Histological and three dimensional organizations of lymphoid tubules in normal lymphoid organ of Penaeus monodon

The normal lymphoid organ of Penaeus monodon (which tested negative for WSSV and YHV) was composed of two parts: lymphoid tubules and interstitial spaces, which were permeated with haemal sinuses filled with large numbers of haemocytes. There were three permanent types of cells present in the wall of lymphoid tubules: endothelial, stromal and capsular cells. Haemocytes penetrated the endothelium of the lymphoid tubule's wall to reside among the fixed cells. The outermost layer of the lymphoid tubule was covered by a network of fibers embedded in a PAS-positive extracellular matrix, which corresponded to a basket-like network that covered all the lymphoid tubules as visualized by a scanning electron microscope (SEM). Argyrophilic reticular fibers surrounded haemal sinuses and lymphoid tubules. Together they formed the scaffold that supported the lymphoid tubule. Using vascular cast and SEM, the three dimensional structure of the subgastric artery that supplies each lobe of the lymphoid organ was reconstructed. This artery branched into highly convoluted and blind-ending terminal capillaries, each forming the lumen of a lymphoid tubule around which haemocytes and other cells aggregated to form a cuff-like wall. Stromal cells which form part of the tubular scaffold were immunostained for vimentin. Examination of the whole-mounted lymphoid organ, immunostained for vimentin, by confocal microscopy exhibited the highly branching and convoluted lymphoid tubules matching the pattern of the vascular cast observed in SEM.     

Detection of gill-associated virus (GAV) by in situ hybridization during acute and chronic infections of Penaeus monodon and P. esculentus

Chronic and acute gill-associated virus (GAV) infections were examined by in situ hybridization (ISH) using a DNA probe targeting a 779 nucleotide region of the ORF1b-gene. Chronic GAV infections were observed in healthy Penaeus monodon collected from farms and healthy P. esculentus surviving experimental infection. During chronic-phase infections in both species, GAV was detected only in partitioned foci of cells with hypertrophied nuclei (spheroids) within the lymphoid organ. Acute-phase infections were observed in moribund P. monodon and P. esculentus infected experimentally with a high dose of GAV, and in moribund P. monodon collected from farms during outbreaks of disease. During acute experimental infections in P. monodon, ISH detected GAV throughout the lymphoid organ, in gills and in connective tissues throughout the cephalothorax. In moribund P. monodon collected from natural outbreaks of disease, GAV was also detected in the gills and in connective tissues of the cephalothorax, but the distribution of virus within the lymphoid organ varied. In acutely infected P. esculentus, GAV was detected in connective tissues, but was restricted to the inner stromal matrix cells and endothelial cells of intact lymphoid organ tubules. The tissue distribution of GAV identified by ISH suggests that shrimp are able to control and maintain chronic asymptomatic infection by a process involving lymphoid organ spheroids. Acute phase infections and the development of disease appear to be dose-related and involve the systemic distribution of virus in connective tissues throughout the cephalothorax.     

Antiserum to the gp116 glycoprotein of yellow head virus neutralizes infectivity in primary lymphoid organ cells of Penaeus monodon

Yellow head virus (YHV) is an invertebrate nidovirus that has caused mass mortality of cultured Penaeus monodon in Asia. In this study, we investigated whether mouse polyclonal antiserum raised against the YHV gp116 or gp64 structural glycoproteins could neutralize YHV infectivity as determined using an in vitro quantal assay in primary cultures of lymphoid organ cells. Anti-gp116 antiserum showed virus-neutralizing activity whereas anti-gp64 antiserum failed to inhibit infection. The results suggest that gpl16 antiserum blocks binding of virions to cellular receptors to facilitate YHV entry into lymphoid organ cells.     

