Primary infection of dogs with Echinococcus granulosus: systemic and local (Peyer's patches) immune responses

Local and systemic lymphocyte proliferation and antibody production were tested in five dogs 35 days after primary experimental infection with Echinococcus granulosus. A significant cell proliferation was demonstrated by [3H] thymidine incorporation in mesenteric, popliteal and/or Peyer's patches (PPs) cells in response to E. granulosus protoscolex or adult worm antigen in three of five infected dogs, but not in five control animals. In contrast, blood mononuclear cells responded very weakly in only two of the infected dogs to parasite antigens. Elevated levels (compared with preinfection status) of protoscolex- and adult worm antigen-specific serum IgG were detected (ELISA) in four of the five dogs 35 days after infection. Furthermore, slightly elevated levels of parasite-specific IgE and IgA were observed in the sera of three and four in four infected dogs, respectively. Specific serum IgM was not significantly higher 35 days after infection than before infection. Local antibody production was studied in vitro using PPs, mesenteric and popliteal cells isolated from three infected and three uninfected dogs by ELISA using adult worm antigen. In two of three cultures of unstimulated PPs cells of infected dogs, parasite-specific IgG was detectable. Parasite-specific IgA and IgM were detected in one of the unstimulated PPs cell culture derived from an infected dog. Following in vitro stimulation with parasite antigen, PPs cells from two infected dogs showed increased parasite-specific IgG and PPs cells of all three infected dogs produced parasite-specific IgA. PPs cells from uninfected dogs did not produce significant quantities of parasite-specific antibodies and cells from mesenteric and popliteal lymph nodes of infected or uninfected dogs neither produced antibodies whilst in in vitro cultures.     

A short region from the LEU2 gene of Saccharomyces cerevisiae functions as an ARS in the yeast Saccharomyces exiguus Yp74L-3

Human cytomegalovirus (HCMV) infection can result in neurological symptoms. In vitro replication of the HCMV was studied in primary cultures of microglial cells from the central nervous systems (CNS) of human embryos. The microglial cells were infected with various amounts of either the AD169 laboratory HCMV strain or a clinical HCMV isolate. A specific cytopathic effect occurred within 24 h and persisted for two months. Immunocytochemical tests for immediate early and late viral antigens done one and three days after the infection demonstrated that 60% to 80% of the microglial cells were infected and that 3% to 8% were the site of viral DNA replication. Kinetic studies showed accumulation of viral particles in the supernatant during the first two weeks after the infection. Prestimulation of the cells by PMA 24 h before the infection was associated with increased release of viral particles and with an increased percentage of cells expressing late viral antigens. The microglial cells of the human embryonic CNS are fully permissive targets for the HCMV. The in vitro HCMV model used in this study may prove useful for investigating the pathophysiology of HCMV encephalitis, in particular after mother-to-fetus transmission of the virus.     

The effect of simian immunodeficiency virus infection in vitro and in vivo on the cytokine production of isolated microglia and peripheral macrophages from rhesus monkey

Microglia are the major target for human immunodeficiency virus (HIV) infection within the central nervous system. Because only a few cells are productively infected, it has been suggested that an aberrant cytokine production by this cell population may be an indirect mechanism leading to the development of neurological disorders in HIV-infected patients. Therefore we decided to study the secretion pattern of several interleukins (IL) by microglial cells and peripheral blood macrophages isolated from uninfected and simian immunodeficiency virus (SIV)-infected Rhesus monkeys. We found that uninfected, unstimulated primate microglia produce more IL-6 and less TNF alpha than peripheral blood macrophages, but generate comparable levels of IL-1 beta and IL-8. After infection with SIV in vitro, synthesis of all cytokines tested is increased compared to uninfected cultures and to peripheral blood macrophages. Microglia isolated from infected animals produce more IL-8 and TNF alpha than the uninfected cultures and display a strongly increased capacity to secrete TNF alpha upon stimulation with lipopolysaccharide. In addition, production of IL-6 by in vivo-infected microglia increases with time in culture to very high levels despite the fact that only a few cells contained replicating virus. These findings clearly show that the cytokine production of microglia is impaired after SIV infection both in vitro and in vivo and that a low level of viral replication is sufficient for these alterations to occur. In conclusion, the results of this study further support a possible role of cytokines in the pathogenesis of neuro-AIDS.     

