Life cycle, growth characteristics and host cell response of Rickettsia helvetica in a Vero cell line.

Rickettsia helvetica, a spotted fever rickettsia and emerging pathogen with Ixodes ricinus ticks as the main vector, is an agent of human disease and may cause febrile illness as well as meningitis. In three parallel series the isolated standard type of R. helvetica, obtained from a PCR-positive I. ricinus tick, was high-passaged and propagated in a Vero cell line. By using quantitative real-time PCR, the generation time from inoculation to stationary phase of growth was calculated to 20-22 h. In the static cultivation system the stationary phase was observed from the seventh day after inoculation, and there was no observed degradation of R. helvetica DNA during the 14 days studied. Microscopy showed that the organisms invaded the host cells rapidly and were primarily found free in the cytoplasm and only occasionally located in the nucleus. Four days after inoculation some of the host cells were broken and many indifferent stages of cytoplasmic organic decomposition were seen. However the R. helvetica organism did not show any morphologic alterations and the number of organisms was stable after the replication peak which may indicate that R. helvetica is adapted to growth in a Vero cell line and/or that the phase of degradation occurs later than the 14 days studied. The findings differ from what has been reported for other rickettsiae of the spotted fever group and may be of importance for invasiveness and virulence of R. helvetica.

Vaccination against the extra domain-B of fibronectin as a novel tumor therapy.

Monoclonal antibody-based therapies have made an important contribution to current treatment strategies for cancer and autoimmune disease. However, the cost for these new drugs puts a significant strain on the health-care economy, resulting in limited availability for patients. Therapeutic vaccination, defined as induction of immunity against a disease-related self-molecule, is therefore an attractive alternative. To analyze the potential of such an approach, we have developed a vaccine against the extra domain-B (ED-B) of fibronectin. This 91-aa domain, inserted by alternative splicing, is expressed during vasculogenesis in the embryo, but essentially undetectable under normal conditions in the adult. However, ED-B is highly expressed around angiogenic vasculature, such as in tumorigenesis. Here, we demonstrate that it is possible to break self-tolerance and induce a strong antibody response against ED-B by vaccination. Nineteen of 20 vaccinated mice responded with production of anti-ED-B antibodies and displayed a 70% reduction in tumor size compared to those lacking anti-ED-B antibodies. Analysis of the tumor tissue revealed that immunization against ED-B induced several changes, consistent with an attack by the immune system. These data show that tumor vascular antigens are highly interesting candidates for development of therapeutic vaccines targeting solid tumors.

Effect of a single dose of tobramycin on systemic inflammatory response-induced acute kidney injury in a 6-hour porcine model.

OBJECTIVE: To evaluate whether the addition of tobramycin further compromises renal function in inflammatory response-induced acute kidney injury. Effective antibiotic treatment in septic shock is crucial for the outcome. The combination of aminoglycosides with different beta-lactam antibiotics offers a broad antimicrobial coverage, rapid bacterial killing, synergistic effects, and low antibiotic-induced endotoxin release. However, aminoglycosides have nephrotoxic effects that may aggravate sepsis-induced acute kidney injury. DESIGN: Prospective, randomized, placebo-controlled experimental study. SETTING: University research unit. SUBJECTS: Twenty-four healthy pigs. INTERVENTIONS: The animals were anesthetized and randomized to four groups. Groups I (n = 8) and II (n = 8) received endotoxin infusion for 6 hrs, whereas groups III (n = 4) and IV (n = 4) received saline. Groups I and III received 7 mg/kg of tobramycin 20 mins after the initiation of the protocol, whereas groups II and IV received saline. MEASUREMENTS AND MAIN RESULTS: The renal elimination rate of a bolus dose of cefuroxime was chosen as the primary end point. Renal function was also evaluated by urine output, creatinine clearance, plasma cystatin C, plasma urea, and urine NAG (N-acetyl-beta-D-glucoaminidase). After 3 hrs, there were significantly lower cefuroxime elimination rates in the two endotoxin groups than in the nonendotoxin groups. No difference in cefuroxime elimination rates between groups I and II could be detected at any time point. Similarly, there were changes indicating acute kidney injury in urine output, creatinine clearance, and plasma cystatin C in the endotoxin groups with no differences between groups I and II. Plasma urea and urine NAG did not differ between any of the groups. CONCLUSIONS: The result of this study does not lend any support to the hypothesis that a single dose of tobramycin enhances the risk of acute renal failure in cases with systemic inflammatory response-induced acute kidney injury.

alphaB-crystallin promotes tumor angiogenesis by increasing vascular survival during tube morphogenesis.