Proteomic analysis of altered proteins in lymphoid organ of yellow head virus infected Penaeus monodon

A comparative proteomic analysis was employed to identify altered proteins in the yellow head virus (YHV) infected lymphoid organ (LO) of Penaeus monodon. At 24 h post-infection, the infected shrimps showed obvious signs of infection, while the control shrimps remained healthy. Two-dimensional electrophoresis of proteins extracted from the LO revealed significant alterations in abundance of several proteins in the infected group. Protein identification by MALDI-TOF MS and nanoLC-ESI-MS/MS revealed significant increase of transglutaminase, protein disulfide isomerase, ATP synthase beta subunit, V-ATPase subunit A, and hemocyanin fragments. A significant decrease was also identified for Rab GDP-dissociation inhibitor, 6-phosphogluconate dehydrogenase, actin, fast tropomyosin isoform, and hemolymph clottable protein. Some of these altered proteins were further investigated at the mRNA level using real-time RT-PCR, which confirmed the proteomic data. Identification of these altered proteins in the YHV-infected shrimps may provide novel insights into the molecular responses of P. monodon to YHV infection.     

Differences in susceptibility of palaemonid shrimp species to yellow head virus (YHV) infection

Five species of palaemonid shrimp, Macrobrachium rosenbergii, M. lanchesteri, M. sintangense, Palaemon styliferus and P. serrifer, were collected from Penaeus monodon farming areas in Thailand. Some of each species were artificially infected with yellow head virus (YHV) by injection and then monitored by RT-PCR and by immunohistochemistry using monoclonal antibodies specific to 116 kDa, 64 kDa, and 20 kDa proteins of YHV. Natural YHV infections were not detected in any of the shrimp examined. In YHV injection experiments, a high proportion of P. serrifer, P. styliferus and M. sintangense exhibited mild to moderate YHV infections at 3 d post-injection. The severity of infection was reduced in shrimp that survived to 10 and 30 d post-injection. Using immunohistochemistry and RT-PCR, a small proportion of M. lanchesteri showed very mild YHV infections at Day 3 but no infections at Days 10 and 30. No YHV infections resulted in M. rosenbergii. The evidence suggested that M. sintangense, P. styliferus and P. serrifer are susceptible to YHV and carry it for some time. In contrast, M, rosenbergii and M. lanchesteri appear to resist YHV infection and eliminate YHV efficiently. Because they display a range of responses to YHV, palaemonid shrimp may serve as a good model for studying YHV defense mechanisms in shrimp.     

Evidence for apoptosis correlated with mortality in the giant black tiger shrimp Penaeus monodon infected with yellow head virus

Histological, cytochemical and ultrastructural changes in giant black tiger shrimp Penaeus monodon were investigated at various time intervals after injection with yellow head virus (YHV). Hemocytes, lymphoid organs (LO) and gills were the main focus of the study. After injection with YHV, onset of mortality varied from 36 h onward. By normal hematoxylin and eosin staining, the 3 tissues showed clear and increasing prevalence of nuclear condensation, pyknosis and karyorrhexis from approximately 36 h post-injection (p.i.) until death, although pathology was evident in the LO as early as 12 h p.i. in some shrimp. By nuclear DNA staining with 4',6-diamidino-2-phenylindole (DAPI) and by specific labeling of 3'-OH ends of nuclear DNA using a technique called terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick-end labeling (TUNEL), cells of the 3 tissues showed evidence of chromatin condensation and DNA fragmentation, respectively. Both are generally considered to be characteristic of apoptosis. In addition to TUNEL labeling, evidence for DNA fragmentation was supported by the appearance of approximately 200 base pair DNA ladders at approximately 48 h p.i. in hemocytes of YHV-infected but not uninfected shrimp. Transmission electron microscopy (TEM) of LO tissue revealed features of apoptosis in tissues of YHV-infected shrimp only. These included marginated, condensed and fragmented chromatin without concurrent cytoplasmic damage. Histological, cytochemical, ultrastructural and biochemical data were consistent with the hypothesis that widespread and progressive apoptosis occurred in susceptible shrimp infected with YHV. Although no specific tests were carried out to determine whether this purported apoptosis was the cause of mortality, moribund shrimp had extensive deterioration of vital tissues such as the hemolymph, gills, heart and LO, suggesting that many essential bodily functions had been severely compromised. This probably resulted in the gross signs of lethargy and weakness seen, and it is reasonable to suggest that further, progressive deterioration could have led to the collapse of vital functions followed by death.     

