Synergistic effect of human lactoferrin and recombinant murine interferon-gamma on disease progression in mice infected with the polycythemia-inducing strain of the Friend virus complex.

Mice infected with the polycythemia-inducing strain of the Friend virus complex (FVC-P) have been used as a leukemic mouse model. In the present study, purified iron-saturated human lactoferrin (LF) and recombinant murine (rmu) interferon-gamma (IFN-gamma), alone or in combination, were used to influence disease progression in virally infected mice. DBA/2 mice were injected i.v. with FVC-P, and were treated s.c. with 100 micrograms LF at day 7, and/or rmuIFN-gamma at 5 x 10(4) units/day for 3 days beginning at day 6 after viral infection. Mice were assessed for survival, and also 14 days after virus inoculation, the mice were killed and spleen extracts were assessed for spleen focus forming virus (SFFV) titers by spleen focus forming unit (SFFU) assay, SFFV mRNA and genomic DNA expression, and natural killer (NK) cell activity. Treatment with LF or rmuIFN-gamma alone had little or no effect on SFFU numbers or SFFV mRNA or genomic DNA expression. However, dramatically decreased SFFV titers and levels of SFFV mRNA and genomic DNA were observed in mice treated with the combination of LF and rmuIFN-gamma. NK cell activity decreased by FVC-P was returned to normal levels by LF and rmuIFN-gamma. The combined treatment also enhanced the survival rates of FVC-P-infected mice. The results suggest synergistic suppressive effects of LF with rmuIFN-gamma on disease progression in FVC-P-infected mice. This information might be of significance as a potential therapy for patients with leukemia and those infected with retroviruses.     

Pancreatitis and altered glucose tolerance in mice infected with coxsackie B4 virus

Summary CD₁ mice infected with Coxsackie B₄ virus showed an early polymorph infiltration of the pancreas, which later changed to a mononuclear exudate. A state of glucose intolerance developed concurrently and both events coincided with the inhibition of migration of spleen cells in the presence of the virus. The relevance of these findings to human juvenile-onset diabetes mellitus is discussed.     

Prostaglandin E counteracts the gamma interferon induction of major histocompatibility complex class-II antigens on U937 cells and induction of responsiveness of U937 colony-forming cells to suppression by lactoferrin, transferrin, acidic isoferritins, and prostaglandin E

The established human monoblast or early monocyte cell line, U937, was evaluated for modulating influences of prostaglandin E2 (PGE2) on human gamma interferon (HuIFN gamma) induction of MHC class-II (Ia) antigens on U937 cells and the HuIFN gamma induction of responsiveness of U937 colony-forming cells (CFC) to inhibition by lactoferrin (LF), transferrin (TF), acidic isoferritins (AIF), and prostaglandin E (PGE). U937 CFC were induced to a state of responsiveness to the suppressive influences of PGE by HuIFN gamma. When MHC class-II antigens were induced on U937 cells and the cells sorted on the fluorescence activated cell sorter (FACS) IV into positive and negative cells, colony formation by the MHC class-II antigen+ population of cells was suppressed by LF, TF, AIF, and PGE2. Colony formation by the sorted population of MHC class-II antigen- cells was not influenced significantly by LF, TF, AIF, or PGE2. When PGE was present in the suspension culture for 72 h with U937 cells exposed to HuIFN gamma plus indomethacin, it blocked the induction of MHC class-II antigens as well as the associated inhibition of U937 CFC by LF, TF, AIF, and PGE2.     

Identification of altered integrin alpha/beta chain expression on T cells from old mice infected with Mycobacterium tuberculosis

During the natural aging process there is a gradual acquisition of T cell surface markers normally associated with cellular activation and memory. Because of this it is difficult to identify cells which are capable of responding to new antigenic challenge in older individuals, which therefore hinders the study of the immune response to infectious diseases. In this study we demonstrate that during natural aging there was an expansion in T cells that expressed the beta1 and beta2 integrins. Fewer CD8 T cells from old mice expressed beta7 integrins, however, they remained unchanged on CD4 T cells. In this study we also measured the expression of alphabeta integrin chains on the surface of T cells from mice of increasing age in response to an infection with Mycobacterium tuberculosis. We found that VLA-2 expression was increased on CD8 T cells from old mice however, the majority of integrins were unchanged in response to infection, in consequence of the increased expression of these molecules normally found in un-infected old mice. These findings are consistent with the hypothesis that age-associated changes occur in the number of cells that express molecules that allow T cells to traffic to inflammatory sites.     