Selective targeting of endothelial cells in tumor vessels requires delineation of key molecular events in formation and survival of blood vessels within the tumor microenvironment. To this end, proteins transiently up-regulated during vessel morphogenesis were screened for their potential as targets in antiangiogenic tumor therapy. The molecular chaperone alphaB-crystallin was identified as specifically induced with regard to expression level, modification by serine phosphorylation, and subcellular localization during tubular morphogenesis of endothelial cells. Small interfering RNA-mediated knockdown of alphaB-crystallin expression did not affect endothelial proliferation but led to attenuated tubular morphogenesis, early activation of proapoptotic caspase-3, and increased apoptosis. alphaB-crystallin was expressed in a subset of human tumor vessels but not in normal capillaries. Tumors grown in alphaB-crystallin(-/-) mice were significantly less vascularized than wild-type tumors and displayed increased areas of apoptosis/necrosis. Importantly, tumor vessels in alphaB-crystallin(-/-) mice were leaky and showed signs of caspase-3 activation and extensive apoptosis. Ultrastructural analyses showed defective vessels partially devoid of endothelial lining. These data strongly implicate alphaB-crystallin as an important regulator of tubular morphogenesis and survival of endothelial cell during tumor angiogenesis. Hereby we identify the small heat shock protein family as a novel class of angiogenic modulators.

FGFR-1 regulates angiogenesis through cytokines interleukin-4 and pleiotrophin.

The role of fibroblast growth factors (FGFs) in blood vessel formation has remained unclear. We used differentiating stem-cell cultures (embryoid bodies) and teratomas to show that FGF receptor-1 (FGFR-1) exerts a negative regulatory effect on endothelial cell function in these models. Embryoid bodies lacking expression of FGFR-1 as a result of gene targeting (Fgfr-1(-/-)) displayed increased vascularization and a distinct, elongated vessel morphology. Teratomas derived from FGFR-1-deficient stem cells were characterized by an increased growth rate and abundant, morphologically distinct vessels. Transmission electron microscopy of the Fgfr-1(-/-) teratomas showed a compact and voluminous but functional endothelium, which anastomosed with the host circulation. The increased vascularization and altered endothelial cell morphology was dependent on secreted factor(s), based on the transfer of the Fgfr-1(-/-) vascular phenotype by conditioned medium to Fgfr-1(+/-) embryoid bodies. Antibody and transcript arrays showed down-regulation of interleukin-4 (IL-4) and up-regulation of pleiotrophin in Fgfr-1(-/-) embryoid bodies, compared with the heterozygous cultures. We used neutralizing antibodies to show that IL-4 and pleiotrophin act as negative and positive angiogenic regulators, respectively. We conclude that FGFR-1 negatively regulates endothelial cell function by altering the balance of modulatory cytokines.

Serglycin proteoglycan is required for secretory granule integrity in mucosal mast cells.

