class I antigen promoter. Cells can be grown for extended periods in vitro simply by growing them at 33 degrees C in the presence of interferon, while still retaining the capacity to undergo normal differentiation in vivo and in vitro. In addition, it appears that cell lines expressing mutant phenotypes can readily be generated by preparing cultures from appropriate offspring of matings between H-2KbtsA58 transgenic mice and mutant mice of interest.

Roles of hepatocyte growth factor/scatter factor and the met receptor in the early development of the metanephros.

Several lines of evidence suggest that hepatocyte growth factor/scatter factor (HGF/SF), a soluble protein secreted by embryo fibroblasts and several fibroblast lines, may elicit morphogenesis in adjacent epithelial cells. We investigated the role of HGF/SF and its membrane receptor, the product of the c-met protooncogene, in the early development of the metanephric kidney. At the inception of the mouse metanephros at embryonic day 11, HGF/SF was expressed in the mesenchyme, while met was expressed in both the ureteric bud and the mesenchyme, as assessed by reverse transcription PCR, in situ hybridization, and immunohistochemistry. To further investigate the expression of met in renal mesenchyme, we isolated 13 conditionally immortal clonal cell lines from transgenic mice expressing a temperature-sensitive mutant of the SV-40 large T antigen. Five had the HGF/SF+/met+ phenotype and eight had the HGF/SF-/met+ phenotype. None had the HGF/SF+/met- nor the HGF/SF-/met- phenotypes. Thus the renal mesenchyme contains cells that express HGF/SF and met or met alone. When metanephric rudiments were grown in serum-free organ culture, anti-HGF/SF antibodies (a) inhibited the differentiation of metanephric mesenchymal cells into the epithelial precursors of the nephron; (b) increased cell death within the renal mesenchyme; and (c) perturbed branching morphogenesis of the ureteric bud. These data provide the first demonstration for coexpression of the HGF/SF and met genes in mesenchymal cells during embryonic development and also imply an autocrine and/or paracrine role for HGF/SF and met in the survival of the renal mesenchyme and in the mesenchymal-epithelial transition that occurs during nephrogenesis. They also confirm the postulated paracrine role of HGF/SF in the branching of the ureteric bud.

The biological clock that measures the mitotic life-span of mouse embryo fibroblasts continues to function in the presence of simian virus 40 large tumor antigen.

Normal mammalian fibroblasts cultured in vitro undergo a limited number of divisions before entering a senescent phase in which they can be maintained for long periods but cannot be induced to divide. In rodent fibroblasts senescence can be prevented by expression of simian virus 40 large tumor antigen (T antigen). Cells expressing T antigen can proliferate indefinitely; however, such cells are absolutely dependent upon continued expression of T antigen for maintenance of growth; inactivation of T antigen results in a rapid and irreversible entry into a postmitotic state. To determine when, after the initial expression of T antigen, fibroblasts become dependent upon it for continued growth, we serially cultivated embryonic fibroblasts prepared from H-2Kb-tsA58 transgenic mice. We show that these fibroblasts become dependent upon T antigen for maintenance of proliferation only when their normal mitotic life-span has elapsed and that the biological clock that limits the mitotic potential continues to function normally, even in cells expressing this immortalizing gene. Our results suggest that random accumulation of cellular damage is unlikely to be the factor that limits fibroblast division but support the hypothesis that senescence is regulated via a genetic program.

Myogenic cell lines derived from transgenic mice carrying a thermolabile T antigen: a model system for the derivation of tissue-specific and mutation-specific cell lines.

Skeletal myoblasts cloned from limb muscles of H-2Kb-tsA58 transgenic mice remained proliferative through at least 80 generations under conditions permissive for expression and function of the tsA58 gene product. When switched to nonpermissive conditions or implanted into muscles of nude mdx mice they underwent differentiation but, in one clonal cell line, a small proportion appeared to become quiescent muscle precursors in vivo. H-2Kb-tsA58 X mdx/mdx F1 male mice yielded dystrophin-deficient myoblasts. By such simple genetic crosses, H-2Kb-tsA58 transgenic mice provide a valuable tool for the rapid isolation of cell lines, myogenic or otherwise, bearing mutations of interest.

A model for primitive neuroectodermal tumors in transgenic neural transplants harboring the SV40 large T antigen.

Using retrovirus-mediated transfer of the SV40 virus large T antigen into neural transplants, we have observed a high incidence of primitive neuroectodermal tumors (PNET). These neoplasms developed in 8 of 14 (57%) neural grafts after latency periods of 176 to 311 days. Histopathologically, the tumors exhibited features of human PNET such as formation of neuroblastic rosettes and immunocytochemical evidence for neuronal differentiation, synaptogenesis, and focal astrocytic differentiation. All neoplasms showed a striking migratory potential. The presence of the large T gene in the tumors was demonstrated by polymerase chain reaction-mediated amplification of a specific 242 bp segment of large T and DNA sequence analysis. Large T antigen was identified in tissue sections using an immunocytochemical reaction with the monoclonal antibody Pab 108. Cell lines were established from several tumors and subjected to G418 selection. Secondary tumors induced by intracerebral transplantation of these cells retained the characteristic morphological and immunocytochemical properties of PNETs. These experiments demonstrate a considerable transforming potential of SV40 large T antigen for neural precursor cells. The long latency period suggests that neoplastic transformation initiated by the large T gene requires additional spontaneous mutations of cooperating cellular genes. Because the mechanism of transformation by large T antigen appears to involve complex formation with and inactivation of cellular tumor suppressor gene products, these cell lines may serve as an interesting tool to search for novel neural tumor suppressor genes.

The characterization of astrocyte cell lines that display properties of glial scar tissue.

The glial scar has been proposed to be a major impediment to regeneration in the adult CNS. Analysis of glial scars in vivo is complicated, however, by the large number of cell types present in such lesions. We have attempted to simplify analysis of the glial scar environment by deriving a series of conditionally immortal astrocyte cell lines that display several properties expressed by glial scar tissue in vitro. The astrocyte lines, which were derived from H-2KbtsA58 transgenic mice, expressed macromolecules associated with glial scars in vivo and were significantly less effective than neonatal astrocytes at promoting neurite outgrowth from postnatal central and peripheral neurons. The astrocyte lines also inhibited migration of oligodendrocyte type-2 astrocyte progenitor cells in vitro. We propose that certain properties shown previously to be expressed by glial scars may be reconstituted in vitro by astrocytes alone.