Macrophages as effector cells of protective immunity in murine schistosomiasis. III. Loss of susceptibility to macrophage-mediated killing during maturation of S. mansoni schistosomula from the skin to the lung stage

Newly transformed skin-stage and lung-stage schistosomula were compared in terms of their susceptibility to killing mediated by activated mouse macrophages in vitro. Although skin-stage schistosomula were readily killed by macrophages activated as a consequence of either BCG or Schistosoma mansoni infection and used either as cell monolayers or in suspension, lung-stage larvae appeared to be totally resistant to this effector mechanism and survived normally when reinjected into mice. Resistance of schistosomula to in vitro damage by macrophages was evident as early as 18 hr after host infection and was complete in worms recovered at 42 hr. The insusceptibility of lung-stage larvae is apparently not due to a defect in effector cell-target contact, because the induction of extensive macrophage adherence to the worms by the addition of anti-mouse red blood cell antisera to the cultures had no effect on parasite viability. These findings provide additional support for the concept that schistosomula during their development to the lung stage undergo a generalized change affecting their susceptibility to a variety of different immunologic effector mechanisms.     

Bone marrow monocyte/macrophages are an early cellular target of pathogenic and nonpathogenic isolates of simian immunodeficiency virus (SIVmac) in rhesus macaques

BACKGROUND: Hematopoietic abnormalities are a common complication of human immunodeficiency virus infection in humans. However, the pathogenesis of these abnormalities remains unclear. Simian immunodeficiency virus (SIV) infection of rhesus macaques is a well-recognized animal model for acquired immunodeficiency syndrome. Our previous studies have determined that in early SIV infection, rhesus macaques develop peripheral blood and bone marrow pathologic changes within the first 14 days after intravenous inoculation. Further investigations were initiated to determine the onset of bone marrow viral infection and the identity of in vivo viral cellular targets in bone marrow during the primary phase of infection in macaques infected with three different strains of SIVmac. EXPERIMENTAL DESIGN: Rhesus macaques experimentally infected with pathogenic uncloned biologic SIVmac, molecularly cloned pathogenic SIVmac-239, or nonpathogenic SIVmac-1A11 were studied at 3, 7, and 14 days postinoculation. Bone marrow samples taken at necropsy were examined to identify early in vivo cellular targets of SIVmac in bone marrow and to correlate hematopathologic lesions with viral infection. In the first 2 weeks after intravenous inoculation, cellular targets of viral infection were identified by a combined in situ hybridization/immunohistochemical technique; changes in bone marrow monocyte/macrophage and CD3+ T lymphocyte populations were evaluated by immunohistochemical techniques. RESULTS: SIV-infected monocyte/macrophages were detected on days 3, 7, and 14 days postinoculation in bone marrow of all monkeys regardless of the viral isolate, whereas only a few SIV-infected CD3+ T lymphocytes were detected in 5 of 18 monkeys. The bone marrow morphologic changes associated with acute SIV infection included macrophage hyperplasia and apparent macrophage activation, diminution of bone marrow T lymphocytes, appearance of lymphoid aggregates, and myeloid and megakaryocytic hyperplasia. CONCLUSIONS: We conclude that bone marrow monocyte/macrophages are an important early cellular target in SIV infection regardless of viral pathogenicity and in vitro cellular tropism. SIV-infected bone marrow monocyte/macrophages may play a key role in the pathogenesis of bone marrow lesions and further dissemination and persistence of virus infection.     

Stimulation of reticuloendothelial system and toxicity to macrophages of Staphylococcus aureus cell wall, peptidoglycan, and teichoic acid

Staphylococcus aureus cell wall possesses several biological activities. It is removed from the blood by reticuloendothelial system (RES) and persists there for a long time. The influence of cell wall components on RES cells in vivo and in vitro was investigated. RES activity was studied in mice by carbon clearance method. Intravenous injection of 10 microgram of cell walls or peptidoglycan caused early stimulation and subsequent suppression of RES activity, while teichoic acid was inactive. Four hundred microgram of peptidoglycan caused RES stimulation with maximum after three days, whereas 400 microgram of cell walls caused no such stimulation. Viability of mouse peritoneal macrophages was studied after four days of culture in the presence of cell walls, peptidoglycan, and teichoic acid. Fifty microgram/ml of cell walls or peptidoglycan caused death of all or 68% of macrophages respectively. Teichoic acid was inactive, exhibiting toxic effects at 400 microgram/ml level.     