Efficacy of recombinant human macrophage colony-stimulating factor in combination with whole-body hyperthermia in the treatment of mice infected with the polycythemia-inducing strain of the Friend virus complex

Macrophage colony-stimulating factor (M-CSF, CSF-1) and whole-body hyperthermia (WBH) were evaluated, alone or in combination, for their capability to influence disease progression in mice inoculated with the polycythemia-inducing strain of the Friend virus complex (FVC-P). DBA/2 mice were injected i.v. with FVC-P and were treated with 20 micrograms/dose M-CSF s.c. twice a day for 5 days beginning 6 days after injection of FVC-P and/or with WBH (between 38.8 degrees C and 40.2 degrees C) given on days 5 and 12 after FVC-P injection. Fourteen days after viral inoculation, mice were sacrificed and spleen cells evaluated for: 1) spleen focus-forming virus (SFFV), by the spleen focus-forming unit assay (SFFU); 2) SFFV mRNA and genomic DNA using, respectively, Northern and Southern analysis with a B-E-SFFV DNA probe; and 3) natural killer (NK) cell activity, by 51Cr-release assay. Treatment with M-CSF or WBH alone had a small effect on SFFU numbers but little or no effect on SFFV mRNA expression and SFFV-specific DNA. However, dramatically decreased levels of SFFU and SFFV mRNA and specific DNA fragments were observed in mice treated with M-CSF in combination with WBH, and NK cell activity was restored to normal. These results suggest the possibility that M-CSF may have a therapeutic effect in combination with WBH in the in vivo treatment of certain hematologic malignancies and/or retroviral infections.     

Selection of peptide inhibitors of interactions involved in complex protein assemblies: association of the core and surface antigens of hepatitis B virus.

As an example for studies of contacts involved in complex biological systems, peptide ligands that bind to the core antigen of hepatitis B virus (HBcAg) have been selected from a random hexapeptide library displayed on filamentous phage. Affinity-purified phage bearing aa sequence LLGRMK, or some related sequences, bound full-length or truncated HBcAg but did not bind denatured HBcAg. The long (L), but not the short (S), hepatitis B virus envelope polypeptide, when synthesized in an in vitro system, bound firmly to HBcAg, indicating that interaction between HBcAg and the pre-S region of the L polypeptide is critical for virus morphogenesis. This interaction was inhibited by peptide ALLGRMKG, suggesting that this and related small molecules may inhibit viral assembly.     

Genetic association of vasoactive intestinal peptide receptor with rheumatoid arthritis: altered expression and signal in immune cells

OBJECTIVE: Vasoactive intestinal peptide (VIP) has been shown to be one of the endogenous factors involved in the maintenance of immune tolerance. Administration of VIP ameliorates clinical signs in various experimental autoimmune disorders. This study was undertaken to investigate whether the exacerbated inflammatory autoimmune response in rheumatoid arthritis (RA) might result directly from altered expression and/or signaling of VIP receptors in immune cells. METHODS: The effect of specific agonists of different VIP receptors on collagen-induced arthritis in mice was investigated by clinical and histologic assessment and measurement of cytokine and chemokine production. Expression of VIP receptor type 1 (VPAC1) in synovial cells and monocytes from RA patients was determined by flow cytometry. Potential associations of VPAC1 genetic polymorphisms with RA susceptibility were investigated. RESULTS: A VPAC1 agonist was very efficient in the treatment of experimental arthritis, and deficient expression of VPAC1 in immune cells of RA patients was associated with the predominant proinflammatory Th1 milieu found in this disease. Immune cells derived from RA patients were less responsive to VIP signaling than were cells from healthy individuals and showed reduced VIP-mediated immunosuppressive activity, rendering leukocytes and synovial cells more proinflammatory in RA. A significant association between multiple-marker haplotypes of VPAC1 and susceptibility to RA was found, suggesting that the reduced VPAC1 expression in RA-derived immune cells is associated with the described VPAC1 genetic polymorphism. CONCLUSION: These findings are highly relevant to the understanding of RA pathogenesis. They suggest that VIP signaling through VPAC1 is critical to maintaining immune tolerance in RA. In addition, the results indicate that VPAC1 may be a novel therapeutic target in RA.     