SG (serglycin) PGs (proteoglycans) are strongly implicated in the assembly of MC (mast cell) granules. However, this notion has mainly been on the basis of studies of MCs of the connective tissue subtype, whereas the role of SG PG in mucosal MCs has not been explored. In the present study, we have addressed the latter issue by using mice with an inactivated SG gene. Bone marrow cells were differentiated in vitro into the mucosal MC phenotype, expressing the markers mMCP (mouse MC protease) -1 and -2. Biosynthetic labelling experiments performed on these cells revealed an approximately 80% reduction of 35SO4(2-) incorporation into PGs recovered from SG-/- cells as compared with SG+/+ counterparts, indicating that SG is the dominating cell-associated PG of mucosal MCs. Moreover, the absence of SG led to defective metachromatic staining of mucosal MCs, both in vivo and in the in vitro-derived mucosal MCs. Ultrastructural analysis showed that granules were present in similar numbers in SG+/+ and SG-/- cells, but that their morphology was markedly affected by the absence of SG, e.g. with electron-dense core formation only seen in SG+/+ granules. Analysis of the MC-specific proteases showed that mMCP-1 and mMCP-7 were completely independent of SG for storage, whereas mMCP-2 showed a partial dependence. In contrast, mMCP-4 and -6, and carboxypeptidase A were strongly dependent on SG for storage. Together, our data indicate that SG PG is of crucial importance for assembly of mature mucosal MC granules, but that the specific dependence on SG for storage varies between individual granule constituents.

Serglycin-deficient cytotoxic T lymphocytes display defective secretory granule maturation and granzyme B storage.

Cytotoxic T lymphocytes eliminate infected and tumor cells mainly by perforin/granzyme-induced apoptosis. Earlier studies suggested that serglycin-proteoglycans form macromolecular complexes with granzymes and perforin in the cytotoxic granule. Serglycin-proteoglycans may also be involved in the delivery of the cytolytic machinery into target cells. We have developed a serglycin-deficient mouse strain, and here we studied the importance of serglycin-proteoglycans for various aspects of cytotoxic T lymphocyte function. 35SO4(2-) radiolabeling of serglycin-deficient cells demonstrated a dramatic reduction of incorporated label as compared with wild type cells, indicating that serglycin is by far the dominating proteoglycan species produced by the cytotoxic T lymphocyte. Moreover, lack of serglycin resulted in impaired ability of cytotoxic T lymphocytes to produce secretory granule of high electron density, although granule of lower electron density were produced both in wild type and serglycin-deficient cells. The serglycin deficiency did not affect the mRNA expression for granzyme A, granzyme B, or perforin. However, the storage of granzyme B, but not granzyme A, Fas ligand, or perforin, was severely defective in serglycin-deficient cells. Serglycin-deficient cells did not display defects in late cytotoxicity toward target cell lines. Taken together, these results point to a key role for serglycin in the storage of granzyme B and for secretory granule maturation but argue against a major role for serglycin in the apoptosis mediated by cytotoxic T lymphocytes.

Tissue factor produced by the endocrine cells of the islets of Langerhans is associated with a negative outcome of clinical islet transplantation.

There are strong indications that only a small fraction of grafts successfully engraft in clinical islet transplantation. One explanation may be the instant blood-mediated inflammatory reaction (IBMIR) elicited by tissue factor, which is produced by the endocrine cells. In the present study, we show that islets intended for islet transplantation produce tissue factor in both the transmembrane and the alternatively spliced form and that the membrane-bound form is released as microparticles often associated with both insulin and glucagon granules. A low-molecular mass factor VIIa (FVIIa) inhibitor that indirectly blocks both forms of tissue factor was shown in vitro to be a promising drug to eliminate the IBMIR. Thrombin-antithrombin complex (TAT) and FVIIa-antithrombin complex (FVIIa-AT) were measured in nine patients who together received 20 infusions of isolated human islets. Both the TAT and FVIIa-AT complexes increased rapidly within 15-60 min after infusion. When the initial TAT and FVIIa-AT levels were plotted against the increase in C-peptide concentration after 7 days, patients with an initially strong IBMIR showed no significant increase in insulin synthesis after 7 days. In conclusion, tissue factor present in both the islets and the culture medium and elicits IBMIR, which affects the function of the transplanted islets.

Evidence of Rickettsia spp. infection in Sweden: a clinical, ultrastructural and serological study.

