Lytic growth of human herpesvirus 8: morphological aspects

The human herpesvirus 8 (HHV-8), also known as Kaposi's sarcoma-associated herpesvirus, is a gamma herpesvirus associated with AIDS-related body cavity-based lymphomas (BCBL), also called primary effusion lymphomas (PEL). These are a rare form of non-Hodgkin lymphomas in which HHV-8 is present, often associated with Epstein-Barr virus (EBV) infection. HHV-8 is also present in a latent state or in a state of low-level persistence in different primary effusion lymphoma-derived cell lines, such BCBL-1 cells, that lack EBV infection. This cell line was induced to produce mature virions by treatment with 12- O -tetradecanoyl phorbol-13-acetate (TPA) and the characteristic ultrastructural features of HHV-8 lytic replication were identified and compared to those of the other members of Herpesviridae family.     

Morphogenesis of HHV8 in Primary Human Dermal Microvascular Endothelium and Primary Effusion Lymphomas

This study elucidates the morphology of HHV8 replication in human dermal endothelial cells and primary effusion lymphomas (PEL) and compares it to that seen in Kaposi sarcoma. Primary human dermal microvascular endothelial cells (DMVEC) exposed to the cell-filtered supernatant of the PEL JSC1 and PEL cell lines (KS-1, BCBL-1, BC-1, BC-3) were cultured in the presence or absence of 12- O -tetradecanoyl-phorbol-13-acetate (TPA) or butyrate. Cells were fixed in neutral-buffered glutaraldehyde, gelled in cooled agar, and processed for TEM. There was a quantitative, but not a qualitative difference in viral expression associated with no treatment or exposureto TPA orbutyrate of HHV8 in DMVEC and PEL. Two types of viral-induced intranuclear inclusions (INI) were visible at the light and ultrastructural levels. The more common INI had lighter staining material filling the nucleus, except for a rim of dense chromatin, and could be seen even beforeviral nucleocapsids (NC) werevisible. The second type of INI resembled a target formed by condensation of electron-dense material surrounded by a lighter halo and marginated heterochromatin and containing NC. Collections of coalescing electron-dense granules resembling starbursts were often present in nuclei containing either type of INI. Next to appear in productively infected cells were mature enveloped particles that formed mostly by the budding of NC into cytoplasmic vacuoles. Mature particles were also seen free on the plasma membrane. Tufts of electron-dense intermediate filaments were associated with maturing particles. Mature virions lacked an electron-dense tegument. Viral production was ultimately associated with cell lysis. It appears that HHV8 propagate in DMVEC, with and without stimulation, and have a similar morphogenesis to that seen in PEL cell lines and Kaposi sarcoma lesions. Several unique features characterize cells productively infected by HHV8.     

Morphological Features of Nerve Terminal Degeneration as Part of the Remodeling Process in the Motor Endplate in Adult Muscles

With the exception of signs of retraction and withdrawal, there have been few morphological data concerning degenerated neural profiles in adult motor endplates. Here, investigation into the ultrastructure of the soleus motor endplates of adult rats (4 months old) turned up particular axonal degeneration in approximately 3% of the subjects. These axons occur as synaptic debris in the synaptic matrix of the motor endplate, adjacent to thin processes of the perisynaptic cells occupying the outer most layer of the motor endplate and were devoid of basal lamina. They often possessed dense-cored vesicles (50-80 nm). Axonal debris released from Schwann cell processes occurred during the period of acute sciatic neurectomy, when nerve terminals progressively disrupted within the motor endplate associated Schwann cells. Finally, immunohistochemical staining for antibodies to label macrophages (ED1 or ED2) has shown that nerve fiber-associated macrophages are located near the motor endplate. The results suggest that during the course of endplate remodeling, a few parts of the terminal branches are disposed of through spontaneous collapse, subsequent release from the Schwann cell investment, and eventual ingestion by macrophages in the perisynaptic space.     

