Two novel sample preparation procedures have been developed to visualize morphological structures in embryonic and fetal tissues. In the first, optimization of the sample preparation allows for the visualization of embryos with a thickness in excess of 500 microns. The morphology of the internal structures in these embryos is observed by optical sectioning. The general sample preparation procedures included paraformaldehyde fixation, methanol dehydration and clearing with either benzyl alcohol/benzyl benzoate or methyl salicylate. The dyes (i.e. LysoTracker Red, YoPro) are incorporated into living or fixed embryos to indicate a specific biological function or embryo morphology. Using these stained embryos, the detection of apoptosis and its subsequent quantification were made in addition to the observation of morphology.

In the second technique, the confocal microscope has been adapted to produce images similar to those generated by a scanning electron microscope. For this application, the embryos are coated with a dilute solution of acridine orange resulting in opaque surface fluorescence.

The ultrastructural pathology of HIV infection is a broad subject, owing to the fact that the transmission electron microscope has played an integral part in our understanding of HIV disease. The subject can be divided into three basic areas: HIV pathogenesis, opportunistic infections (OI), and neoplastic disease. Once HIV was determined to be a retrovirus, TEM confirmed its membership in the Lentivirus family and showed that it has a conical nucleoid. TEM has played a key role in our understanding of: 1) the CNS as a target of HIV, 2) the macrophage as a “trojan horse” for HIV, 3) how lymphocytes and macrophages differ in their HIV expression, 4) attachment of virions to the processes of follicular dendritic cells via complement and Fc receptors, and 5) the formation of multinucleated giant cells.

The course of HIV infection necessarily involves the initial establishment of the virus in a receptive host environment. HIV disease involves active intracellular replication of the virus and spread of the virus from the initial compartment of the infection. Finally, from the virus’ perspective _ virions must enter a compartment suitable for eventual host-host transmission. In the vernacular of HIV epidemiology this implies some sort of “exchange of bodily fluids”.

Recently, our research focus has been on tracking HIV during the early phases of infection. The goal is to piece together a full chronology of the disease at the tissue level. The emphasis will be on changes in lymphoid architecture that might ultimately be important in the pathogenesis of AIDS malignancies and offer insight into the dysfunctional aspects of immunity in the setting of retroviral infection. Because HIV disease often ultimately features a severe immunodeficiency, other pathogens by necessity play a role in shaping the “visible” immune response.

The World Health Organization estimates by the year 2000 that the cumulative total of HIV-1 infected adults and children will be about 30 million worldwide. Disease of the central nervous system (CNS) in association with AIDS is common. 20-30% of AIDS patients will develop varying degrees of cognitive and motor deficits designated the “AIDS dementia Complex (ADC)” or “HIV-1-associated cognitive/motor deficit”. Progression of symptoms is usually slow and irregular. In the end-stage, the patient is severely impaired with mutism, incontinence and prominent motor impairment. Neuroradiologic studies show generalized cortical atrophy and diffuse white matter changes. HIV can be cultured from the CSF. Neurologic involvement in pediatric AIDS is very common with CNS complications usually occurring within the first two postnatal years. Given the severeness of the dementia and motor dysfunction in ADC, one is struck by the paucity of neuropathological features. Hematoxylin and eosin stained sections reveal only three neuropathological features. First, there are small numbers of microglial nodules scattered in the white matter and sometimes in grey matter (Fig. 1). However, microglial nodules occur in virtually all viral infections of the CNS, including CMV micronodular encephalitis in AIDS patients.

TRS and CCC are endoplasmic reticulum membrane-derived structures seen in HIV-infected individuals in a variety of cell types. TRS (synonym: tubuloreticular inclusions) are 24-25 nm in diameter, branching tubules which are short or long and associated with the granular or smooth endoplasmic reticulum, the Golgi complex, the perinuclear cistern, and annulate lamellae (Fig. 1-3, 8). The tubules are noticeably more stretched out in Kaposi's sarcoma endothelial cells, presumably by an increase of matrix material within the reticulum sac (Fig. 3). TRS consist of membranous components, including polypeptides, but lack nucleic acid.

CCC (test tube and ring-shaped forms; curvilinear membranes) are formed by a concentric stacking of two or three cisterns of endoplasmic reticulum resembling nuclear membrane in mitosis (Fig. 7), but with the interposition between the opposing membranes of a layer of electron-dense material that is resistant to lipid solvents (Fig. 4,5,8).

