Ultrastructural pathology and interorganelle cross talk in hepatotoxicity.

Mitochondria, endoplasmic reticulum (ER), cytoplasmic lipid droplets (CLD), and Golgi vesicles use cross talk to control hepatocyte metabolism, growth, and stress. Interpretation of ultrastructural change requires knowledge of how cross talk pathways function, how differential activation of hepatocellular signals influences organelle structure, and how organelles position themselves to become central hubs for stress responses. Mitochondria, by coupling energy production to pathways for protection, form critical platforms for innate signaling. Mitochondrial outer and inner membranes activate channels and signals to translocate peptides that drive oxidative phosphorylation, ß-oxidation of fatty acids, and calcium ion (Ca(2+)) flux. In cell stress, mitochondrial signals initiate fusion and fission, reactive oxygen species (ROS) control, autophagy, apoptosis, and senescence. Specialized tethering proteins tie mitochondria to ER to support translocation of metabolites. For Ca(2+) translocation, ER pores are connected to mitochondrial voltage-dependent anion channels, and for mitochondrial fission, unique membrane proteins pull ER to mitochondria. In toxic injury, cytosolic cytokines translocate to alter metabolism. Toxic effects on ER lipid synthesis lead to Golgi vesicle reduplication and transport of perilipin and other protein cargos into CLDs. How cellular proteostasis, oxidative homeostasis, and ion balance are maintained depend upon the effectiveness of mitochondrial ROS defense responses, unfolded protein responses in mitochondria and ER, and other organelle defenses.

Gliomatosis cerebri in two dogs.

A 3.5 yr old Saint Bernard was evaluated for nonambulatory tetraparesis and cranial nerve dysfunction, and a 7 yr old rottweiler was evaluated for progressive paraparesis. Clinical signs of left-sided vestibular and general proprioceptive ataxia and cranial nerve VII dysfunction in the Saint Bernard suggested a lesion affecting the brain stem. Signs in the rottweiler consisted of general proprioceptive/upper motor neuron paraparesis, suggesting a lesion involving the third thoracic (T3) to third lumbar (L3) spinal cord segments. MRI was normal in the Saint Bernard, but an intra-axial lesion involving the T13-L2 spinal cord segments was observed in the rottweiler. In both dogs, the central nervous system (CNS) contained neoplastic cells with features consistent with gliomatosis cerebri (GC). In the Saint Bernard, neoplastic cells were present in the medulla oblongata and cranial cervical spinal cord. In the rottweiler, neoplastic cells were only present in the spinal cord. Immunohistochemistry disclosed two distinct patterns of CD18, nestin, and vimentin staining. GC is a rarely reported tumor of the CNS. Although GC typically involves the cerebrum, clinical signs in these two dogs reflected caudal brainstem and spinal cord involvement.

Investigation of antigen-specific T-cell responses and subcutaneous granuloma development during experimental sensitization of calves with Mycobacterium avium subsp paratuberculosis.

OBJECTIVE: To characterize the early cellular immune response to Mycobacterium avium subsp paratuberculosis (MAP) infection and evaluate the development of granulomatous inflammation at the SC injection site in experimentally inoculated calves. ANIMALS: Forty-eight 4-week-old calves. PROCEDURE: Calves received an SC injection of MAP strain 19698 (n = 25), sterile saline (0.9% NaCl) solution (20), or a commercial paratuberculosis vaccine (3); the inoculation site tissue and associated draining lymph node were excised at postinoculation day (PID) 0 (n = 36), 7 (14), 14 (6), 21 (8), and 60 (32). Sections of inoculation site tissues were evaluated immunohistochemically for T-cell subsets; lymph node mononuclear cells (LNMCs) were assessed for T-cell surface markers and for intracellular interferon-gamma via flow cytometry. RESULTS: At MAP inoculation sites, calves developed mild, focal granulomatous inflammation by PID 7; by PID 60, areas of inflammation contained macrophages with numerous lymphocytes. Compared with control calves, there was increased antigen-specific LNMC proliferation in MAP- and vaccine-inoculated calves at PID 60, although proliferation among lymphocyte subsets was not significantly different between MAP-inoculated and control calves; in vaccine-inoculated calves, CD4+ T-cells predominated. In MAP-inoculated and control calves, antigen-specific interferon-gamma production by LNMCs did not differ significantly; vaccine-inoculated calves had marked interferon-gamma expression by CD4+ T-cells. CONCLUSIONS AND CLINICAL RELEVANCE: In calves, SC administration of MAP resulted in granulomatous inflammation at inoculation sites and an antigen-specific T-cell proliferative response. Results suggest that this experimental system can be used to reproducibly generate antigen-specific T-cells during MAP infection for functional analysis.

Clinical and pathologic comparison of acute leptospirosis in dogs caused by two strains of Leptospira kirschneri serovar grippotyphosa.