细粒棘球绦虫感染小鼠肝脏髓源抑制性细胞 与调节性T细胞比例动态变化

目的　分析细粒棘球蚴感染小鼠肝脏髓源抑制性细胞（MDSCs）和调节性T（Treg）细胞比例动态变化，探讨其可能的生物学意义。方法　将30只6 周龄雌性BALB/c 小鼠随机分为感染组和对照组，每组15只。感染组每只小鼠腹腔注射细粒棘球绦虫原头节约2 000个，对照组注射等体积生理盐水。感染后3、6、12个月（感染早、中、晚期）收集小鼠肝脏白细胞，采用流式细胞术检测其中MDSCs 及其亚型M?MDSCs、PMN?MDSCs 与Treg细胞比例。结果　感染组小鼠感染后3、6、12个月肝脏白细胞中MDSCs 比例分别为（1.61 ± 0.36）%、（5.68 ± 0.69）%和（16.18 ± 0.69）%，对照组分别为（2.19 ± 0.42）%、（0.99 ± 0.07）%和（4.18 ± 0.84）%，感染后6个月和12个月两组差异均有统计学意义（P均 < 0.01）；感染组小鼠感染3、6、12个月后肝脏白细胞中M?MDSCs 比例分别为（0.69 ± 0.27）%、（5.30 ± 0.72）%和（10.75 ± 0.29）%，对照组分别为（0.42 ± 0.24）%、（0.69 ± 0.02）%和（2.12 ± 0.13）%，感染后6个月和12个月两组差异均有统计学意义（P 均< 0.01）；感染组小鼠感染后3、6、12个月肝脏白细胞中PMN?MDSCs 比例分别为（0.93 ± 0.23）%、（0.32 ± 0.02）%和（5.14 ± 1.03）%，对照组分别为（1.77 ± 0.26）%、（0.28 ± 0.05）%和（1.99 ± 0.90）%，感染后3个月和12个月两组差异均有统计学意义（P均 < 0.05）。感染组小鼠感染后3、6、12个月肝脏白细胞中Treg细胞比例分别为（3.35 ± 0.14）%、（6.24 ± 0.38）%和（3.41 ± 0.07）%，对照组分别为（3.48 ± 0.46）%、（3.65 ± 0.45）%和（3.12 ± 0.12）%，感染后6个月和12个月两组差异均有统计学意义（P均＜0.01）。结论　小鼠感染细粒棘球蚴6个月和12个月后，其肝脏白细胞中MDSCs与Treg细胞比例增加，前者比例变化更加明显，以M?MDSCs为主；以上提示M?MDSCs可能在小鼠感染细粒棘球蚴中后期发挥主要免疫抑制作用。     

Effect of actinomycin D and busulphan on stem cells in normal and friend virus infected mice

Summary Busulphan, Actinomycin D, and a combination of both were used to treat normal NMRI and DBA/2 mice and mice with Friend virus induced polycythemia. In FV-P infected mice a new cell type is found after virus infection, which gives rise to erythropoietic colonies (CFU_E) in vitro without addition of Erythropoietin (Ep), which completely replace normal Ep dependent CFU_E. After treatment, CFU_s, CFU_c, and CFU_E were studied. Busulphan (30 mg/kg p.o.) did reduce CFU_s and CFU_c growth in virus infected and control mice to the same extent. Six days after Busulphan in the bone marrow, but not in the spleen, some Ep dependent CFU_E were observed in the virus infected animals, not seen before treatment. Actinomycin D (2×120 g/kg s.c.) suppressed the CFU_E growth in normal mice more effectively in the spleen than in the bone marrow. In FV-P infected mice Act D induced a total suppression of CFU_E colony growth in both organs. During regeneration no normal Ep-dependent CFU_E growth was observed. The combination of both drugs completely suppressed CFU_E growth for at least 4 days in the marrow and for 9 days in the spleen. During the regeneration all CFU_E growth was Ep independent. The results are discussed with respect to the origin of the Ep independent colonies within the stem cell compartments and the possibilities of chemotherapy.Supported by the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 112, Project Bl     