Rapid expression of the CD69 antigen on T cells and natural killer cells upon antigenic stimulation of peripheral blood mononuclear cell suspensions

The CD69 antigen has been identified as the earliest activation marker on the surface of cytokine- or mitogen-activated lymphocytes. The expression of this molecule may be a useful early marker of antigen- or allergen-specific activation of lymphocytes in vitro. We evaluated the expression of the CD69 and CD25 antigens on antigen- or allergen-stimulated lymphocytes and the proliferative responses as detected by thymidine incorporation. Peripheral blood mononuclear cells (PBMC) of allergic patients sensitized to Dermatophagoides pteronyssinus, bovine casein, or nickel sulfate were cultured in the absence or presence of clinically relevant allergens, tetanus toxoid, or recombinant interleukin (IL)-2. The respective binding of CD69 or CD25 antibodies to PBMC and thymidine incorporation were measured. An early expression of CD69, but not of CD25, antigen was detectable after 24-72 h of stimulation on up to 80% of natural killer (NK) cells and up to 10% of CD4+ T cells in PBMC cultures. Anti-IL-2 antibodies inhibited these increases of CD69 on NK cells and T cells by up to 60%. After 6 days of antigenic stimulation, the rates of both CD25+ and CD69+ lymphocytes were higher. Seventy-four percent of the CD25+ PBMC but only 55% of the CD69+ cells were CD3+ T lymphocytes at this time. No qualitative differences were detectable in allergen- or tetanus-toxoid-stimulated PBMC from allergic patients. The high expression of CD69 on NK cells in antigen-stimulated cultures suggests that these cells are easily activated by cytokines from antigen-stimulated T cells. CD69+ NK cells may serve as early-indicator cells in cultures with antigen- or allergen-stimulated mononuclear cells.     

In vivo effects of a recombinant vaccinia virus expressing a mouse mammary tumor virus superantigen

Early after infection, the mouse mammary tumor virus (MMTV) expresses a superantigen (SAg) at the surface of B lymphocytes. Interaction with the T-cell receptor Vbeta domain induces a polyclonal proliferative response of the SAg-reactive T cells. Stimulated T cells become anergic and are deleted from the T-cell repertoire. We have used a recombinant vaccinia virus encoding the MMTV(GR) SAg to dissect the effects of the retroviral SAg during an unrelated viral infection. Subcutaneous infection with this recombinant vaccinia virus induces a very rapid increase of Vbeta14 T cells in the draining lymph node. This stimulation does not require a large Plumber of infectious particles and is not strictly dependent on the expression of the major histocompatibility complex class II I-E molecule, as it is required after MMTV(GR) infection. In contrast to MMTV infection during which B cells are infected, we do not observe any clonal deletion of the reactive T cells following the initial stimulation phase. Our data show that contrary to the case with MMTV, macrophages but not B cells are the targets of infection by vaccinia virus in the lymph node, indicating the ability of these cells to present a retroviral SAg. The altered SAg expression in a different target cell observed during recombinant vaccinia virus infection therefore results in significant changes in the SAg response.     

Lymphocyte apoptosis during classical swine fever: implication of activation-induced cell death

Infection of pigs with classical swine fever virus (CSFV), a member of the Flaviviridae family, causes a severe leukopenia, particularly notable with the lymphocytes. The goal of this study was to analyze mechanisms behind this CSFV-induced lymphopenia. To this end, the kinetics of leukocyte depletion, the appearance of apoptotic cells, and virus infection of leukocytes after infection of pigs with the virulent CSFV strain Brescia were analyzed. Depletion of B and T lymphocytes was noted as early as 1 day postinfection (p.i.). Circulating viable lymphocytes with reduced mitochondrial transmembrane potential--a particular early marker for apoptosis--were also detectable as early as 1 day p.i. When isolated peripheral blood mononuclear cells were cultured for 6 h, significantly more sub-G1 cells with reduced DNA content were detected among the lymphocytes from CSFV-infected animals, again as early as 1 to 3 days p.i. The first time virus was first found in the plasma, as well as infection of leukocytes, was 3 days p.i. However, throughout the observation time of 1 week, <3% of the circulating leukocytes and no lymphocytes contained virus or viral antigen. Further analysis of the T lymphocytes from infected animals demonstrated an increase in CD49d, major histocompatibility complex class II, and Fas expression. An increased susceptibility to apoptosis in vitro was also observed, particularly after addition of concanavalin A as well as apoptosis-inducing anti-Fas antibody to the cultures. Taken together, these results imply that activation-induced programmed cell death was the mechanism behind lymphopenia during classical swine fever.     