Microscopy has had a major role in the analysis of renal disorders associated with human immunodeficiency virus (HIV) infection, both as a diagnostic method and as a means of studying pathogenic mechanisms. In the diagnostic realm, microscopic analysis of renal tissue obtained at biopsy and autopsy is a mainstay for the detection of a wide range of glomerular, vascular, and tubulointerstitial diseases. As an investigative tool, microscopy has made an important, albeit somewhat controversial, contribution to our understanding of the pathogenesis of at least one HIV-associated renal lesion.

A variety of ultrastructural alterations have been documented in association with HIV infection. These include tubuloreticular inclusions (most commonly seen in vascular endothelial cells and mononuclear leukocytes)(Fig. la), cylindrical confronting cisternae (typically found in mononuclear leukocytes) (Fig. lb), and nuclear abnormalities such as nuclear bodies and granular change (most often in tubulointerstitial cells) (Fig. lc).

Use of TEM in the diagnosis of opportunistic infections in AIDS patients is indicated in three situations. First, when an opportunistic infection is suspected, but an infectious agent is not visible in tissue sections studied by light microscopy (LM). This situation is illustrated by the following case.(1) A 33-year-old man with AIDS presented with fever, nightsweats, generalized lymphadenopathy, and a 30 lb. weight loss. An inguinal lymph node biopsy was performed. There was no consensus as to the diagnosis among the pathologists who studied H&E sections of the biopsy. An obscure infectious disease, a B-cell lymphoma with amyloid stroma, and possibly Hodgkin's disease were the major considerations. TEM study revealed unexpected clusters of small, pleomorphic, coccobacillary bacteria in vascular walls typical of cat scratch disease. These bacteria were later further identified by polymerase chain reaction as belonging to the genus Rochalimaea(now Bartonella), an important cause of cat scratch disease.

Infection with human immunodeficiency virus (HIV) eventually causes a profound decrease in the body's ability to eradicate or control infections with microorganisms, including viruses. Some infections in AIDS patients are due to common organisms which are of little significance in immunocompetent individuals. Other organisms can be harbored continuously, occasionally causing disease, but normally being suppressed after a heightened immune defense; in AIDS patients, these infections can be life-threatening. Further, practices that predispose to HIV infection also permit entry of other organisms, such as hepatitis and herpesviruses. Electron microscopy is beneficial as an adjunct to other modalities for viral detection. Methods for identifying viruses, both in fluids by negative staining and in tissues by thin sectioning, have been published. Some viral pathogens, including HIV itself, are best documented by other means.

HIV has been demonstrated by EM in infected individuals, but because it destroys and makes scarce the cells for which it has an affinity, it is difficult to find them.

Introduction

Chimeric Simian/Human Immunodeficiency Viruses (SHIV) causes persistent infection characterized by CD4+ T cell lymphopenia in rhesus monkeys. The SHIV model infection closely resembles HIV-1 infection and disease in humans, except that it progresses at an accelerated rate. During the early stages of HIV-1 infection, viral replication is at its highest rate and there is a rapid decline of the immune system. Cell death induced by apoptosis, a feature in the pathogenesis of acquired immunodeficiency syndrome (AIDS) and AIDS-like syndromes of many lentiviral infections, is thought to be important in the progressive decline of lymphocytes and the cell mediated immunity of infected individuals. The apoptosis is not specific for lymphocytes, this phenomenon also occurs in numerous cell types of the immune system, including the polymorphonuclear leukocytes. Abnormalities of neutrophil function and number because of HIV-1 and other retroviral infections have been recognized.

Microsporidia are obligate intracellular parasites that lack mitochondria. Until recently, their importance was primarily limited to fish and insects; but with the onset of the AIDS epidemic, understanding their role as human pathogens has become increasingly important. Encephalitozoon microsporidia develop and mature within a membrane bound parasitophorous vacuole (pv). Following initial infection of the host cell, the developing stages (meronts) of the parasite proliferate in close proximity to the pv membrane. Later, they differentiate through a sporogonial stage and finally into environmentally resistant mature spores located more centrally in the vacuole.

Previous work done in our laboratory has shown that the pv membrane does not act as a significant diffusion barrier to the movement of ions between the host cell cytoplasm and the parasite, permitting the free-acid form of calcein, a fluorescent dye, loaded into the host cell to enter the pv.

Since the emergence of HIV and its counterpart,acquired immunodeficiency syndrome (AIDS), rare opportunistic organisms have become increasingly prevalent. This increased prevalence has provided an opportunity for ultrastructural evaluation of these infectious organisms. Herein is presented a case of intestinal Mycobacterium avium-intracellulare (MAI) in a middle-aged male with uncertain HIV status at diagnosis.

