Waddlia chondrophila induces systemic infection, organ pathology, and elicits Th1-associated humoral immunity in a murine model of genital infection

Waddlia chondrophila is a known bovine abortigenic Chlamydia-related bacterium that has been associated with adverse pregnancy outcomes in human. However, there is a lack of knowledge regarding how W. chondrophila infection spreads, its ability to elicit an immune response and induce pathology. A murine model of genital infection was developed to investigate the pathogenicity and immune response associated with a W. chondrophila infection. Genital inoculation of the bacterial agent resulted in a dose-dependent infection that spread to lumbar lymph nodes and successively to spleen and liver. Bacterial-induced pathology peaked on day 14, characterized by leukocyte infiltration (uterine horn, liver, and spleen), necrosis (liver) and extramedullary hematopoiesis (spleen). Immunohistochemistry demonstrated the presence of a large number of W. chondrophila in the spleen on day 14. Robust IgG titers were detected by day 14 and remained high until day 52. IgG isotypes consisted of high IgG2a, moderate IgG3 and no detectable IgG1, indicating a Th1-associated immune response. This study provides the first evidence that W. chondrophila genital infection is capable of inducing a systemic infection that spreads to major organs, induces uterus, spleen, and liver pathology and elicits a Th1-skewed humoral response. This new animal model will help our understanding of the mechanisms related to intracellular bacteria-induced miscarriages, the most frequent complication of pregnancy that affects one in four women.

Murine coronavirus ubiquitin-like domain is important for papain-like protease stability and viral pathogenesis

Ubiquitin-like domains (Ubls) now are recognized as common elements adjacent to viral and cellular proteases; however, their function is unclear. Structural studies of the papain-like protease (PLP) domains of coronaviruses (CoVs) revealed an adjacent Ubl domain in severe acute respiratory syndrome CoV, Middle East respiratory syndrome CoV, and the murine CoV, mouse hepatitis virus (MHV). Here, we tested the effect of altering the Ubl adjacent to PLP2 of MHV on enzyme activity, viral replication, and pathogenesis. Using deletion and substitution approaches, we identified sites within the Ubl domain, residues 785 to 787 of nonstructural protein 3, which negatively affect protease activity, and valine residues 785 and 787, which negatively affect deubiquitinating activity. Using reverse genetics, we engineered Ubl mutant viruses and found that AM2 (V787S) and AM3 (V785S) viruses replicate efficiently at 37°C but generate smaller plaques than wild-type (WT) virus, and AM2 is defective for replication at higher temperatures. To evaluate the effect of the mutation on protease activity, we purified WT and Ubl mutant PLP2 and found that the proteases exhibit similar specific activities at 25°C. However, the thermal stability of the Ubl mutant PLP2 was significantly reduced at 30°C, thereby reducing the total enzymatic activity. To determine if the destabilizing mutation affects viral pathogenesis, we infected C57BL/6 mice with WT or AM2 virus and found that the mutant virus is highly attenuated, yet it replicates sufficiently to elicit protective immunity. These studies revealed that modulating the Ubl domain adjacent to the PLP reduces protease stability and viral pathogenesis, revealing a novel approach to coronavirus attenuation. IMPORTANCE Introducing mutations into a protein or virus can have either direct or indirect effects on function. We asked if changes in the Ubl domain, a conserved domain adjacent to the coronavirus papain-like protease, altered the viral protease activity or affected viral replication or pathogenesis. Our studies using purified wild-type and Ubl mutant proteases revealed that mutations in the viral Ubl domain destabilize and inactivate the adjacent viral protease. Furthermore, we show that a CoV encoding the mutant Ubl domain is unable to replicate at high temperature or cause lethal disease in mice. Our results identify the coronavirus Ubl domain as a novel modulator of viral protease stability and reveal manipulating the Ubl domain as a new approach for attenuating coronavirus replication and pathogenesis.

Response profiles of murine spiral ganglion neurons on multi-electrode arrays.