Monofunctional and Polyfunctional CD8+ T Cell Responses to Human Herpesvirus 8 Lytic and Latency Proteins

Human herpesvirus 8 (HHV-8) is the etiological agent of Kaposi''s sarcoma, primary effusion lymphoma, and multicentric Castleman''s disease. It is postulated that CD8⁺ T cell responses play an important role in controlling HHV-8 infection and preventing development of disease. In this study, we investigated monofunctional and polyfunctional CD8⁺ T cell responses to HHV-8 lytic proteins gB (glycoprotein B) and K8.1 and latency proteins LANA-1 (latency-associated nuclear antigen-1) and K12. On the basis of our previous findings that dendritic cells (DC) reveal major histocompatibility complex (MHC) class I epitopes in gB, we used a DC-based system to identify 2 novel epitopes in gB, 2 in K8.1, 5 in LANA-1, and 1 in K12. These new HHV-8 epitopes activated monofunctional and polyfunctional CD8⁺ T cells that produced various combinations of gamma interferon, interleukin 2, tumor necrosis factor alpha, macrophage inhibitory protein 1β, and cytotoxic degranulation marker CD107a in healthy HHV-8-seropositive individuals. We were also able to detect HHV-8-specific CD8⁺ T cells in peripheral blood samples using HLA A*0201 pentamer complexes for one gB epitope, one K8.1 epitope, two LANA-1 epitopes, and one K12 epitope. These immunogenic regions of viral lytic and latency proteins could be important in T cell control of HHV-8 infection.Human herpesvirus 8 (HHV-8), also referred to as Kaposi''s sarcoma-associated herpesvirus, is a gammaherpesvirus that causes Kaposi''s sarcoma (KS), primary effusion lymphoma, and multicentric Castleman''s disease. The importance of developing effective prevention and treatment for HHV-8 infection is evident in that KS, a neoplasm of endothelial origin, continues to be the most common cancer among human immunodeficiency virus (HIV)-infected patients (8). KS is also the leading cause of cancer in children in sub-Saharan Africa (7). Although the incidence of KS in HIV-infected persons declined with the advent of antiretroviral therapy (ART) (10), KS can occur in persons on ART with suppressed HIV infection and high CD4⁺ T cell counts (25).The immune responses responsible for controlling HHV-8 infection and preventing KS are not clear. CD8⁺ T cell immunity likely plays a significant role in HHV-8 infection, as these cells have been shown to be crucial in controlling infection caused by the other human gammaherpesvirus, i.e., Epstein-Barr virus (EBV) (11, 14). In support of this hypothesis, our laboratory (40-42) and others (4-6, 12, 19, 23, 26-28, 31, 32, 36, 37, 43, 44) have shown that CD8⁺ T cells produce gamma interferon (IFN-γ) in response to HHV-8 immunodominant epitopes presented by major histocompatibility complex class I (MHC-I) in HHV-8-seropositive individuals. Little is known whether T cells produce other immune mediators in response to HHV-8 infection. Indeed, polyfunctional T cells, i.e., single cells producing two or more immune mediators, have been linked to control of HIV and other persistent infections (1, 24, 29, 33) and could play a role in controlling HHV-8 infection. In one recent study, HHV-8 epitope-specific, polyfunctional T cells were detected in patients with multicentric Castleman''s disease, but these cells did not differ in number from those in healthy controls (13). Another study has found that patients with controlled KS had HHV-8-specific CD8⁺ T cells that secreted IFN-γ and tumor necrosis factor alpha (TNF-α) but that patients with progressive disease had weaker and less polyfunctional CD8⁺ T cells (2).HHV-8 epitope-specific monofunctional and polyfunctional T cell immunity could be important in development of HHV-8 vaccines that induce T cell responses that target these viral epitopes. In the present study, we therefore investigated CD8⁺ T cell responses to two HHV-8 lytic proteins, gB (glycoprotein B) and K8.1, and two latency proteins, LANA-1 (latency associated nuclear antigen-1) and K12. We previously showed that optimal induction of T cell reactivity to the HHV-8 protein gB required 1 week of stimulation with peptide-loaded, autologous, mature, monocyte-derived dendritic cells (DC) (40). Using this enhanced DC-T cell stimulation system, we now have revealed several new epitopes for these four lytic and latency HHV-8 proteins in healthy HHV-8-seropositive individuals, which induce both monofunctional and polyfunctional CD8⁺ T cells. These regions of HHV-8 could be critical in understanding HHV-8 immunopathogenesis and in vaccine development.     

