Anti-LcrV antibody inhibits delivery of Yops by Yersinia pestis KIM5 by directly promoting phagocytosis

LcrV of Yersinia pestis is a major protective antigen proposed for inclusion in subunit plague vaccines. One way that anti-LcrV antibody is thought to protect is by inhibiting the delivery of toxins called Yops to host cells. The present study characterizes the relation between this inhibition and the phagocytosis of the bacteria. J774A.1 cells were infected with Y. pestis KIM5 in the presence of a protective polyclonal anti-LcrV antibody or a nonprotective polyclonal anti-YopM antibody, and delivery of YopH and YopE into the cytoplasm was assayed by immunoblotting. The ability to inhibit the delivery of these Yops depended upon having antibody bound to the cell surface; blocking conditions that prevented the binding of antibody to Fc receptors prevented the inhibition of Yop delivery. Anti-LcrV antibody also promoted phagocytosis of the yersiniae, whereas F(ab')(2) fragments did not. Further, anti-LcrV antibody could not inhibit the delivery of Yops into cells that were unable to phagocytose due to the presence of cytochalasin D. However, Yops were produced only by extracellular yersiniae. We hypothesize that anti-LcrV antibody does not directly inhibit Yop delivery but instead causes phagocytosis, with consequent inhibition of Yop protein production in the intracellular yersiniae. The prophagocytic effect of anti-LcrV antibody extended to mouse polymorphonuclear neutrophils (PMNs) in vitro, and PMNs were shown to be critical for protection: when PMNs in mice were ablated, the mice lost all ability to be protected by anti-LcrV antibody.     

Antibody against V antigen prevents Yop-dependent growth of Yersinia pestis

The V antigen (LcrV) of the plague bacterium Yersinia pestis is a potent protective antigen that is under development as a vaccine component for humans. LcrV is multifunctional. On the bacterial surface it mediates delivery of a set of toxins called Yops into host cells, and as a released protein it can cause production of the immunosuppressive cytokine interleukin-10 (IL-10) and can inhibit chemotaxis of polymorphonuclear neutrophils. It is not known how these mechanisms of LcrV operate, what their relative importance is, when they function during plague, and which are critical to protection by antibody. This study investigated several of these issues. C57BL/6 mice, mice unable to express IL-10, or mice with the macrophage lineage eliminated were treated with a protective anti-LcrV antibody or a nonprotective antibody against YopM and infected intravenously by Y. pestis KIM5 or a strain that lacked the genes encoding all six effector Yops. Viable bacterial numbers were determined at various times. The data indicated that Yops were necessary for Yersinia growth after the bacteria had seeded liver and spleen. Anti-LcrV antibody prevented this growth, even in IL-10-/- mice, demonstrating that one protective mechanism for anti-LcrV antibody is independent of IL-10. Anti-LcrV antibody had no effect on persistence in organs of Y. pestis lacking effector Yops, even though the yersiniae could strongly express LcrV, suggesting that Yops are necessary for building sufficient bacterial numbers to produce enough LcrV for its immunosuppressive effects. In vitro assays showed that anti-LcrV antibody could partially block delivery of Yops and downstream effects of Yops in infected macrophage-like J774A.1 cells. However, cells of the macrophage lineage were found to be dispensable for protection by anti-LcrV antibody in spleen, although they contributed to protection in liver. Taken together, the data support the hypothesis that one protective effect of the antibody is to block delivery of Yops to host cells and prevent early bacterial growth. The findings also identified the macrophage lineage as one host cell type that mediates protection.     

Chromatin decondensation, pronucleus formation, metaphase entry and chromosome complements of human spermatozoa after intracytoplasmic sperm injection into hamster oocytes

Obtaining karyotypes from human spermatozoa after microinjection into Syrian golden hamster oocytes is difficult and the hitherto reported results are unsatisfactory. This may be related to the injection and culture technique or to the high susceptibility of the hamster oocytes to undergo parthenogenetic activation or both. Therefore, we investigated the hamster oocyte-human sperm microinjection model using the following two approaches: (i) application of contemporary techniques for injection (touching the sperm tail) and culture (hamster embryo culture medium, HECM-3, 10% CO2) and (ii) omission of Ca2+ from the injection medium. Thus, in the first series of experiments, 252 hamster oocytes were injected with human spermatozoa. Among the 219 (87%) oocytes that survived the injection procedure, the mean percentages of male pronucleus formation [two pronuclei (2PN), two polar bodies (PB)], mitotic metaphase entry and sperm chromosome spreads were 41.4, 27.8 and 18.2% respectively. Analysis of the oocytes which failed to develop the male pronucleus following injection revealed that most of them had developed only the hamster female PN while the sperm nuclei were either intact or swollen (partially decondensed), indicating that failure of oocyte activation was not the likely reason for the failure of male PN formation in these oocytes. In the next series of experiments, sibling oocytes were alternately injected with spermatozoa suspended either in the regular (1.9 mM Ca2+) or Ca2+-free injection medium (experiment set 2, n=278). A significant improvement was noted in the mean percentages of oocytes with 2PN, 2PB, metaphase entry and sperm chromosome spreads in the Ca2+-free group versus the regular group (2PN, 2PB: 51 versus 36.6%, metaphase entry: 36.3 versus 26.9% and sperm chromosome spreads: 28 versus 20.4%; all P < 0.04). Thus, parthenogenetic activation appears to be one of the contributing factors for the failure of male PN formation after heterospecific hamster ICSI. From these experiments it can be concluded that application of the advanced injection and culture techniques and omission of Ca2+ from the injection medium are promising for the routine application of the hamster oocyte microinjection for karyotyping of human spermatozoa with poor fertilizing capacity.      