Assessment of the expression of p53, MIB-1 (Ki-67 antigen), and argyrophilic nucleolar organizer regions in carcinoma of the extrahepatic bile duct

Group B streptococci (GBS) are an important cause of neonatal sepsis, pneumonia and meningitis. In the early phase of infection, macrophages and polymorphonuclear cells (PMN) are the first immune cells that interact with GBS. In this in vitro study, to gain insight into GBS-macrophage interaction in the absence of type-specific antibodies, we examined the features of GBS survival in thioglycollate-elicited murine peritoneal macrophages and the effect of GBS on the protein kinase C (PKC)-dependent transduction pathway. Our results demonstrate that type Ia GBS, strain 090 (GBS-Ia) and type III GBS strain COH 31r/s (GBS-III), after in vitro phagocytosis survive and persist intracellularly in macrophages for up to 24 and 48 hr, respectively. However, macrophage activation by interferon-gamma (IFN-gamma) and lipopolysaccharide from Escherichia coli (LPS) caused a significant reduction in the time of intracellular persistence. Macrophage activation by IFN-gamma and LPS seems to be a multifactorial event involving multiple intracellular signal pathways also including PKC. Since PKC is one of the components in the signal network leading to macrophage activation and an important target for several intracellular micro-organisms, we wondered whether PKC could have a role in intracellular GBS survival. Both PKC depletion by treatment with phorbol 12-myristate 13-acetate (PMA) for 18 hr and PKC inhibition by Calphostin C rendered macrophages more permissive for the intracellular GBS survival. Furthermore, GBS-infected macrophages were unable to respond to PMA and LPS, activators of PKC, by inducing antimicrobial activity. The ability of GBS to impair PKC-dependent cell signalling was also demonstrated by the reduced c-fos gene expression in GBS-infected macrophages with respect to control macrophages, after LPS stimulation. In conclusion, our results indicate that GBS survive in macrophages and impairment of PKC signal transduction contributes to their intracellular survival.     

Intestinal sleeve anastomosis: a comparative study with end-to-end anastomosis

This study examines the utility of a sleeve anastomosis with comparison to conventional end to end anastomosis. Thirty New Zealand white rabbits were randomized to sleeve (n = 15) or end-to-end (n = 15) small bowel anastomosis. Five rabbits of each group were sacrificed at 3 days, 7 days, and 6 weeks. Anastomoses were assessed for integrity, bursting strength, and stenosis and examined histologically. Ten control specimens of small bowel were tested for bursting pressure. Three rabbits died postoperatively (1 sleeve and 2 end-to-end). A fourth rabbit (sleeve) was sacrificed early at 3 weeks and had a total stenosis at the anastomosis. The remaining 26 rabbits were reoperated at the prescribed times. There was no evidence of infection or dehiscence in any of these rabbits. Both end-to-end and sleeve anastomoses were equivalent for bursting pressure at all times and, at 7 days and 6 weeks, were similar to controls. The stenotic index revealed no evidence of proximal dilation suggestive of obstruction in the 26 rabbits. For sleeve anastomoses the length of the projected bowel into the lumen persisted at the 6-week stage. Histologically there was good evidence of healing in both the sleeve and end-to-end anstomoses and the serosal surface of the sleeved bowel had epithelialized. Sleeve anastomosis has been demonstrated to heal well and to be as strong as conventional end-to-end anastomosis. Further studies are warranted to determine its role in intestinal anastomosis and potential as a valve.     

