Calcium loss and muscle atrophy are two of the main metabolic changes experienced by astronauts and crew members during exposure to microgravity in space. To investigate the effects of spaceflight on calcium-dependent secretion and cytoskeletal formation in a less complex system we utilized sea urchin eggs and embryos which were fertilized and cultured under spaceflight conditions during the STS-77 shuttle mission. Sea urchin eggs were fertilized and cultured in the newly developed aquatic research facility (ARF) which allowed culture of eggs and embryos in microgravity and in a 1g centrifuge in space. This allowed analysis of the comparison of microgravity and 1g spaceflight treatments with samples cultured on ground. Eggs and embryos were maintained in Standard Container Assemblies (SCAs) with identical sets prepared for culture in microgravity, and at 1g in the middeck compartment of the shuttle Endeavor, as well as for ground observations at the Kennedy Space Center.

A combination of microscopical methods (TEM, SEM, freeze fracture, confocal, immunoEM) are utilized to demonstrate functional interactions between somatic cells and the germline in the model organism, C. elegans.A variety of signaling mechanisms are required to coordinate activity of maturing oocytes and the somatic sheath of the gonad. Communication via gap junctions allows direct coordination of sheath cell motility and oocyte development. Gap junctions link adjoining sheath cells, and link the sheath to the most mature oocyte. Yolk protein produced in the intestine passes from the pseudocoelom through the basal lamina of the proximal gonad and then through fenestrations (sheath pores) in the gonad sheath before being endocytosed into the most mature oocytes. Myoepithelial specializations anchored by hemi-adherens junctions in the proximal sheath act to squeeze the primary oocyte out of the gonad to the spermatheca for fertilization.

A persistent problem in elucidating the anatomy of the peripheral nervous system has been an inability to stain both myelinated and unmyelinated nerve fibers. To overcome this problem, our laboratory developed two workmg protocols for reliably and differentially labeling and staining the peripheral nervous system and combined them with an enzyme clearing and staining procedure for the simultaneous visualization of bone and cartilage.

One protocol uses anti-acetylated α-tubulin immunohistochemistry to follow the course, peripheral branching, and origin of the ventral spinal nerve innervating the axial musculature and a second uses anterograde and retrograde transport of selectively applied 3000 molecular weight (MW) biotin dextran amines and/or biocytin to identify specific afferent and efferent projections and their cell bodies. Both procedures can be combined with an enzyme clearing and staining procedure for the simultaneous visualization of bone (alizarin red S) and cartilage (alcian blue) in whole-mount preparations.

Because of their great size, availability, and capacity to develop autonomously in culture, amphibian eggs have become a productive system for a wide variety of experimental studies of animal cells. Their use has advanced our understanding of fundamental molecular and cellular processes such as transcription and translation, regulation of the cell cycle, and the function of the cytoskeleton. The amphibian egg, particularly that of the frog Xenopus laevis,has further served as an excellent system for studying complex developmental phenomena, including the roles of growth factors, signal transduction, and transcription regulation in cell determination, morphogenesis, and tissue differentiation.

Despite these advantages, the frog egg's great size and opacity have made it a challenging subject for microscopy. Thus, many problems in early development, such as how maternal cytoplasmic determinants become localized during cell division, have resisted traditional optical approaches.

The early vertebrate embryo develops a characteristic tube-within-a-tube body plan. This plan is realized through a series of cell movements and cell-cell interactions that collectively result in tissue shaping and the formation of the three-dimensional body plan. Tissue shaping is a highly choreographed process that is under the control of the organizer--a specialized region of the embryo that is both sufficient and required for formation of the body plan. Recent technical advances have greatly increased our understanding of the role of the organizer in vertebrate embryogenesis. Such advances include the use of new cellular, molecular, genetic, and embryological approaches.

A hallmark of embryogenesis is its dynamic nature. Classically, embryos were studied in three major ways. 1) With morphological/descriptive analysis, initially involving histological procedures (stained whole mounts and serial sections cut in the three cardinal axes) and more recently electron microscopy.

The embryonic lens contains two populations of cells, lens epithelial cells and lens fiber cells. As the growth of the human lens is linear from the age of 10 to 90, lens epithelial cells divide and differentiate into fiber cells at a uniform rate during adulthood. Any defects that arise during fiber formation will remain in the lens. We postulate that regulation of early lens growth is critical for the onset of cataracts that usually occur later in life. Slowing the growth of the lens could therefore provide a strategy to suppress the formation of cataracts. However, the factor(s) that control lens growth in vivo have not been identified.

We have identified 18 receptor and cytoplasmic kinases present in lens epithelial cells. Using these as potential targets for factors controlling growth, we tested corresponding ligands in a lens epithelial cell proliferation assay. Platelet-derived growth factor (PDGF) proved to promote a growth activity similar to, but distinct from, that of embryo serum.

Over the past several years the central roles of several different growth factors in the normal development of the embryonic heart have been identified through a variety of techniques involving microscopy. Initially, most studies consisted of descriptions of gross changes in cardiac morphology associated with naturally occurring mutations that affected growth factor function. More recently the development of specific probes for growth factor receptors that can be used in confocal microscopy have aided in the identification of changes in the temporal and spatial distributions of receptors at various stages of development. The correlation of these changes with developmental events such as valve formation and trabeculation in the heart, in conjunction with biochemical studies and blocking agents for the growth factors have significantly increased our understanding of growth factor function in cardiac development.

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.