A 56 year-old black male presented to University of South Alabama Medical Center complaining of shortness of breath, anorexia, nausea and vomiting, six month weight loss, and bloody diarrhea. His evaluation was negative for stool ova and parasites and fecal white blood cells. Stool culture and hepatitis viral panel was negative. Iron deficiency anemia and multiple calcified granulomas in both lungs were identified. On his third hospital day, he developed an acute abdomen. At exploratory celiotomy, segmental resection of jejunum and a liver biopsy were performed. Post-operatively the patient's cardiopulmonary status steadily declined and he died three days later.

Vascular endothelial cells (EC) forms a continuous monolayer, a selective barrier that coats the inner surface of the vascular system, representing the primary anatomical site between blood and tissue. Under closed conditions (shear stress, hypoxia-reoxygenation..), EC are activated and the level of adhesion molecules (ICAM-1) expression increased. A technique of fluorescence quenching by oxygen in cell membrane was used to study molecular transport processes on a larger scale1-4. Collisional fluorescence quenching is described by the Stern-Volmer equation : τ0 / τ = 1 + kd.PO2 where τ0 and τ are respectively the excited state lifetimes in the absence and in the presence of oxygen, Kd the quenching dynamic constant and PO2 the oxygen partial pressure. The main purpose of the present study was to investigate the relation a) between the plasma viscosity and PO2 across human EC monolayer by Time-Resolved Fluorescence Microscopy (TRFM5-6 ) between the PO2 calculated and the level of ICAM-1 expression by flow cytometry (FCM)7 and Conventional Optical Scanning Microscopy (COSM8).

Complexes formed during interaction of cationic liposomes with poly-nucleotides such as DNA (CLDC) display a variety of polymorphic and metastable structures. These include multilamellar structures of alternating lipid bilayers and DNA monolayers; fibrillar structures, among them spaghetti-liketubules (Figure 1), and map-pin-structures(Figure 2), and, finally, non-bilayer lipid arrangements, such as hexagonal (HII) (Figure 3) and cubic phases.

In order to find out the “active” structure(s) in terms of transfection, we investigated the transfection activity both in vivoand in vitroof CLDC composed of the lipid DDAB (dimethyl-dioctadecylammonium bromide) and Choi (cholesterol) or DOPE (l,2-dioleoyl-sn-glycerol-3- phosphoethanolamine) as helper lipids. In parallel we studied their morphology by freeze-fracture electron microscopy.The in vivostudies were carried out in mice following i.v. injection and therefore the morphology of the CLDC was investigated in mouse serum.

Taxol has been used as anti-cancer compound against prostate, ovarian, and metastatic breast cancer. While the most obvious effect of taxol is bundeling of microtubules and mitotic arrest, recent studies have demonstrated that taxol is able to induce intranucleosomal DNA fragmentation and typical morphological features of apoptosis in a number of solid tumor cells. These results indicate that taxol may exert its anti-tumor effects via secondary mechanisms which may or may not be related to its primary effects on microtubules. It has been shown that taxol-induced microtubular changes and G2/M arrest are associated with the release of the electron transfer protein cytochrome C from mitochondria into the cytosol. Cytochrome C then binds to APAF-1 (a human homolog of the ced-4 gene of C. elegans), which binds, cleaves, and activates caspase- 9, ultimately resulting in the cleavage and activity of caspase-3. We investigated the effects of taxol (100nM) on microtubules, on DNA, and on the pre-apoptotic mitochondrial events using LNCaP and DU145 prostate cancer cells.

The usefulness of Differential Contrast Interference (DIC) light microscopy for observing fine details within transparent specimens is well known. However, when viewed by the eye or by recording with photographic emulsion, fine structural detail at the limit of resolution is often not visible because of lack of contrast. To overcome this problem, electronic contrast enhancement capabilities of video cameras have been used to enhance structural details that would otherwise be invisible. The technique, commonly referred to as VE-DIC (Video Enhanced DIC), uses first analog contrast enhancement of the image with a video camera followed by a real-time digital image processor to further enhance the image with. We are exploring the feasibility of achieving fine structural detail of live cells by directly acquiring digital images of them with a high resolution CCD camera.

High resolution cooled slow-scan 12-bit CCD cameras are well suited for DIC microscopy because of their greater dynamic range than video CCD cameras that are normally 8-bits or lower.