Association of cell cycle expression of Ia-like antigenic determinations on normal human multipotential (CFU-GEMM) and erythroid (BFU-E) progenitor cells with regulation in vitro by acidic isoferritins

An association has been established between human Ia-like antigenic determinants, expression during DNA synthesis on multipotential (CFU- GEMM) and erythroid (BFU-E) progenitor cells, and the regulatory action of acidic isoferritins in vitro. Treatment of human bone marrow cells with monoclonal anti-Ia (NE1-011) plus complement inhibited colony formation of CFU-GEMM) and BFU-E by 50%-70%. Reduction of colonies was similar whether bone marrow cells were exposed to anti-Ia plus complement, high specific tritiated thymidine (3HTdr), or acidic isoferritins. No further decrease was apparent with 3HTdr or acidic isoferritins after Ia-antigen+ CFU-GEMM or BFU-E were removed, or with anti-Ia plus complement or acidic isoferritins after S-phase CFU-GEMM or BFU-E were removed. Anti-Ia, in the absence of complement, had no effect on colony formation but blocked the inhibition of CFU-GEMM and BFU-E by acidic isoferritins. Demonstration of Ia-antigens on BFU-E and inhibition of BFU-E by acidic isoferritins appeared to require the presence of phytohemmagglutinin leukocyte conditioned medium (PHA-LCM) in the culture medium during the 14-day incubation period. these results implicate Ia-antigen+ cells, acidic isoferritins, and PHA-LCM in the regulation of multipotential and erythroid progenitor cells in vitro.     

Distribution of virus structural proteins and protein-protein interactions in plasma membrane of baby hamster kidney cells infected with Sindbis or vesicular stomatitis virus.

The plasma membrane of baby hamster kidney (BHK-21) cells infected with either Sindbis or vesicular stomatitis virus was isolated by a technique involving the ingestion of latex beads by the cells. Plasma membrane isolated from Sindbis virus-infected cells contained only one (E1) of the three (E1, E2, and C) structural proteins of this virus. When the latex beads were pretreated with either polylysine or DEAE-dextran, plasma membrane obtained from Sindbis virus-infected cells contained all three structural proteins and PE2, a precursor to one of the structural proteins. In pulse-chase radiolabeling experiments with Sindbis virus-infected cells, it was possible to follow the appearance of the precursor protein (PE2) i the plasma membrane and its eventual conversion to E2. The appearance of Sindbis virus membrane proteins PE2 and E1 in the purified plasma membrane was not affected by the drug tunicamycin, an inhibitor of glycosylation. These experiments imply the following: (i) Cleavage of the Sindbis virus precursor polypeptide PE2 to E2 is not a prerequisite for its transport to the cell plasma membrane; (ii) transport of virus membrane proteins to the cell surface does not depend on glycosylation; and (iii) although all Sindbis virus structural proteins are associated with the plasma membrane, a generally accepted pairing of PE2-E1 or E2-E1 in the plasma membrane either does not exist or, if it does exist, involves a very weak interaction. The procedures used in this study also resulted in the successful isolation of plasma membrane from vesicular stomatitis virus-infected cells containing the glycoprotein, the matrix protein, and the nucleocapsid protein, a result that suggests that these proteins are located on the media side of baby hamster kidney cells grown in monolayer.     

Biochemical and ultrastructural changes in A and B cells of the islets of Langerhans of mice infected with EMC virus