Preliminary study on haemocyte response to white spot syndrome virus infection in black tiger shrimp Penaeus monodon

White spot syndrome virus (WSSV) has been a major cause of shrimp mortality in aquaculture in the past decade. In contrast to extensive studies on the morphology and genome structure of the virus, little work has been done on the defence reaction of the host after WSSV infection. Therefore, we examined the haemocyte response to experimental WSSV infection in the black tiger shrimp Penaeus monodon. Haemolymph sampling and histology showed a significant decline in free, circulating haemocytes after WSSV infection. A combination of in situ hybridisation with a specific DNA probe for WSSV and immuno-histochemistry with a specific antibody against haemocyte granules in tissue sections indicated that haemocytes left the circulation and migrated to tissues where many virus-infected cells were present. However, no subsequent haemocyte response to the virus-infected cells was detected. The number of granular cells decreased in the haematopoietic tissue of infected shrimp. In addition, a fibrous-like immuno-reactive layer appears in the outer stromal matrix of tubule walls in the lymphoid organ of infected shrimp. The role of haemocytes in shrimp defence after viral infection is discussed.     

Role of an arbovirus nonstructural protein in cellular pathogenesis and virus release

The insect-borne Bluetongue virus (BTV) is considered the prototypic Orbivirus, a member of the Reovirus family. One of the hallmarks of Orbivirus infection is the production of large numbers of intracellular tubular structures of unknown function. For BTV these structures are formed as the polymerization product of a single 64-kDa nonstructural protein, NS1, encoded by the viral double-stranded RNA genome segment 6. Although the NS1 protein is the most abundant viral protein synthesized in infected cells, its function has yet to be determined. One possibility is that NS1 tubules may be involved in the translocation of newly formed viral particles to the plasma membrane, and NS1-specific monoclonal antibodies have been shown to react with viral particles leaving infected cells. In the present study we generated a mammalian cell line that expresses a recombinant single-chain antibody fragment (scFv) derived from an NS1-specific monoclonal antibody (10B1) and analyzed the effect that this intracellular antibody has on BTV replication. Normally, BTV infection of mammalian cells in culture results in a severe cytopathic effect within 24 to 48 h postinfection manifested by cell rounding, apoptosis, and lytic release of virions into the culture medium. However, infection of scFv-expressing cells results in a marked reduction in the stability of NS1 and formation of NS1 tubules, a decrease in cytopathic effect, an increased release of infectious virus into the culture medium, and budding of virions from the plasma membrane. These results suggest that NS1 tubules play a direct role in the cellular pathogenesis and morphogenesis of BTV.     

Immunodetection of hemocytes, peneidins and α2-macroglobulin in the lymphoid organ of white spot syndrome virus infected shrimp

Viral diseases restrict the development of the world shrimp industry and there are few studies on cell response to the presence of viral infections. We performed immunohistochemistry assays to characterize hemocytes subpopulations involved in the immune process occurring in the LO of Litopenaeus vannamei shrimp. Tissue sections of animals that increased their LO spheroids and hemocytes infiltration after WSSV induced infection, were used. Three MABs namely, 40E10 (recognizing small granule hemocytes), 40E2 (recognizing large granule hemocytes), and 41B12, which recognize α(2)-macroglobulin were used. Additionally one polyclonal antibody was used against the penaeidins antimicrobial peptides, and to detect WSSV a commercial immunohistochemistry kit (DiagXotics) was used. Numerous small granule hemocytes were detected in the stromal matrix of LO tubules, whereas large granule hemocytes were less numerous and located mainly in hemal sinuses. The exocytosis of two molecules, which have been related to the phagocytosis process, i.e. penaeidins, and α(2)-macroglobulin, was detected in the external stromal matrix and the outer tubule walls. α(2) -macroglobulin inhibits phenoloxidase activity and its strong release in LO tissue may explain the absence of melanization in the immune processes occurring in it. The immunolabeling of vesicles within the LO spheroids with MABs 41B12 40E10 and antipenaedin antibody suggests that LOS are formed by phagocytic cells derived from small granule and hyaline hemocytes, with a possible role of peneidins and α(2)-macroglobulin acting as opsonines.     