Morphogenesis of HHV8 in primary human dermal microvascular endothelium and primary effusion lymphomas

This study elucidates the morphology of HHV8 replication in human dermal endothelial cells and primary effusion lymphomas (PEL) and compares it to that seen in Kaposi sarcoma. Primary human dermal microvascular endothelial cells (DMVEC) exposed to the cell-filtered supernatant of the PEL JSC1 and PEL cell lines (KS-1, BCBL-1, BC-1, BC-3) were cultured in the presence or absence of 12- O -tetradecanoyl-phorbol-13-acetate (TPA) or butyrate. Cells were fixed in neutral-buffered glutaraldehyde, gelled in cooled agar, and processed for TEM. There was a quantitative, but not a qualitative difference in viral expression associated with no treatment or exposureto TPA orbutyrate of HHV8 in DMVEC and PEL. Two types of viral-induced intranuclear inclusions (INI) were visible at the light and ultrastructural levels. The more common INI had lighter staining material filling the nucleus, except for a rim of dense chromatin, and could be seen even beforeviral nucleocapsids (NC) werevisible. The second type of INI resembled a target formed by condensation of electron-dense material surrounded by a lighter halo and marginated heterochromatin and containing NC. Collections of coalescing electron-dense granules resembling starbursts were often present in nuclei containing either type of INI. Next to appear in productively infected cells were mature enveloped particles that formed mostly by the budding of NC into cytoplasmic vacuoles. Mature particles were also seen free on the plasma membrane. Tufts of electron-dense intermediate filaments were associated with maturing particles. Mature virions lacked an electron-dense tegument. Viral production was ultimately associated with cell lysis. It appears that HHV8 propagate in DMVEC, with and without stimulation, and have a similar morphogenesis to that seen in PEL cell lines and Kaposi sarcoma lesions. Several unique features characterize cells productively infected by HHV8.     

Xanthogranulomatous appendicitis--an incidental finding of localized pathology

The clinical, histopathological, and electron microscopic features of an unusual case of xanthogranulomatous appendicitis are reported. The patient, a 37-year-old female, presented with typical signs of acute appendicitis and the appendix appeared slightly dilated at laparatomy. The histopathological sections showed numerous xanthoma cells mixed with inspissated fecaliths. Electron microscopy disclosed the presence of xanthoma cells filled with electron-lucent lipid droplets of variable size. The ultrastructural characteristics of these cells enabled the distinction of two types of lipid-laden histiocytes, in relationship to the size of the lipid droplets. Since the lipid droplets were seen also in cells other than histiocytes, it appears that these changes are secondary to a common mechanism,comprising factors such as obstruction, hemorrhage, inflammation,and local hypoxia.     

Morphological features of nerve terminal degeneration as part of the remodeling process in the motor endplate in adult muscles

With the exception of signs of retraction and withdrawal, there have been few morphological data concerning degenerated neural profiles in adult motor endplates. Here, investigation into the ultrastructure of the soleus motor endplates of adult rats (4 months old) turned up particular axonal degeneration in approximately 3% of the subjects. These axons occur as synaptic debris in the synaptic matrix of the motor endplate, adjacent to thin processes of the perisynaptic cells occupying the outer most layer of the motor endplate and were devoid of basal lamina. They often possessed dense-cored vesicles (50-80 nm). Axonal debris released from Schwann cell processes occurred during the period of acute sciatic neurectomy, when nerve terminals progressively disrupted within the motor endplate associated Schwann cells. Finally, immunohistochemical staining for antibodies to label macrophages (ED1 or ED2) has shown that nerve fiber-associated macrophages are located near the motor endplate. The results suggest that during the course of endplate remodeling, a few parts of the terminal branches are disposed of through spontaneous collapse, subsequent release from the Schwann cell investment, and eventual ingestion by macrophages in the perisynaptic space.     