Summary Streptozotocin-induced diabetes in mice was associated with a marked decrease in size and nucleated cell content of the thymus and spleen. Thymocytes were reduced by 96% compared to the number observed in normal age- and sex-matched mice; recovery was not observed within 81 days following Streptozotocin administration. The degree of hyperglycaemia produced by a single dose of Streptozotocin (200mg/kg, IV) in mice 30 days after treatment was inversely related to the percentage of lymphocytes in peripheral blood. The number of nucleated spleen cells remaining 9–81 days after Streptozotocin treatment was 26–47% of the number observed for normal mice. The blastogenic responses of spleen cells from streptozotocin-diabetic mice did not parallel the continued depression in the number of lymphocytes when the number of cells in the in vitro assays was normalised. Spleen cells from 9-day streptozotocin-diabetic mice had depressed thymidine incorporation in mixed lymphocyte culture responses, an in vitro correlate of graft rejection responses induced when allogeneic lymphocytes are cultured together. Spleen cells from 20–81 day Streptozotocin animals did not differ significantly from normal. The generation of cytotoxic effector cells in vitro, however, was depressed for spleen cells obtained from 22- and 48-day Streptozotocin mice. When UV-induced syngeneic tumour fragments were implanted in normal and Streptozotocin mice, the diabetic animals supported the progressive growth of the tumour while normal mice did not. In contrast, Streptozotocin mice that had been lethally irradiated and reconstituted with normal syngeneic bone marrow and spleen cells prevented tumour growth as well as normal control mice. Insulin treatment of Streptozotocin mice (1 unit NPH/day, SC) did not change tumour rejection by diabetic animals, although a recovery in the size of the thymus was observed. These results indicated that Streptozotocin directly depresses some cell-mediated immune responses independent of its diabetogenic actions.     

Schistosoma mansoni: evidence that immunity in vaccinated and chronically infected CBA/Ca mice is sensitive to treatment with a monoclonal antibody that depletes cutaneous effector cells

Naive mice, mice vaccinated 4 weeks previously with radiation-attenuated cercariae of Schistosoma mansoni and mice infected 16 weeks previously with normal S. mansoni cercariae were treated with a rat monoclonal antibody (NIMP-R14), that has been reported elsewhere to recognize neutrophils selectively. The establishment of a primary schistosome population in naive mice was not affected by the administration of this reagent. In contrast, immune-dependent challenge elimination was reduced in both vaccinated and chronically infected mice following treatment with NIMP-R14. Maximal suppression of resistance in both vaccinated (67% mean reduction) and chronically infected (44% mean reduction) mice was achieved when NIMP-R14 was injected intraperitoneally on the day of challenge. The monoclonal was markedly less effective when administered on or after day 3. Analysis of the blood leucocyte profiles of vaccinated/NIMP-R14 treated mice showed that the monoclonal totally abrogated neutrophils from the peripheral circulation between days 1 and 6. Histological examination of the skin site of challenge revealed, however, that NIMP-R14 treatment had reduced the number of eosinophils and macrophages as well as neutrophils in the cutaneous tissues of vaccinated mice. The reaction site thus more closely resembled that of naive/challenged mice than that of untreated vaccinated/challenged mice. Although we have not been able to identify a specific effector cell from these studies, we have demonstrated clearly that a skin-located cellular effector mechanism contributes to immune resistance in both murine models.     

Differentiation and virus expression in BALB/Mo mice: endogenous Moloney leukemia virus is not activated in hematopoietic cells.

The endogenous Moloney leukemia virus (M-MuLV) in the BALB/Mo substrain of mice is activated during the first week after birth. Virus replication occurs in cells of the lymphatic system. Lymphoid cells therefore represent the target cells for virus replication and leukemic transformation. To investigate whether virus activation occurs during lymphoid cell differentiation, hematopoietic stem cells carrying the endogenous Mov-1 genome were transplanted to sublethally irradiated BALB/c mice. Effective colonization of the recipients was demonstrated by Southern DNA hybridization. No activation of the endogenous Mov-1 genome occurred during a 4-month observation period after the transplantation. Thus the first step in development of disease in BALB/Mo mice involves activation of the Mov-1 locus in a nonhematopoietic cell followed by superinfection of lymphatic cells and subsequent virus replication and virus spread.     