Sweden is an area potentially endemic for spotted fever rickettsioses. Rickettsia helvetica has been isolated from its tick vector Ixodes ricinus, and in a handful of cases linked to human disease. This study demonstrates for the first time in Sweden the transmission of rickettsial infection after a tick bite and the attack rate in an endemic area. We present three cases of documented rickettsial infection and a prospective serological study of Swedish recruits who were trained in the area where the patients lived and showed seroconversion to spotted fever rickettsiae. All patients showed a four-fold increase in antibody titer to the spotted fever rickettsia, R. helvetica, and immunohistochemical examination revealed rickettsia-like organisms in the walls of skin capillaries and veins. Electron microscopy showed organisms resembling R. helvetica and immunogold labeling with two anti-rickettsial antibodies demonstrated specific labeling of the rickettsial organisms in the skin biopsy specimens. Eight of the thirty-five recruits showed a four-fold increase in IgG titer reflecting a high rate of exposure. The results of this study demonstrate that spotted fever rickettsioses should be taken into consideration in the diagnosis of tick-transmitted infections in Sweden.

Development and characterization of two human tumor sublines expressing high-grade resistance to the cyanoguanidine CHS 828.

The cyanoguanidine CHS 828 has shown promising antitumor properties and is currently in early clinical trials, although the mechanism of action still is largely unknown. In this study, resistant sublines of the histiocytic lymphoma cell line U-937 GTB and the myeloma line RPMI 8226 were developed by culturing under gradually increasing concentrations of CHS 828 until reaching 25 times the parental line EC50s. The new phenotypes demonstrate more than 400-fold resistance to CHS 828 and cross-resistance to six cyanoguanidine analogs, but no resistance to nine standard drugs of different mechanistic classes or to the cytotoxic guanidines m-iodobenzylguanidine and methylglyoxal-bis(guanylhydrazone). The resistant phenotypes were stable for several months even if cultivated in drug-free medium and no difference in proliferation, ultrastructural or morphologic appearance in the sublines could be detected. Neither was decreased accumulation of tritium-labeled CHS 828 observed. Furthermore, the new U-937 phenotype was not accompanied by changes in differentiation or an altered cell-cycle distribution. In the myeloma cell line, esterase activity was shown to be moderately enhanced. Two-dimensional protein electrophoresis was undertaken to unmask possible resistance-mediating proteins and/or the target molecule(s) for CHS 828. In the myeloma cell line, lambda light chain immunoglobulin (down-regulated) and a fatty acid-binding protein (up-regulated) were identified. The findings presented here indicate that development of specific cellular alterations is responsible for the gained CHS 828 resistance.

Cellular expression and specific intragranular localization of chromogranin A, chromogranin B, and synaptophysin during ontogeny of pancreatic islet cells: an ultrastructural study.

INTRODUCTION AND AIMS: To get more insight into the differentiation patterns of pancreatic islet neuroendocrine cells and granules during ontogeny, the expression and localization of chromogranin A (CgA), chromogranin B (CgB), synaptophysin, and insulin were ultrastructurally studied with the immunogold technique in porcine and human pancreatic islet neuroendocrine cells. METHODOLOGY AND RESULTS: In porcine pancreas at early fetal stage, CgA was visualized throughout the immature granules in all neuroendocrine cells. Later, CgB also was expressed with the same pattern in most granules in all types of cells. In neonatal islets, CgA was localized in the periphery of immature alpha- and beta-cell granules and throughout the matrix of delta-cell granules; CgB was distributed throughout the matrix of these granules. In adult islets, alpha-cell granules stored CgA in the halo and CgB in both the core and the halo, beta-cell granules stored both CgA and CgB in their cores, and in delta-cell granules, both substances were mixed throughout the matrix. In all ontogenetic stages, synaptophysin was demonstrated in all cell types and granules. Insulin was expressed in early fetal cells of both pigs and humans, and colocalization with CgA, CgB, and synaptophysin was demonstrated. CONCLUSION: The early expression of CgA and synaptophysin may reflect a role at early fetal stages, and the individual localization of CgA and CgB upon differentiation indicates individual functions.