In vivo delivery of interleukin-4 by a recombinant vaccinia virus prevents tumor development in mice

To study the immunotherapeutic potential of interleukin-4 (IL-4) delivered in vivo via a recombinant vaccinia virus, a thymidine kinase-negative (TK-) vaccinia virus that expressed the murine IL-4 gene (VV1/IL-4) was constructed. When mice were inoculated with 10(7) plaque-forming units (pfu) of VV1/IL-4 subcutaneously (s.c.), 10(5) pfu/cm2 were found in skin, and smaller numbers in liver and kidney between 1 and 7 days after infection; few viral pfu were found in spleen and lung, or in any organ after intravenous infection. This suggested that recombinant vaccinia viruses might be most efficient at delivery of cytokine genes to the skin. Because IL-4 has recently been found to have potent anti-tumor activity, the effect of recombinant virus infection on the development of s.c. tumors was studied. A single s.c. inoculation with VV1/IL-4 delayed the development of NCTC 2472 tumors, but when VV1/IL-4 was inoculated s.c. weekly for 8 weeks, tumor development was completely prevented in 93% of mice. Similarly, the development of M-3 melanoma tumors was also prevented by weekly s.c. inoculations of VV1/IL-4. About 40% of mice treated with control VV2/beta gal by the same regimen also failed to develop tumors. Weekly virus treatment did not prevent NCTC 2472 tumor development in athymic nu/nu mice, suggesting that mature T cells are required for expression of VV1/IL-4 induced antitumor activity. Thus, recombinant vaccinia viruses may be especially well suited for convenient therapeutic delivery of immunomodulator genes to skin-related sites.     

Release of superoxide anion from activated mouse peritoneal macrophages during Mycobacterium tuberculosis infection

Mouse peritoneal macrophage monolayers infected with M. tuberculosis were cultured in RPMI up to 7 days. Release of superoxide was assayed on different days in presence or absence of Phorbol myristate acetate (PMA), a known stimulator of NADPH oxidase which is involved superoxide production. Basal level of superoxide release was significantly higher in M. tuberculosis infected peritoneal mouse macrophages (P < 0.01) as compared to normal mouse macrophages. When normal and tuberculoid macrophage cultures were stimulated with PMA, increased superoxide anion release was observed in both the cultures but the increase of superoxide was significantly higher in normal macrophages as compared to tuberculoid stimulated macrophages. Superoxide release was maximum in 4 day old cultured macrophages and gradually it declined in older cultures by day 7, both in vitro and in vivo. A defective macrophage function in killing of M. tuberculosis bacilli was observed after 4 days of in vitro and in vivo cultures.     

Human gamma delta T-cell recognition of Yersinia enterocolitica

The cellular immune response after an experimental oral infection with Yersinia enterocolitica (serotype 0:3, biotype 4, harbouring the virulence plasmid-p YV) was studied in pigs. A maximal stimulation of the T cell population in the thymus, spleen and peripheral blood was stated on the 15th day post infection (p.i.) by the rosette forming cell (RFC) test. The hemolysins (produced by B cells and detected by the plaque forming cell test-PFC) were significantly increased on the 15th day p.i. among the thymus and spleen lymphocytes and on the 25th day p.i. among the blood lymphocytes. Blood and thymus lymphocytes were activated faster by the infectious agent in comparison to the spleen cells. The electronmicroscopic studies revealed an intracellular presence of the bacteria in alveolar macrophages (aMa) and peritoneal macrophages (pMa) as well as in Peyer's patches and tonsils as early as on the 4th day p.i. Extracellularly located bacteria were observed, too. The results have shown that inspite of the activation of T and B cell immune response, this infectious agent persisted in the porcine organism.     

The accessory cell effects of liver macrophages in concanavalin A stimulation of rat spleen lymphocytes

Liver and peritoneal macrophages under similar test conditions behaved in an identical manner with regard to accessory cell effects in the lymphocyte response to concanavalin A. When present in low concentrations (less than or equal to 3.3%) they stimulate lymphocytes, and when present in high concentrations (greater than or equal to 10%) they inhibit lymphocyte proliferation. These two effects are, however, mediated through totally different mechanisms. Stimulation was an early effect, required viable cells, was not affected by enzymatic treatment of macrophages, and was similar to the effect of 2-mercaptoethanol, allogeneic macrophages, and even non-macrophages. Inhibition occurred at a larger stage of lymphocyte transformation, was sensitive to collagenase and pronase treatment of macrophages, was more specifically due to macrophages, was reduced with allogeneic macrophages, and persisted after freeze-thawing of macrophages. Removal of Fc receptors or related segments of the surface of macrophages greatly reduced their inhibitory capacity, whereas removal of foreign surface receptors apparently had no consequence.     