OBJECTIVE: To develop a method for inducing acute leptospirosis in dogs. ANIMALS: 31 nine-week-old female Beagles. PROCEDURE: Beagles were randomly assigned to 2 inoculation groups or a control group. Dogs were inoculated on 3 successive days by conjunctival instillation of 5 x 10(7) cells of Leptospira kirschneri serovar grippotyphosa strain 82 (12 dogs) or strain RM 52 (14 dogs). Control dogs (n = 5) were similarly inoculated with sterile leptospiral culture media. Clinical signs, clinicopathologic variables, anti-leptospiral antibody titers, and evidence of leptospires in tissues and body fluids were evaluated. Dogs were euthanatized and necropsied on days 7, 14, 22, or 28 after inoculation or as required because of severe illness. RESULTS: Clinical signs in infected dogs included conjunctivitis, lethargy, diarrhea, dehydration, vomiting, and icterus. Consistent clinicopathologic alterations included azotemia, hyperphosphatemia, increased anion gap, hyperbilirubinemia, and an increase in alkaline phosphatase activity. Leptospires were cultured from the kidneys (11/12), urine (6/9), aqueous humor (9/12), blood (12/12), and liver (12/12) of dogs inoculated with strain 82. Only 3 of 14 dogs became infected after inoculation with strain RM 52. Histopathologic lesions in infected dogs included interstitial nephritis, renal tubular degeneration and necrosis, pulmonary hemorrhage, and hepatic edema and perivasculitis. CONCLUSIONS AND CLINICAL RELEVANCE: Conjunctival exposure to L kirschneri serovar grippotyphosa strain 82 resulted in acute leptospirosis in all inoculated dogs, but only 3 of 14 dogs inoculated with strain RM 52 became infected. This method of infection by serovar grippotyphosa can be used to study the pathogenesis and prevention of leptospirosis in dogs.

Absence of Mycobacterium avium subspecies paratuberculosis components from Crohn's disease intestinal biopsy tissues.

BACKGROUND: Crohn's disease is a chronic human intestinal inflammatory disorder for which an etiologic agent has not been identified. Johne's disease is a similar chronic enteric granulomatous disease of ruminant species and has been used as a model of Crohn's disease. Johne's disease has been proven to be caused by Mycobacterium avium subspecies paratuberculosis (M. avium ss paratuberculosis). It has been proposed that M. avium ss paratuberculosis may also cause Crohn's disease. This is of particular concern because the organism may be spread to humans through inadequately pasteurized dairy products. OBJECTIVE: We sought to determine whether M. avium ss paratuberculosis could be detected using identical techniques in paraffin-embedded tissue samples of bovine Johne's disease and human Crohn's, ulcerative colitis and diverticular diseases. Samples were obtained for analysis from national tissue banks. DESIGN: Cross-species and cross-disease sample comparisons by multiple detection techniques. METHODS: Histology, immunocytochemistry and polymerase chain reaction (PCR) were utilized to test and compare the presence of M. avium ss paratuberculosis components. Insertion sequence IS900, present in multiple copies and found only in M. avium ss paratuberculosis, was utilized in both PCR and immunocytochemical analyses. RESULTS: The IS900 sequence was demonstrable in all samples of confirmed positive Johne's disease tissue. The sequence was not identified in the 35 Crohn's, 36 ulcerative colitis, and 21 diverticular disease samples. CONCLUSION: M. avium ss paratuberculosis was not associated with the lesions in these Crohn's disease samples, using these methods.

Cytokine effects on maturation of the phagosomes containing Mycobacteria avium subspecies paratuberculosis in J774 cells.

Mycobacterium avium subspecies paratuberculosis (M. a. ptb) is an intracellular pathogen of macrophages. Intracellular survival of several species of pathogenic mycobacteria is dependent on inhibition of maturation of the phagosomes containing these pathogens into functional phagolysosomes. In activated macrophages, however, this capacity is reduced, leading to increased bacterial killing. It is the hypothesis of this study that there is increased acidification and maturation of the phagosome containing M. a. ptb in interferon gamma and lipopolysaccharide (IFN-gamma/LPS) activated macrophages. In activated macrophages colocalization of M. a. ptb with either a marker of acidic compartments (Lysotracker Red) or compartments containing a late phagosome maturation marker lysosome-associated membrane protein-1 (Lamp-1) were evaluated by laser confocal microscopy. Intracellular survival of M. a. ptb in activated macrophages was evaluated directly using differential fluorescent live/dead staining. The results of this study demonstrated increased colocalization of both Lysotracker Red and Lamp-1 with FITC labeled M. a. ptb, which correlated with decreased survival of M. a. ptb within activated macrophages.

Segmented filamentous bacteria interact with intraepithelial mononuclear cells.

Segmented filamentous bacteria (SFB) are found in multiple species and play an important role in the development of mucosal immunity. The mechanism by which the bacteria interact with the immune system has not been well defined. We provide morphologic evidence of direct interaction between SFB and intraepithelial mononuclear cells.