Morphological Evidence of Neutrophil-Tumor Cell Phagocytosis (Cannibalism) in Human Gastric Adenocarcinomas

The phenomenon of neutrophil-tumor cell emperipolesis or phagocytosis has been documented by light microscopy in various human carcinomas, but little is known about the cellular pathological processes and the morphological changes involved. In an attempt to clarify the nature of this phenomenon, the authors' ultrastructural studies on the relationships among neutrophils and tumor cells in human gastric carcinomas are reviewed and analyzed. At the electron microscopy level, apoptotic neutrophils were found within vacuoles of adenocarcinoma cells in 2 cases. They showed either early apoptotic morphology with perinuclear chromatin aggregation but cytoplasm integrity or late apoptotic morphology with uniform, collapsed nucleus and tightly packed cytoplasmic granules. A light microscopy review of 200 cases of resected gastric carcinomas identified 22 cases (11%) that were characterized by neutrophil-tumor cell phagocytosis (cannibalism). TUNEL staining confirmed the presence of apoptotic neutrophils within the cytoplasm of the tumor cells. This study provides light and electron microscopic evidence of apoptotic neutrophils phagocytosed by gastric adenocarcinoma cells. The morphological features of neutrophil-tumor cell phagocytosis (cannibalism) would suggest a particular mechanism of tumor-immune escape in human gastric carcinoma.     

Xanthogranulomatous Appendicitis - An Incidental Finding of Localized Pathology

The clinical, histopathological, and electron microscopic features of an unusual case of xanthogranulomatous appendicitis are reported. The patient, a 37-year-old female, presented with typical signs of acute appendicitis and the appendix appeared slightly dilated at laparatomy. The histopathological sections showed numerous xanthoma cells mixed with inspissated fecaliths. Electron microscopy disclosed the presence of xanthoma cells filled with electron-lucent lipid droplets of variable size. The ultrastructural characteristics of these cells enabled the distinction of two types of lipid-laden histiocytes, in relationship to the size of the lipid droplets. Since the lipid droplets were seen also in cells other than histiocytes, it appears that these changes are secondary to a common mechanism,comprising factors such as obstruction, hemorrhage, inflammation,and local hypoxia.     

Correlation between Enzyme-Linked Immunosorbent Assay and Immunofluorescence Assay with Lytic Antigens for Detection of Antibodies to Human Herpesvirus 8

The purpose of this study was to evaluate, in Kaposi's sarcoma patients, the correlation between antibody titers to the lytic antigens of human herpesvirus 8, as assessed by immunofluorescence assay, and values obtained by an enzyme immunoassay. The methods showed a stringent correlation, r = 0.625 (P < 0.001).     

循环内皮细胞的基本形态学特性研究

近年来，国外开展的循环内皮细胞（ＣＥＣ）检测能结合临床研究血管内皮细胞（ＶＥＣ）的变化，具有较高的临床应用价值，但对其基本形态学特性尚待阐明。本文采用细胞培养、免疫组化及扫描电镜等手段，对ＣＥＣ的数量、形态、ⅧＲ：Ａｇ分布及ＣＥＣ的形成过程进行了研究。结果表明：（１）我国健康人群的外周血中存在ＣＥＣ，其值为１．３±０．７／０．９μｌ；（２）ＣＥＣ具有独特的形态特点（与循环血中其它正常血细胞相比），如体大、无核、细胞呈皱缩、拆叠或扭曲状；（３）在脐静脉和ＥＣ培养片中均存在与ＣＥＣ形态极为相似的脱落ＥＣ，提示健康人群外周血中的ＣＥＣ可能来源于正常代谢过程中衰老的ＶＥＣ；（４）从ＶＥＣ向ＣＥＣ过渡是一个渐变的过程，其形态变化主要有细胞肿大、胞膜破坏和胞核脱落等。     