Selective expansion and continuous culture of macrophages from adult pig blood

Macrophages were selectively expanded and continuously cultured from adult pig blood. One-half ml of heparinized adult pig blood was inoculated directly into the medium overlaying a feeder layer of STO mouse fibroblasts. After attachment to the feeder cells for 24 h, the culture was washed several times with the medium to remove most of any unattached blood cells and re-fed. Approximately 7 x 10(4) blood monocytes were initially detected and enumerated by specific binding of DiI-labeled acetylated low density lipoprotein (DiI-Ac-LDL). Macrophage outgrowths appeared in the primary culture after 6-7 days. The macrophages grew to relatively high density in 2-3 weeks (2-3 x 10(6) cells/T25 flask), and the culture was passaged on to fresh STO feeder layers to begin secondary culture. Over 2-3 months of culture the macrophage replication produced as many as 1.4 x 10(9) DiI-Ac-LDL-positive cells. The macrophages grew on top of the feeder cells in two forms: either a semi-attached, round morphology, or a closely adherent, flat ameboid morphology with several extended pseudopods. Electron microscopic examination revealed the cells to be uniformly of macrophage character and that 4-5% were giant cells. The macrophages were phagocytic and expressed CD14 on their surfaces. They also reacted positively with pig macrophage-specific monoclonal antibody (mAb), and were negative for reactivity with pig T- and B-cell-specific mAb. This simple method for isolating and propagating macrophages may indicate the replicative capacity of either adult pig blood monocytes or circulating blood stem cells, and it may be useful in providing macrophages for general research, virological assay, adoptive-immunotherapy models, and somatic gene therapy models.     

Influence of vitamin D3 infusion and dietary calcium on secretion of interleukin 1, interleukin 6, and tumor necrosis factor in mice infected with Mycobacterium paratuberculosis

OBJECTIVE: To study the effects of 1,25(OH)2D3 and calcium (Ca) on splenocyte cytokine secretion during Mycobacterium paratuberculosis infection. DESIGN: Mice were assigned to the following treatments: 1-noninfected, 2-infected, 3-noninfected/1,25(OH)2D3, 4-infected/1,25(OH)2D3, and 5-infected/low-Ca diet (0.15%). ANIMALS--Male beige mice averaging 6 weeks of age and 20 g in body weight. PROCEDURE: After acclimation to their diets, mice in treatments 2, 4, and 5 were inoculated IV with 10(8) colony-forming units of M paratuberculosis. At 1, 6, and 12 months after infection, mice in treatment groups 3 and 4 had miniosmotic pumps implanted subcutaneously that delivered 13 ng of 1,25(OH)2D3/day for 14 days. Treatment 5 was included as a control for comparison with treatment 4 because low dietary Ca should increase endogenous 1,25(OH)2D3 values. Splenocytes were isolated from mice at 1, 6, and 12 months and stimulated in vitro with medium alone (nonstimulated), lipopolysaccharide (LPS), concanavalin A, and M paratuberculosis whole-cell sonicate (MpS). RESULTS: Production of interleukin 6 after stimulation with LPS, concanavalin A, or MpS was higher (P < 0.05) for splenocytes isolated from mice fed the low-Ca diet, compared with control infected mice 1 and 6 months after infection. Interleukin 1 and tumor necrosis factor activities were increased (P < 0.05) in splenocytes cultured with LPS and MpS after isolation from mice of the low-Ca group. Mice infused with 1,25(OH)2D3 had higher (P < 0.05) interleukin 1 secretion after stimulation of splenocytes with LPS and higher (P < 0.05) tumor necrosis factor production after incubation with MpS. CONCLUSION: 1,25(OH)2D3 and low dietary Ca increase cytokine secretion in mice infected with M paratuberculosis.     

Estramustine and estrone analogs rapidly and reversibly inhibit deoxyribonucleic acid synthesis and alter morphology in cultured human glioblastoma cells