Primary stage of feline immunodeficiency virus infection: viral dissemination and cellular targets.

The objective of this study was to identify cellular and organ targets of acute feline immunodeficiency virus (FIV) infection in vivo. Tissues of FIV-infected cats were studied at eight time points during the first 3 months after experimental infection. FIV nucleic acids were first detected by in situ hybridization 21 days after infection, approximately 1.5 weeks after lymph node enlargement was first observed and 3 weeks before the primary acute flu-like illness. The majority of FIV-infected cells were present in lymphoid organs, though low numbers of infected cells were noted in nonlymphoid organs as well. Germinal centers harbored many of the FIV-infected cells within lymphoid tissues. The thymic cortex was also a major site of early infection. Combined in situ hybridization and immunohistochemistry revealed that T lymphocytes were the primary target of early FIV infection in tissues of cats before the onset of clinical signs of acute illness. An unidentified population of mononuclear cells and a few macrophages were also infected. During the ensuing acute flu-like illness, the proportion of FIV-infected macrophages in tissues increased dramatically. This early shift in the predominant cellular localization of FIV from T lymphocytes to macrophages may be important for establishing viral persistence.     

Phosphorylation is required for myosin regulatory light chain (PmMRLC) to control yellow head virus infection in shrimp hemocytes

The cellular signal-transduction process is largely controlled by protein phosphorylation. Shrimp infected with yellow head virus show dramatic changes in their hemocyte phosphoproteomic patterns, and aberrant activation of phosphorylation-based signaling networks has been implicated in a number of diseases. In this study, we focused on phosphorylation of Penaeus monodon myosin regulatory light chain (PmMRLC) that is induced at an early hour post YHV infection and is concomitant with cellular actin remodeling. In shrimp cell cultures, this phosphorylation was inhibited by the myosin light chain kinase (MLCK) inhibitors ML-7 and ML-9, suggesting that PmMLC phosphorylation is MLCK pathway-dependent. Blocking PmMRLC phosphorylation resulted in increased replication of YHV and reduction of phagocytic activities of shrimp hemocytes called semigranular cells (SGC) and granular cells (GC). Injection of MLCK inhibitors prior to YHV challenge resulted in dose-dependent elevation in quantity of YHV-positive GC and cytoplasmic YHV protein, coincident with high shrimp mortality. Altogether, we demonstrated that PmMRLC phosphorylation increases after YHV infection in shrimp and that inhibition of the phosphorylation leads to increased YHV replication, reduced hemocyte phagocytic activity (probably through actin remodeling) and subsequent shrimp death. Thus, further studies on the MLCK activation pathway may lead to new strategies in development and implementation of therapy for YHV infections in shrimp.     

Establishment of early lymphoid organ infrastructure in transplanted tumors mediated by local production of lymphotoxin alpha and in the combined absence of functional B and T cells