OBJECTIVE Cochlear implants (CIs) have become the gold standard treatment for deafness. These neuroprosthetic devices feature a linear electrode array, surgically inserted into the cochlea, and function by directly stimulating the auditory neurons located within the spiral ganglion, bypassing lost or not-functioning hair cells. Despite their success, some limitations still remain, including poor frequency resolution and high-energy consumption. In both cases, the anatomical gap between the electrode array and the spiral ganglion neurons (SGNs) is believed to be an important limiting factor. The final goal of the study is to characterize response profiles of SGNs growing in intimate contact with an electrode array, in view of designing novel CI devices and stimulation protocols, featuring a gapless interface with auditory neurons. APPROACH We have characterized SGN responses to extracellular stimulation using multi-electrode arrays (MEAs). This setup allows, in our view, to optimize in vitro many of the limiting interface aspects between CIs and SGNs. MAIN RESULTS Early postnatal mouse SGN explants were analyzed after 6-18 days in culture. Different stimulation protocols were compared with the aim to lower the stimulation threshold and the energy needed to elicit a response. In the best case, a four-fold reduction of the energy was obtained by lengthening the biphasic stimulus from 40 μs to 160 μs. Similarly, quasi monophasic pulses were more effective than biphasic pulses and the insertion of an interphase gap moderately improved efficiency. Finally, the stimulation with an external electrode mounted on a micromanipulator showed that the energy needed to elicit a response could be reduced by a factor of five with decreasing its distance from 40 μm to 0 μm from the auditory neurons. SIGNIFICANCE This study is the first to show electrical activity of SGNs on MEAs. Our findings may help to improve stimulation by and to reduce energy consumption of CIs and thereby contribute to the development of fully implantable devices with better auditory resolution in the future.

Impact of platelet rich plasma and adipose stem cells on lymphangiogenesis in a murine tail lymphedema model.

BACKGROUND Lymphedema is an underdiagnosed pathology which in industrialized countries mainly affects cancer patients that underwent lymph node dissection and/or radiation. Currently no effective therapy is available so that patients' life quality is compromised by swellings of the concerned body region. This unfortunate condition is associated with body imbalance and subsequent osteochondral deformations and impaired function as well as with an increased risk of potentially life threatening soft tissue infections. METHODS The effects of PRP and ASC on angiogenesis (anti-CD31 staining), microcirculation (Laser Doppler Imaging), lymphangiogenesis (anti-LYVE1 staining), microvascular architecture (corrosion casting) and wound healing (digital planimetry) are studied in a murine tail lymphedema model. RESULTS Wounds treated by PRP and ASC healed faster and showed a significantly increased epithelialization mainly from the proximal wound margin. The application of PRP induced a significantly increased lymphangiogenesis while the application of ASC did not induce any significant change in this regard. CONCLUSIONS PRP and ASC affect lymphangiogenesis and lymphedema development and might represent a promising approach to improve regeneration of lymphatic vessels, restore disrupted lymphatic circulation and treat or prevent lymphedema alone or in combination with currently available lymphedema therapies.

The Topoisomerase I Inhibitor Irinotecan and the Tyrosyl-DNA Phosphodiesterase 1 Inhibitor Furamidine Synergistically Suppress Murine Lupus Nephritis

OBJECTIVE The treatment of lupus nephritis is still an unmet medical need requiring new therapeutic approaches. Our group found recently that irinotecan, an inhibitor of topoisomerase I (topo I), reversed proteinuria and prolonged survival in mice with advanced lupus nephritis. While irinotecan is known to stabilize the complex of topo I and DNA, the enzyme tyrosyl-DNA phosphodiesterase 1 (TDP-1) functions in an opposing manner by releasing topo I from DNA. Therefore, we undertook this study to test whether the TDP-1 inhibitor furamidine has an additional effect on lupus nephritis when used in combination with irinotecan. METHODS NZB/NZW mice were treated with low-dose irinotecan and furamidine either alone or in combination beginning at age 26 weeks. DNA relaxation was visualized using gel electrophoresis. Binding of anti-double-stranded DNA (anti-dsDNA) antibodies to DNA modified by topo I, TDP-1, and the topo I inhibitor camptothecin was determined by enzyme-linked immunosorbent assay. RESULTS Compared to treatment with either agent alone, simultaneous treatment with low-dose irinotecan and furamidine significantly improved survival of NZB/NZW mice. Similar to what has been previously shown for irinotecan alone, the combination treatment did not change the levels of anti-dsDNA antibodies. In vitro, recombinant TDP-1 increased topo I-mediated DNA relaxation, resulting in enhanced binding of anti-dsDNA antibodies. In combination with topo I and camptothecin, TDP-1 reversed the inhibitory effects of camptothecin on DNA relaxation and anti-dsDNA binding. CONCLUSION Affecting DNA relaxation by the enzymes topo I and TDP-1 and their inhibitors may be a promising approach for the development of new targeted therapies for systemic lupus erythematosus.