Development of spontaneous arthritis in beta2-microglobulin-deficient mice without expression of HLA-B27: association with deficiency of endogenous major histocompatibility complex class I expression

OBJECTIVE: Mice deficient in beta2-microglobulin (beta2m), but expressing the human major histocompatibility complex (MHC) class I molecule HLA-B27, have been reported to develop spontaneous inflammatory arthritis (SA). We sought to determine whether, under certain conditions, beta2m deficiency alone was sufficient to cause SA, and if this might be a result of class I deficiency. METHODS: The following types of mice were produced: mice of the MHC b haplotype genetically deficient in beta2m (beta2m(0)) on several genetic backgrounds (C57BL/6J [B6], BALB/cJ, SJL/J, MRL/MpJ, and B6,129), mice deficient in the transporter associated with antigen processing (TAP1(0)) on a B6,129 background, and HLA-B27-transgenic beta2m(0) mice on a B6 background. Cohorts were transferred from specific pathogen-free (SPF) to conventional (non-SPF) animal rooms, and evaluated clinically and histologically for the development of SA. RESULTS: SA occurred in TAP1(0) and beta2m(0)/class I-deficient mice with a mixed B6,129 genome at a frequency of 30-50%, while 10-15% of B6, SJL/J, and BALB/cJ beta2m(0) mice developed this arthropathy. MRL/ MpJ beta2m(0) mice were unaffected. Expression of B27 did not increase the frequency of SA in B27-transgenic B2m(0) B6 mice compared with that in beta2m(0) B6 controls. CONCLUSION: Class I deficiency is sufficient to cause SA in mice. The frequency of disease, as well as B27-specific SA, is markedly dependent on a non-MHC genetic background. These results suggest that class I deficiency in a genetically susceptible mouse can mimic B27-associated arthropathy.     

Necrotizing myocarditis in mice infected with Western equine encephalitis virus: Clinical, electrocardiographic, and histopathologic correlations

Western equine encephalitis (WEE) virus was found in myocardial tissue of adult mice during the first five days after inoculation of the virus, with a peak titer (5.0 log plaque-forming units/g) at 24 hr. Light microscopy revealed a multifocal necrotizing myocarditis with a prominent inflammatory response and hyaline and granular degeneration of myofibers. Electron microscopy showed cytoplasmic viral nucleoids and budding and free mature WEE viral particles. Serial electrocardiograms showed the development of disturbances of rate and rhythm, defects in conduction, marked elevation in the ST segment, and low voltage. Myocarditis has not been previously recognized as a complication of alphavirus infection in humans. and we found no evidence for myocardial damage in 11 persons with acute WEE virus infections studied electrocardiographically in 1975. Demonstration of myocarditis in the WEE virus-infected mouse, however, suggests the need to monitor human patients for possible cardiac involvement during future epidemics of WEE virus infection.     

翻白草油对RSV感染宿主HeLa细胞Fas/FasL蛋白表达的影响

目的通过检测呼吸道合胞病毒(RSV)感染宿主HeLa细胞Fas蛋白和FasL蛋白的变化,探讨翻白草油的抗RSV作用。方法用RSV感染宿主HeLa细胞,采用Reed-Muench法求出RSV对细胞半数感染量(CCID50);将翻白草油与HeLa细胞共孵育,MTT法检测翻白草油对Hela细胞的毒性作用;选取最大无毒浓度的翻白草油和100CCID50的RSV病毒进行抗病毒试验,在药物4种作用方式下(对RSV病毒的直接灭活作用,在生物合成阶段对RSV病毒的作用,对RSV病毒吸附的作用,药物预处理作用),采用免疫荧光和Western blot方法检测宿主HeLa细胞Fas/FasL蛋白的表达。结果 RSV培养液对HeLa细胞中的毒力为105.8 CCID50/L;翻白草油对HeLa细胞的最小毒性浓度是0.10mg/L。翻白草油(除药物预处理作用方式外)Fas蛋白和FasL蛋白表达的荧光强度分别为1.851±0.022、1.830±0.044、1.801±0.038和1.832±0.034、1.767±0.022、1.816±0.024,RSV病毒对照组荧光强度分别为2.112±0.048和1.904±0.025,翻白草油(除药物预处理作用方式)组与病毒组比较,宿主HeLa细胞Fas蛋白和FasL蛋白的表达显著降低(P<0.05);翻白草预处理组Fas蛋白和FasL蛋白表达的荧光强度分别为2.098±0.075和1.882±0.055,与RSV病毒对照组比较差异无统计学意义(P>0.05)。结论翻白草油在直接灭活阶段、病毒复制阶段、病毒吸附阶段的抗RSV作用与调节宿主HeLa细胞Fas/FasL蛋白的表达有关。