Three-Dimensional Structure of Oncocyte Mitochondria in Human Salivary Glands: A Scanning Electron Microscope Study

Oncocytes occurring in otherwise normal human salivary glands were examined by scanning electron microscopy after being subjected to a maceration technique that removes all soluble cytoplasmic components and leaves only membranes in place. The three-dimensional images resulting from this procedure confirm that the majority of oncocyte mitochondria have shelf-like cristae that are attached to the inner boundary membrane, often by means of tubular structures. In some mitochondria, the cristae are digitiform and, when transected, give rise to putative vesicles. The oncocytic mitochondria in human salivary glands, by and large, lack the structural irregularity that has been reported for these organelles occurring in oncocytes in other organs.     

Lytic replication of Human herpesvirus 8 and induction of apoptosis

Although many viruses can induce apoptosis in infected cells, large DNA viruses, such as poxviruses, herpesviruses and adenoviruses, usually exhibit the ability to suppress the induction of apoptosis in the infected cells. We investigated the ability of Human herpesvirus 8 (HHV-8) to protect cells from apoptosis induced by the virus. HHV-8 has been shown to harbor genes with anti-apoptotic capacity. However, we demonstrate here that a lytic replication of HHV-8 resulted in induction of apoptosis using different techniques to detect apoptosis. Therefore, despite the presence of anti-apoptotic genes in its genome, HHV-8 could complete its cycle of productive infection while inducing apoptosis in infected cells. This finding might have implications for the pathobiology of HHV-8 and other gamma herpesviruses in vivo.     

Age-specific seroprevalence of Human Herpesvirus 8 in Mediterranean regions

Objective   Human herpesvirus 8 (HHV8) is believed to be transmitted mainly by sexual contact; epidemiological data from Africa show, however, that non-sexual transmission routes may also play an important role. To evaluate better the distribution of HHV8 infection in the Mediterranean area, we performed an age-specific seroprevalence study.
Methods   Sera were collected from subjects from different geographical areas. The sera were analyzed by immunofluorescence assay (IFA) and enzyme-linked immunosorbent assay (ELISA). A total of 1083 patients were studied, 667 patients from various regions of Italy and 416 from Albania. The patients were stratified into six age groups. Multivariate logistic regression was used to evaluate associations between HHV8 and demographic data.
Results   An overall seropositivity rate of 17.6% was observed. The highest rate was observed in Sardinia (25.0%) and the lowest was found in Albania (13.9%). The prevalence rate increased linearly with age, from 9.7% in patients belonging to the 0–14 years age group to 26.3% for patients more than 59 years old. Seropositivity for HHV8 was significantly associated with membership of the 59 years-plus age group. Rates of seropositivity were significantly higher in patients from central southern Italy (OR = 1.7) and Sardinia (OR = 1.8) than in patients from Albania.
Conclusions   The data suggest that HHV8 is widespread in the Mediterranean area, including regions like Albania that have not been previously investigated. The statistically significant association between HHV8 seropositivitity and increasing age suggests that non-sexual transmission routes may be involved in the spread of the virus.     