Lymphoid organogenesis is a highly coordinated process involving orchestrated expression of a number of genes. Although the essential role of lymphotoxin alpha (LTalpha) for the normal development of secondary lymphoid organs is well established, it is not clear to which extent it depends upon cooperation with T and B lymphocytes for lymphoid neo-organogenesis. To determine whether LTalpha is sufficient to mediate recruitment of basic elements needed for lymphoid organogenesis, we made use of a LTalpha-transfected cell line as an experimental tool and established tumors in nude and SCID mice. Our data showed that high endothelial venules formed and follicular dendritic cells accumulated and differentiated in response to LTalpha in the absence of lymphocytes. A CD4(+)CD3(-)CD11c(+) cell population that is found in the secondary lymphoid organ was also recruited into tumors expressing LTalpha. Furthermore, in nude mice, B cells migrated in response to LTalpha and formed intratumoral follicles. These B cell follicles were structurally well equipped with follicular dendritic cell networks and high endothelial venules; however, they were not functionally active; e.g., those B cells specific for a surrogate Ag expressed by the tumor were found in the spleen, but not in the tumor. We show that, even in the absence of functional T and B lymphocytes, local expression of LTalpha in transplanted tumors induced typical stromal characteristics of lymphoid tissue, emphasizing that LTalpha is a critically important cytokine for formation of lymphoid organ infrastructure.     

Renal coccidiosis in interior Canada geese, Branta canadensis interior Todd, of the Mississippi Valley population

Kidneys from 309 Interior Canada geese from three locations in the Mississippi Flyway were examined for renal coccidia. Oocysts and/or young zygotes of Eimeria sp. were found in 6.8% of goose kidneys sampled. Only one type of renal coccidian oocyst was observed. Significantly more immature geese were infected than adults; however, there was no significant difference observed between the prevalences of infection in male and female birds. A host cellular response to zygotes and oocysts was noted in the majority of infected adult geese. Heavily infected kidneys were hypertrophic with minute foci on the surface of the organ. Histological examinations showed large numbers of unsporulated oocysts accumulated in distended collecting tubules, resulting in pressure necrosis to adjacent tissue and urate retention. Zygotes were observed in the cytoplasm of tubule cells and extracellularly in interstitial tissue. Infected tubule cells were characterized by the peripheral location of the nuclei, cytoplasmic basophilia, and cellular hypertrophy. This is the first report of an Eimeria sp. in the kidneys of Canada geese of the Mississippi Valley population.     

Fibroblast EXT1-Levels Influence Tumor Cell Proliferation and Migration in Composite Spheroids

Background

Stromal fibroblasts are important determinants of tumor cell behavior. They act to condition the tumor microenvironment, influence tumor growth, support tumor angiogenesis and affect tumor metastasis. Heparan sulfate proteoglycans, present both on tumor and stromal cells, interact with a large number of ligands including growth factors, their receptors, and structural components of the extracellular matrix. Being ubiquitously expressed in the tumor microenvironment heparan sulfate proteoglycans are candidates for playing central roles in tumor-stroma interactions. The objective of this work was to investigate the role of heparan sulfate expressed by stromal fibroblasts in modulating the growth of tumor cells and in controlling the interstitial fluid pressure in a 3-D model.

Methodology/Principal Findings

We generated spheroids composed of fibroblasts alone, or composite spheroids, composed of fibroblasts and tumor cells. Here we show that stromal fibroblasts with a mutation in the heparan sulfate elongating enzyme Ext1 and thus a low heparan sulfate content, formed composite fibroblast/tumor cell spheroids with a significant lower interstitial fluid pressure than corresponding wild-type fibroblast/tumor cell composite spheroids. Furthermore, immunohistochemistry of composite spheroids revealed that the cells segregated, so that after 6 days in culture, the wild-type fibroblasts formed an inner core and the tumor cells an outer layer of cells. For composite spheroids containing Ext1-mutated fibroblasts this segregation was less obvious, indicating impaired cell migration. Analysis of tumor cells expressing the firefly luciferase gene revealed that the changes in tumor cell migration in mutant fibroblast/tumor cell composite spheroids coincided with a lower proliferation rate.

Conclusions/Significance

This is the first demonstration that stromal Ext1-levels modulate tumor cell proliferation and affect the interstitial fluid pressure in a 3-D spheroid model. Learning how structural changes in stromal heparan sulfate influence tumor cells is essential for our understanding how non-malignant cells of the tumor microenvironment influence tumor cell progression.