Acute Cytomegalovirus Hepatitis in an Immunocompetent Host as a Reason for Upper Right Abdominal Pain

Cytomegalovirus infections are widely distributed with a seroprevalence of up to 100%. The majority of the cases take a silent course or deal with unspecific clinical symptoms. Complications in immunocompetent patients are rare but may affect the liver and lead up to an acute organ failure. In this case report, we describe a 35-year-old immunocompetent female with an acute cytomegalovirus infection presenting as acute hepatitis with ongoing upper right abdominal pain after cholecystectomy. Upper right abdominal pain is a common symptom with a wide range of differential diagnoses. If common reasons can be excluded, we want to sensitize for cytomegalovirus infection as a minor differential diagnosis even in immunocompetent patients.

Protective effect of a germline, IL-17-neutralizing antibody in murine models of autoimmune inflammatory disease.

Monoclonal antibodies (mAbs) inhibiting cytokines have recently emerged as new drug modalities for the treatment of chronic inflammatory diseases. Interleukin-17 (IL-17) is a T-cell-derived central mediator of autoimmunity. Immunization with Qβ-IL-17, a virus-like particle based vaccine, has been shown to produce autoantibodies in mice and was effective in ameliorating disease symptoms in animal models of autoimmunity. To characterize autoantibodies induced by vaccination at the molecular level, we generated mouse mAbs specific for IL-17 and compared them to germline Ig sequences. The variable regions of a selected hypermutated high-affinity anti-IL-17 antibody differed in only three amino acid residues compared to the likely germline progenitor. An antibody, which was backmutated to germline, maintained a surprisingly high affinity (0.5 nM). The ability of the parental hypermutated antibody and the derived germline antibody to block inflammation was subsequently tested in murine models of multiple sclerosis (experimental autoimmune encephalomyelitis), arthritis (collagen-induced arthritis), and psoriasis (imiquimod-induced skin inflammation). Both antibodies were able to delay disease onset and significantly reduced disease severity. Thus, the mouse genome unexpectedly encodes for antibodies with the ability to functionally neutralize IL-17 in vivo.

STUDIES ON THE MECHANISM OF ASSEMBLY OF MURINE LEUKEMIA VIRUS

The purpose of this research was to elucidate the mechanism of assembly of retroviruses, specifically of murine leukemia virus, as studied through the treatment of virus-infected cells with interferon and through the use of temperature sensitive (ts) mutants. Our studies have shown a rapid and specific association of Rauscher murine leukemia virus (R-MuLV) precursor polyprotein Pr65('gag) with cytoskeletal elements in infected mouse fibroblasts. The Pr65('gag) associated with Nonidet P-40 (NP40)-insoluble cytoskeletal structures appeared to be subphosphorylated in comparison to NP40-soluble Pr65('gag). The association of Pr65('gag) with skeletal elements could be disrupted by extraction of the cytoskeleton with sodium deoxycholate, an ionic detergent. Both the skeleton-associated Pr65('gag) and its NP40-soluble counterpart were labeled with {('3)H}-palmitate, indicating their probable association with lipids presumably in the plasma membrane. Pr65('gag) molecules bound to skeletal elements in the infected cell appeared to be more stable to proteolytic processing than NP40-soluble Pr65('gag). Our studies with certain ts mutants of murine leukemia virus, defective in virus assembly, including Mo-MuLV ts3 and R-MuLV ts17, ts24, ts25 and ts26, have shown that virions released at 39(DEGREES)C (nonpermissive temperature) had high levels of uncleaved Pr65('gag) relative to that seen in virions released at 33(DEGREES)C (permissive temperature). Examination of cell extracts revealed that Pr54('gag) was more stable to processing at 39(DEGREES)C than at 33(DEGREES)C, whereas the 'env' and glycosylated 'gag' proteins were processed to the same extent at both temperatures. Detergent extraction of pulse-labeled cells to generate an NP40-insoluble cytoskeleton-enriched fraction showed that in ts3-, ts17- and ts24-infected cells, Pr65('gag) accumulated in the cytoskeleton-enriched fraction. In contrast, cells infected with ts25 or ts26 showed no preferential localization of Pr65('gag) in the cytoskeleton in a short pulse, but instead, Pr65('gag) accumulated in both the NP40-soluble and -insoluble fractions during a chase-incubation. The association of Pr65('gag) with cytoskeletal elements in the cell was neither increased nor decreased by blocking virus assembly and release with interferon. Based on these and other results, we have proposed a model for the active role of cytoskeleton-associated Pr65('gag) in retrovirus assembly.^

BIOLOGICAL AND BIOCHEMICAL CHARACTERIZATION OF MURINE-TUMOR SPECIFIC TRANSPLANTATION ANTIGENS (ISOTACHOPHORESIS, IMMUNOGENICITY)