Role of ADP-ribosyl transferase in differentiation of human granulocyte- macrophage progenitors to the macrophage lineage

Nuclear adenosine diphosphate-ribosyl (ADP-ribosyl) transferase is a chromatin-bound enzyme catalyzing the transfer of ADP-ribose from NAD+ to chromatin proteins. The physiologic function of this covalent modification of chromatin has not been fully established, but roles in both DNA repair and in differentiation have been proposed. We demonstrate that three specific inhibitors of ADP-ribosyl transferase (5-methylnicotinamide, 3-methoxybenzamide, 3-aminobenzamide) inhibit differentiation of human granulocyte-macrophage progenitor cells to the macrophage lineage. Differentiation to the neutrophil-granulocyte lineage is much less affected. The inhibition of macrophage differentiation seems to relate to the ability of these compounds to inhibit ADP-ribosyl transferase. A structural analogue (3- methoxybenzoic acid), which is not inhibitory for the enzyme, did not inhibit macrophage differentiation. Additional evidence for a role of ADP-ribosyl transferase in the differentiation of granulocyte- macrophage progenitors was obtained from experiments in which enzyme activity levels were measured in permeabilized marrow cells. Marrow cell ADP-ribosyl transferase activity increased after 3-hr stimulation by the differentiation/proliferation stimulus--granulocyte-macrophage colony-stimulating activity (GM-CSA). Unstimulated marrow cells showed low or undetectable levels of enzyme activity.     

Estrogen-induced microsatellite DNA alterations are associated with Syrian hamster kidney tumorigenesis

Exposure to estrogens is associated with an increase in cancers, including malignancies of the breast and uterus in humans, and of the kidney in hamsters. DNA damage induced by metabolic activation of estrogen has been postulated to result in gene mutations critical for the development of estrogen-induced kidney tumors in hamsters. As part of our examination of the genetic consequences of estrogen-induced DNA damage, we searched for estrogen-induced alterations in microsatellite DNA, a frequent site of mutation in tumors. Genomic DNA isolated from kidney of hamsters treated with estradiol, from estrogen-induced kidney tumors and from untreated age-matched controls, was examined by Southern blot analysis with three multi-locus oligonucleotide probes: (GACA)4, (CAC)6 and (CAG)6. Alterations in DNA fragments containing GACA and CAC tandem repeats were detected in kidney DNA of hamsters treated with hormone for 3 and 4 months, whereas no such effects were seen in control animals. In estrogen-induced tumors, microsatellite alterations were observed in fragments that contain these same two repeat sequences and also CAG repeat sequences. The induction of microsatellite alterations by estradiol in kidney DNA preceding estrogen-induced renal malignancy may play a role in hormone-induced tumorigenesis.      

Cutaneous infection caused by Salmonella typhi

Salmonella typhi is a flagellated, gram-negative bacillus belonging to the family Enterobacteriaceae responsible for typhoid fever, which is a prolonged bacteraemic, systemic illness with minimal, at least initially, diarrhoea. Localized infection is a not uncommon complication of salmonella septicaemia, particularly occurring in immunocompromised patients. However, salmonella localization to the skin presenting as cutaneous ulceration is regarded as a rare event. We report a unique case of such a presentation as the sole clinical manifestation of infection with S. typhi and highlight the possible salmonella aetiology for unusual cutaneous lesions in individuals coming from endemic areas.     

Elephantoid oedema of the eyelids

We describe a male patient with rosacea who had a 2-year history of persistent bilateral oedema of the eyelids, leading to an elephantoid condition with blepharoptosis. An upper eyelid blepharoplasty was performed, but swelling progressively recurred over a few months. Based on the case history, clinical appearance and histological findings, rosaceous lymphoedema was considered to be the diagnosis. The latter is a bilateral, solid oedema of the mid-third of the face, regarded as a rare complication of rosacea. It is thought to occur as a result of chronic inflammation and lymphatic stasis, but its exact aethiopathogenesis remains elusive. Predominant eyelid involvement, causing severe visual impairment as in our patient, is unique.