The active form of platelet derived growth factor (PDGF) is a polypeptide dimer of which there are three isoforms: PDGF-AA, PDGF-BB, and PDGF-AB. Furthermore, two types of PDGF receptors also exist: PDGFRα and PDGFRβ. The β-receptor binds only the PDGF-B chain with high affinity, while the αreceptor can bind either PDGF-A or -B chains. Both PDGF ligands and their receptors have been linked to such cellular responses as mitogenisis, cell migration, chemotaxis, adhesion and differentiation in a variety of cell types.

Several studies have reported fetal cardiac abnormalities associated with disruption of PDGF and its receptors. In spontaneous mutants lacking the PDGF α-receptor (Patchmice), fetal hearts are generally dilated and exhibit valve and outflow tract defects. Similar defects are observed in normal embryos injected with antibodies to block the functioning of the PDGF-A chain. Disruption of PDGF-B function results in ventricular and atrial dilation, reduction of the ventricular wall, and hyper-trabeculation.

Platelets play a major role in hemostasis due to their ability to adhere and aggregate at sites of vessel wall injury. Damage to the inner surface of arteries includes ruptures or tears at the site of atherosclerotic plaques or injury resulting from interventional procedures. Uncontrolled platelet recruitment and aggregation at these sites can lead to complete occlusion of the vessel. Thus the understanding of platelet function and structure has become an important goal in preventing vascular disease. We employed cryo-HRSEM techniques to gain more insight into the surface morphology of both quiescent and thrombin stimulated platelets.

Platelet rich plasma (PRP) was obtained by mixing whole blood (21 ml) drawn from juvenile pigs with 3 ml of acid citrate dextrose and centrifugation at 300g for 10 min. To obtain washed platelets the PRP was washed three times with a citrate wash buffer consisting of 36mM citric acid, 5mM glucose, 5mM KCl, 2 mM CaCl2, 1 mM MgCl2, 90 mM NaCl, pH 6.5.

Spaceflight induced mitochondrial alterations have been reported for muscle and may be associated with altered physiological functions in space. Mitochondrial alterations are also indicative of preapoptotic events which are seen in greater amounts in cells exposed to spaceflight when compared with cells cultured at 1 g. Preapoptotic mitochondrial changes include alterations of processes at the inner mitochondrial membrane and can result in changes in mitochondrial volume. Higher amounts of oxidative stress during space flight may be one of the causes for changes which lead to apoptosis. Jurkat cells flown on the STS-76 space shuttle mission showed an increase in the number of cells with apoptotic bodies early in the mission and a time-dependent, microgravity-related increase in the Fas/APO-1 cell death factor. Here we investigated the morphology of mitochondria in Jurkat cells exposed to spaceflight during the STS-76 mission.

Fibrinogen is composed of three pairs of polypeptide chains, termed Aα, Bβ and γ, respectively. Proteolytic conversion of fibrinogen to fibrin by thrombin releases fibrinopeptide A and exposes an N-terminal polymerization site ('EA’) in the central E domain of the molecule. This site interacts with a constitutive complementary site in each outer D domain ('Da’) to drive the molecular assembly process by end-to-middle domain non-covalent ‘Da:EA’ interactions. End-to-end aligned double-stranded fibrils are formed in coordination with another recently described constitutive self-association site (termed ‘D:D’)fhat is situated at the outer end of each D domain. The D:D site contributes to end-to-end molecular alignment of fibrin molecules within each fibril strand. Concomitant branching and lateral fibril associations to form thick fibers, lead to formation of the mature fibrin clot.

Fibrinogen Marburg is a homozygous hypodysfibrinogenemia lacking amino acid residues Aα461- 610 due to a stop codon at position 461 of the Aα chain.

Leukocytes adherence receptors are the basis of cells interactions1. Flow cytometry (FCM) makes it possible to assess a characterization of the activation level of a given cellular population by receptor quantifying2. That technique, however does not integrate at the same time, all others factors of leucocyte adhesive phenotype regulation, as spatial distribution and molecular conformation. Our study consisted in exploring the main adhesion receptors (CD62L, CDllb/CD18) on the surface of Polymorphonuclear Neutrophils (PMN) that were prepared under identical conditions and simultaneously analyzed by FCM and Conventional Optical Scanning Microscopy/Deconvolution3 (COSM). Methods: PMN cells from whole blood were obtained using Polymorphprep™. Isolated PMN (105 cells /ml) were incubated with Tumor Necrosis Factor (TNFα, 100 UI/ml) at 37°C for one hour. Quantification of cell surface molecules was assessed according to the quantitative indirect immunofluorescence3 method using QIFIKIT® (Dako, France).