Laboratory Diagnosis of Human Herpesvirus 8 Infection in Humans

Human herpesvirus 8 (HHV-8) is causally associated with Kaposi's sarcoma, primary effusion lymphoma and multicentric Castleman's disease. Serological and molecular biology assays are used to investigate the biology of this virus in different populations and diseases. Serological assays are mainly used to study the prevalence of the viral infection and to predict the diagnosis of Kaposi's sarcoma and other HHV-8-associated cancers. The appearance of antibodies against lytic antigens precedes the appearance of antibodies against latent antigens, probably explaining the lower sensitivity of assays based on latent HHV-8 antigens. The lack of international reference serum panels is presently the major bottleneck for further progress in the field of HHV-8 serology. Molecular biological assays are an absolute requirement for both the diagnosis and the follow-up of HHV-8 infection. Qualitative methods have been particularly useful to elucidate the mode of transmission and the causal association between HHV-8 and HHV-8-associated diseases. Quantitative methods have become an essential tool to monitor the progression of the infection and the effects of antiviral therapies. This review analyzes the performance of the different serological and molecular biological assays available at present. The main conclusion is that more research is needed to define the most useful laboratory tests for the diagnosis of HHV-8 infection and to establish the clinical role of such tests. Electronic Publication     

Aggressive angiomyxoma: an ultrastructural study of four cases

The histogenesis of the aggressive angiomyxoma of the vulvo-vaginal region was studied. Four cases of aggressive angiomyxoma were examined by using light microscopy, electron microscopy, and immunohistochemistry. Myofibroblastic from smooth muscle cell differentiation was distinguished by paying close attention to the structures of the neoplastic cell membrane. Aggressive angiomyxomas exhibit subtle features of smooth muscle differentiation, suggesting that the neoplastic cells differentiate from a multipotent perivascular cell.     

Ultrastructural Features of 'Solid Cell Nest' of the Human Thyroid Gland: A Study of 8 Cases

The ultrastructural features of solid cell nests (SCN), made of squamous cells, and associated calcitonin cells (C cells), of the thyroid gland were studied in only a few cases in humans. A study was performed on 8 paraffin-embedded SCN, postembedded in Epon, to look for their ultastructural features. Immunohistochemical analysis using calcitonin antibody was performed on semithin sections of SCN to explore the presence of C cells. Three cases (37.5%) of SCN were positive for calcitonin, and electron-dense secretory granules were observed in the cytoplasm. In two of these cases, an increased number of C cells in the adjacent thyroid parenchyma was observed. The presence of ciliated and lymphoid cells, in addition to intracytoplasmic microvacuolar and microfollicular (microglandular) structures, was noticed. Ciliated cells have already been reported in embryonic rests of human and animals, but ultrastructurally for the first time in human SCN. The presence of microfollicular structures, intracytoplasmic microvacuolar, secretory granules features, and ciliated cells, in addition to lymphoid cell, suggests the existence of a common ultimobranchial stem cell for C cells or for one or more cell types of the thyroid gland.     

Morphological Study of Pancreatic Endocrine in an Experimental Chronic Pancreatitis with Diabetes Induced by Stress and Cerulein

The purpose of this study was to investigate the morphological changes in the islets observed in a new chronic pancreatitis model with diabetes induced by repetition of cerulein injection plus water-immersion stress in rats. The rats of this model were treated with water-immersion stress for 5 h and two intraperitoneal injections of 20 mug/kg body weight of cerulein once a week for 16 weeks. In the stress and cerulein group, 62% of the islets exhibited infiltration of mononucleated cells, and/or peri- and intrainsular fibrosis. On immunohistochemical study, some islets showed reduced density of the insulin immunoreactivity. The glucagon-producing cells decreased in number. With electron microscopy, various endocrine changes were observed, mainly in the B cells. The changes included scattered debris damage with reduction of secretary granules, and vesiculation of the endoplasmic reticulum. Numerous fibroblasts clustered around the islets, and proliferating collagen fibers invaded the islets. The microvascular changes consisted of bleeding and damage to the endothels. In the pancreas treated with stress alone or cerulein alone, significant endocrine damage was not observed. In conclusion, chronic repetitive treatment with stress and cerulein, together with poor islet circulation due to fibrosis and vascular changes, resulted in the endocrine cellular damage.