PACAP is expressed in secretory granules of insulin and glucagon cells in human and rodent pancreas. Evidence for generation of cAMP compartments uncoupled from hormone release in diabetic islets.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is an islet neuropeptide with potent insulinotropic action. The current study investigates PACAP expression in normal human and rat pancreatic islets, and whether it is altered in diabetic state. To that end, PACAP immunoreactivity was studied by immunofluorescence methods enhanced by the catalyzed reporter deposition (CARD) technique. Insulin and cyclic adenosine monophosphate (cAMP) generation induced by PACAP were investigated in islets isolated from the spontaneously diabetic Goto-Kakizaki (GK) rat. PACAP immunoreactivity was observed in virtually all insulin and glucagon cells in both species, but not in somatostatin or pancreatic polypeptide (PP) cells; this co-localization pattern was unaltered in diabetic pancreata. In normal human pancreas, PACAP was further localized ultrastructurally to the secretory granules of insulin and glucagon cells. PACAP significantly potentiated glucose-stimulated insulin release in isolated islets of normal but not of GK rats. PACAP failed to enhance cAMP generation in normal islets, but induced approximately 5-folds exaggeration in the diabetic islets. In conclusion, using improved immunocytochemistry techniques and electron microscopy (EM), PACAP was shown to be expressed both in normal and diabetic islet cells and localized to secretory granules of insulin and glucagon cells. Furthermore, the insulinotropic action of PACAP was markedly impaired in diabetic islets in spite of exaggerated cAMP response.

Presence of Rickettsia helvetica in granulomatous tissue from patients with sarcoidosis.

In samples obtained during the autopsies of 2 patients with sarcoidosis, genetic material from Rickettsia helvetica was detected by polymerase chain reaction, and histologic and immunohistochemical examination (using 3 different antibodies) of the polymerase chain reaction-positive tissues showed different degrees of granuloma formation and presence of rickettsia-like organisms predominantly located in the endothelium and macrophages. Electron microscopic examination clearly identified and demonstrated rickettsia-like organisms within the granuloma, with findings suggestive of ongoing infection. Immunogold labeling with Proteus OX-19 antiserum showed that the gold markers were localized to the rickettsia-like organisms. Paraffin-embedded biopsy specimens from 30 patients with confirmed sarcoidosis were also reexamined, and 26 specimens were judged to be positive for rickettsia-like organisms by histologic and immunohistochemical examination. In a specimen from 1 patient, rickettsia-like organisms also were demonstrated and identified by transmission electron microscopy. These results support the hypothesis that rickettsiae may contribute to a granulomatous process, as is seen in sarcoidosis.

Chlamydia pneumoniae in patients undergoing surgery for thoracic aortic disease.

OBJECTIVE: To investigate if Chlamydia pneumoniae is present in the wall of the thoracic aorta in patients operated on for aneurysm or aortic dissection. DESIGN: Consecutive patients undergoing surgery for thoracic aortic aneurysm (TAA, 32 patients) and for aortic dissection (6 patients) were included in this prospective study. Tissue samples from the aorta were analysed for the presence of C. pneumoniae by polymerase chain reaction (PCR), histopathology, immunohistochemistry and in one aortic tissue sample C. pneumoniae was verified by electron microscopy and immunogold labelling technique. Cultured Hep 2 cells infected with C. pneumoniae were used as a positive control for electron microscopy. Sera for microimmunofluorescence were obtained in 36/38 and throat swabs for C. pneumoniae PCR in 17/38 patients. RESULTS: Chlamydia pneumoniae was detected by PCR in 4 of 32 TAA tissue samples (12%) and in 0 of 6 patients operated on for aortic dissection. Chlamydia pneumoniae inclusion bodies in one of the PCR positive tissue samples were verified by electron microscopy. IgG antibodies to C. pneumoniae were present in 17/31 (55%) and IgA in 15/31 (48%) of the TAA patients and in none of five tested patients with dissection. None of the tested throat swabs was positive. CONCLUSION: In this study we report the presence of C. pneumoniae by PCR and electron microscopy in the wall of TAA. A high prevalence of serum IgA antibodies to C. pneumoniae was found in TAA patients. In contrast no signs of C. pneumoniae were detected in patients with thoracic aortic dissection.