Estramustine is an estradiol-based agent that has been shown to accumulate in human glioma cells, resulting in a concentration-dependent alteration in cell size and shape within minutes and an inhibition of proliferation over 3 to 6 days. We evaluated human glioblastoma cultures with [3H]thymidine incorporation assays to determine estramustine's early effects on deoxyribonucleic acid synthesis in these tumors. Because estramustine shares a common structural motif with other antimicrotubule drugs, we synthesized four A-ring conjugates of estrone that contained a carbamate moiety but lacked nitrogen mustard. These analogs were examined by [3H]thymidine incorporation and compared with vinblastine. Greater than 70% inhibition of [3H]thymidine incorporation occurred within 1 hour of treatment with estramustine at 10(-5) mol/L, which increased to 80% inhibition at 4 hours. Ethyl carbamate JE208 was nearly as effective as estramustine in inhibiting deoxyribonucleic acid synthesis, and both were more effective than vinblastine. The inhibitory effects of estramustine and estrone analogs were reversible; vinblastine was not reversible. Although estramustine and JE208 induced similar antiproliferative and morphological changes in glioblastoma cells that persisted for at least 4 days, there was a modest recovery of morphology and thymidine incorporation with JE208 after prolonged treatment. The common findings with estramustine and JE208 suggest that these agents may have a similar mechanism of action and form the basis for the investigation of new agents that may rapidly and reversibly inhibit glioblastoma.     

Reversal of advanced liver fibrosis in rabbits with Schistosomiasis japonica

Advanced liver fibrosis is generally considered to be irreversible. We studied the reversibility of marked liver fibrosis in rabbits infected with Schistosoma japonicum. We determined liver collagen content, collagen biosynthesis, and collagenase activity using serial biopsy specimens obtained 20, 40, and 60 weeks after infection. Reversibility of this process was investigated in rabbits cured of infection at 21 weeks; control rabbits not cured of infection were also studied. At 20 weeks, liver collagen content was 16-fold greater than normal, with accumulation of collagen types I, III, and V. Synthesis of collagen within fibrotic liver slices was 10-fold greater than normal. Liver collagenolytic activity for a type I substrate was 19-fold greater than normal. After parasitologic cure, a striking morphologic reversal of fibrosis occurred during the subsequent 40 weeks, with the return of liver collagen content to three-fold greater than normal and a 75% decrease in synthetic rates compared with those at 20 weeks (P < 0.01). Collagenolytic activity remained elevated to the same degree noted at 20 weeks. A similar but lesser resolution of fibrosis also occurred in untreated control rabbits, coincident with a spontaneous decrease in new egg deposition known to occur in this model system. We conclude that advanced liver fibrosis in S. japonicum-infected rabbits is slowly reversible after cure or senescence of the infection. A possible mechanism for this reversal is persistently increased collagenolysis as collagen synthesis diminishes.     

A missense mutation in the human connexin50 gene (GJA8) underlies autosomal dominant "zonular pulverulent" cataract, on chromosome 1q

OBJECTIVE: To determine the effect of hemorrhagic shock on spontaneous and endotoxin-induced cytokine release from intestinal mononuclear cells compared with whole portal venous blood and splenic macrophages. DESIGN: Random assignment to either unmanipulated control group, sham operation group, or hemorrhagic shock group. SETTING: University animal laboratory. SUBJECTS: Male Wistar rats, weighing between 300 and 350 g. INTERVENTIONS: Rats were bled to 30 mm Hg for 30 mins by withdrawal/reinfusion of shed blood and Ringer's lactate equivalent to the shed blood volume. MEASUREMENTS: Rats were killed immediately, 4 hrs, or 24 hrs after reperfusion. Portal venous blood, splenic macrophages, and small bowel mononuclear cells were obtained and spontaneous (unstimulated) and endotoxin-induced supernatant tumor necrosis factor (TNF)-alpha (WEHI 164 subclone 13) and interleukin (IL)-6 (B 13-29 clone 9) release was measured by bioassay. MAIN RESULTS: Increased endotoxin-induced TNF release from gut mononuclear cells and portal venous blood was suppressed 4 and 24 hrs after reperfusion compared with sham operated animals (p < .05). TNF release from splenic macrophages could be significantly increased (p < .05) by addition of endotoxin in all groups with no difference between control, sham, and shock animals. In shock animals, endotoxin-stimulated IL-6 release was significantly greater (p < .05) than control and sham operated rats 4 hrs after reperfusion in portal blood and from splenic macrophages. In contrast to splenic macrophages, gut mononuclear cells and portal venous blood demonstrated high spontaneous IL-6 concentrations without further stimulation by endotoxin. CONCLUSIONS: Hemorrhagic shock induced a hyporesponsiveness from gut mononuclear cells and whole portal venous blood 4 and 24 hrs after reperfusion. Spontaneous and endotoxin-induced stimulation of IL-6 indicates a different modulation of cytokines in gut, portal vein and spleen. The differences of gut mononuclear cells and portal blood compared with the splenic macrophages indicate a compartmentalized cytokine response.