Tumor-specific transplantation antigens (TSTA) are individually distinct neoantigens expressed on the cells of chemically-induced neoplasms. TSTA are operationally defined by immunization of syngeneic mice against challenge with viable tumor cells. Immunization with cell surface or extracted TSTA induces specific resistance to transplanted tumor cells. The biological and biochemical nature of TSTA was investigated in the 3-methylcholanthrene-induced fibrosarcomas of female C3H/HeJ mice, MCA-F and MCA-D. Tumor cell suspensions were extracted by treatment with 3M KCl or 2.5% butanol solutions and the TSTA was partially purified by preparative isoelectric focusing. The isoelectric pH of TSTA purified from 3M KCl extracts was 5.8-6.0, and from butanol extracts was 6.4-6.6. Whereas immunization with 10('5) and 10('6) irradiated tumor cells induces complete rejection of tumor cell challenge over a two-fold-log dose range, immunization with ug quantities within a one-fold-log dose range of extracted TSTA induces only partial resistance to tumor challenge. Reduced immunogenicity of extracted TSTA is hypothesized to result from immunization of mice with insufficiently purified TSTA preparations. The hypothesis predicts that immunization with highly purified TSTA, free from interfering substances, induces complete rejection of tumor challenge over a broad dose range. To test the hypothesis preparative isotachophoresis (pITP) was used to purify TSTA from electrofocused TSTA fractions. Significant purification was achieved, as immunization with 15 pg to 1.5 ug (5 logs) of pITP-purified TSTA extracted from the MCA-F, or with 1 pg to 10 ng (4 logs) of TSTA from the MCA-D tumor induced specific resistance to tumor challenge. Despite 50,000 fold purification of TSTA, immunization induced partial, not complete, rejection of transplanted tumor cells. This suggests a clear dissociation of the immunogenicity and purification of extracted TSTA, indicating that the induction of partial immunity to tumor challenge is an intrinsic property of extracted TSTA.^

AUGMENTATION OF RECONSTITUTION OF GUT ASSOCIATED LYMPHOID TISSUE IN THE SYNGENEIC MURINE BONE MARROW TRANSPLANTATION MODEL

Gut was studied as a prototypical mucosal membrane in the murine BDF-1 syngeneic bone marrow transplant model. Measures of jejunal intraepithelial lymphocytes (IELs) and crypt cells were obtained by standard techniques and a method of quantifying gut lamina propria plasma cells (PCs) was developed. The degree of ablation of gut PCs and IELs after 900 rads total body irradiation with ('60)Co, and their repopulation effected by transplantation with 2.0 x 10('5) or 1.0 x 10('6) bone marrow cells demonstrated a prolonged period of profound depression in population levels of these cells which was not reflected by the extent of damage sustained to the epithelium. Differences in the depopulation and recovery of gut PCs and IELs revealed a tendency towards initial differentiation of effector cells. A positive dose response to high bone marrow cell innocula was obtained. Subsequent studies determined that gut IEL and PC repopulation was potentiated by the addition of IELs or buffy coat cells (BCs) to the bone marrow transplant. A method of isolating 1.4 - 4.0 x 10('7) viable IELs per gram of murine small bowel was devised employing intralumenal hyaluronidase digestion of the epithelial layer and centrifugation of the resulting suspension through discontinuous Percoll gradients. Irradiated mice received 2.0 x 10('5) bone marrow cells along with an equal number of IELs or BCs. The extent and duration of depression of numbers of IELs and PCs was markedly reduced by the addition of the IEL isolate to the transplantation innocula, and to a lesser degree by the addition of BCs. The augmentation of repopuation far exceeded that expected by simple lodging of cells suggesting that the additionally transplanted cells contained a subpopulation of mucosal membrane lymphoid stem cells or helper cells. Correlation analysis of PC versus IEL levels suggests a possible feedback mechanism governing the relative size of their populations. Normal ratios of IgA, IgM, and IgG bearing PCs was maintained post transplantation with all of the regimens. ^

CHARACTERIZATION OF SERINE KINASE ACTIVITY ASSOCIATED WITH MOLONEY MURINE SARCOMA VIRUS V-MOS GENE PRODUCTS

Various Moloney murine sarcoma virus (Mo-MuSV) isolates contain a cellular sequence, termed mos, which is responsible for the transforming ability of Mo-MuSV. A serine kinase activity has been found to be associated with mos gene products of several isolates of Mo-MuSV. A mutant of Mo-MuSV strain 124 (designated MuSV ts110) is temperature-sensitive (ts) for transformation and encodes two proteins, P85('gag-mos) (an 85,000 M(,r) protein encoded by the gag and mos genes) and P58('gag), at the permissive temperature (28(DEGREES)C). At the nonpermissive temperature (39(DEGREES)C), only P58('gag) is found in MuSV ts110-infected NRK cells (6m2 cells). Both P85('gag-mos) and P58('gag) were phosphorylated when anti-gag immune complexes containing these proteins were incubated at 22(DEGREES)C with (lamda)-('32)P -ATP and MnCl(,2). The kinase detected in anti-gag complexes from 6m2 cells at permissive temperature was associated with P85('gag-mos) since immune complexes from 39(DEGREES)C 6m2 cells, which lack P85('gag-mos), produced no phosphorylated P58('gag) molecules. In addition, an anti-mos complex (anti-mos 37-55 complexes) allowed in vitro phosphorylation of P85('gag-mos) in the absence of P58('gag). No kinase activity was detectable with other gag gene products (e.g., Mo-MuSV-124 P62('gag)), suggesting that the P85('gag-mos) kinase activity was present within the mos portion of the protein. The P85('gag-mos) kinase activity was very thermolabile upon shifting 6m2 cells from permissive to nonpermissive temperatures (t(, 1/2) for inactivation = 5 min). In contrast, a spontaneous revertant of MuSV ts110 encodes a larger gag-mos protein (termed P100('gag-mos)) which contained a kinase activity stable to 39(DEGREES)C. Using the optimal conditions developed for the P85('gag-mos) kinase, Mo-MuSV-encoded p37('mos) was also found to be associated with a serine kinase activity. Phosphorylation of p37('mos) and a 43 Kd protein (super-phosphorylated p37('mos)) occurred in anti-mos(37-55) complexes from Mo-MuSV-124 acutely-infected NIH 3T3 cells, but neither in mos 37-55 peptide-blocked anti-mos(37-55) complexes nor in immune complexes from uninfected NIH 3T3 cells. Antibodies directed against the C-terminus of v-mos were found to inhibit the in vitro phosphorylation of p37('mos), suggesting that the extreme C-terminal sequence of v-mos may be important for an intrinsic kinase activity. This inhibitory action by antibodies to the C-terminus of p37('mos), when considered with all the other data reported here, provides convincing evidence that the v-mos gene encodes a serine protein kinase activity. ^

STUDIES ON THE CLASS I GLYCOPROTEINS OF THE MURINE MAJOR HISTOCOMPATIBILITY COMPLEX (TRANSPLANTATION ANTIGEN, LYMPHOCYTE, CELL SURFACE MOLECULE)

A series of studies were undertaken to analyze and compare various aspects of murine class I glycoproteins. An initial area of investigation characterized the Qa-1 alloantigens using two-dimensional gel electrophoresis. Analysis of the products of the Qa-1('b), Qa-1('c) and Qa-1('d) alleles indicated that these were distinct molecules as determined by their lack of comigration upon comparative two-dimensional gel analysis. The importance of asparagine-linked glycosylation in the cell surface expression of class I molecules was also examined. These studies employed tunicamycin, an inhibitor of N-linked glycosylation. Tunicamycin treatment of activated T lymphocytes diminished the surface expression of Qa-1 to undetectable levels; the levels of other class I molecules exhibited little or no decrease. These results indicated that N-linked glycosylation has a differential importance in the cell surface expression of various class I molecules. The molecular weight diversity of class I molecules was also investigated. Molecular weight determination of both the fully glycosylated and unglycosylated forms of H-2 and Qa/Tla region encoded molecules established that there is a significant variation in the sizes of these forms of various class I molecules. The most significant difference ((TURN)9,000 daltons) exists between the unglycosylated forms of H-2K('b) and Qa-2, suggesting that the structural organization of these two molecules may be very different. A comparative two-dimensional gel analysis of various class I glycoproteins isolated from resting and activated T and B lymphocytes indicated that class I molecules expressed on activated T cells exhibited an isoelectrophoretic pattern that was distinct from the isoelectrophoretic pattern of class I molecules expessed on the other cell populations. This difference was attributed to a lower sialic acid content of the molecules expressed on activated T cells. Analysis of cell homogenates determined that activated T cells contained a higher level of endogenous neuraminidase activity than was detected in the other populations, suggesting that this may be the basis of the lower sialic acid content. The relationship of the Qa-4 and Qa-2 alloantigens was also examined. It was established that upon mitogen activation, the expression of Qa-4 was greatly decreased, whereas Qa-2 expression was not decreased. However, an anti-Qa-2 monoclonal antibody blocked the binding of an anti-Qa-4 monoclonal antibody to resting cells. These studies established that Qa-4 is a determinant restricted to resting cells, which is closely associated on the surface with the Qa-2 molecule. ^