Anti-CotH3 antibodies protect mice from mucormycosis by prevention of invasion and augmenting opsonophagocytosis

Mucorales are fungal pathogens that cause mucormycosis, a lethal angioinvasive disease. Previously, we demonstrated thatRhizopus, the most common cause of mucormycosis, invades endothelial cells by binding of its CotH proteins to the host receptor GRP78. Loss of CotH3 renders the fungus noninvasive and attenuatesRhizopusvirulence in mice. Here, we demonstrate that polyclonal antibodies raised against peptides of CotH3 protected diabetic ketoacidotic (DKA) and neutropenic mice from mucormycosis compared to mice treated with control preimmune serum. Passive immunization with anti-CotH3 antibodies enhanced neutrophil inlfux and triggered Fc receptor-mediated enhanced opsonophagocytosis killing ofRhizopus delemar. Monoclonal antibodies raised against the CotH3 peptide also protected immunosuppressed mice from mucormycosis caused byR. delemarand other Mucorales and acted synergistically with antifungal drugs in protecting DKA mice fromR. delemarinfection. These data identify anti-CotH3 antibodies as a promising adjunctive immunotherapeutic option against a deadly disease that often poses a therapeutic challenge.

Inhibition of Premature Oocyte Maturation: A Role for Bone Morphogenetic Protein 15 in Zebrafish Ovarian Follicles

Bone morphogenetic protein-15 (BMP-15) is a member of the TGF-β superfamily known to regulate ovarian functions in mammals. Recently, we cloned zebrafish BMP-15 (zfBMP-15) cDNA and demonstrated that it may play a role in oocyte maturation. In this study, we further investigated the role of BMP-15 in zebrafish follicular development and oocyte maturation using an antiserum developed for zfBMP-15 and by microinjection of follicles with antisense zfBMP-15 N-morpholino oligonucleotides or an expression construct containing zfBMP-15 cDNA. Injection with antiserum caused a significant decrease in maturation-incompetent [insensitive to maturation-inducing hormone (MIH)] early growth phase follicles and a concomitant increase in mature follicles in vivo. In vitro maturation assays showed that incubation with antiserum resulted in a significant increase in oocyte maturation as compared with follicles incubated in preimmune serum or media control. Next, early growth phase follicles were collected and preincubated with either antiserum, preimmune serum, or medium control before treatment with MIH or human chorionic gonadotropin (hCG). Antiserum significantly increased oocyte maturation in response to MIH, but not to hCG, and enhanced basal maturation rate in longer-term incubations. Knockdown of BMP-15 in early growth stage follicles with a BMP-15 antisense oligonucleotide resulted in increased oocyte maturation, whereas microinjection of BMP-15 cDNA into oocytes significantly reduced MIH- and hCG-induced oocyte maturation in normally competent, mid-growth-phase follicles. Collectively, these findings suggest that BMP-15 modulates follicular growth and prevents premature oocyte maturation in zebrafish, in part, by suppressing the sensitivity of follicles to MIH.

Enhancement of Clearance of Bacteria from Murine Lungs by Immunization with Detoxified Lipooligosaccharide fromMoraxella catarrhalis Conjugated to Proteins

ABSTRACT Moraxella catarrhalis strain 25238 detoxified lipooligosaccharide (dLOS)-protein conjugates induced a significant rise of bactericidal anti-LOS antibodies in animals. This study reports the effect of active or passive immunization with the conjugates or their antiserum on pulmonary clearance of M. catarrhalis in an aerosol challenge mouse model. Mice were injected subcutaneously with dLOS-tetanus toxoid (dLOS-TT), dLOS–high-molecular-weight proteins (dLOS-HMP) from nontypeable Haemophilus influenzae(NTHi), or nonconjugated materials in Ribi adjuvant and then challenged with M. catarrhalis strain 25238 or O35E or NTHi strain 12. Immunization with dLOS-TT or dLOS-HMP generated a significant rise of serum anti-LOS immunoglobulin G and 68% and 35 to 41% reductions of bacteria in lungs compared with the control (P < 0.01) following challenge with homologous strain 25238 and heterologous strain O35E, respectively. Serum anti-LOS antibody levels correlated with its bactericidal titers against M. catarrhalis and bacterial CFU in lungs. Additionally, immunization with dLOS-HMP generated a 54% reduction of NTHi strain 12 compared with the control (P < 0.01). Passive immunization with a rabbit antiserum against dLOS-TT conferred a significant reduction of strain 25238 CFU in lungs in a dose- and time-dependent pattern compared with preimmune serum-treated mice. Kinetic examination of lung tissue sections demonstrated that antiserum-treated mice initiated and offset inflammatory responses more rapidly than preimmune serum-treated mice. These data indicate that LOS antibodies (whether active or passive) play a major role in the enhancement of pulmonary clearance of different test strains of M. catarrhalis in mice. In addition, dLOS-HMP is a potential candidate for a bivalent vaccine against M. catarrhalis and NTHi infections.

Only low levels of spermadhesin AWN are detectable on the surface of live ejaculated boar spermatozoa

The zona-binding protein family of spermadhesins are constituents of boar seminal plasma that are generally believed to attach to the acrosomal region of spermatozoa and thereby assist sperm interaction with the zona pellucida at fertilization. However, previous studies have yielded conflicting results with respect to amounts of adhesin bound to ejaculated cells, to the distribution of bound adhesin within the sperm population, and the regionalization of binding on the sperm surface. In the present study, spermadhesin AWN in unfixed living suspensions of boar spermatozoa was assessed by means of flow cytometry and immunocytochemistry, using a polyclonal antibody raised in chicken. Direct probing with an Oregon Green conjugate of the antibody was compared with indirect probing using Alexa Fluor-conjugated goat anti-chicken IgG as second antibody. Regardless of staining procedure, the live sub-population showed homogeneously low levels of staining, whereas the dead sub-population showed high (more than 5-fold greater) levels of staining. The live cells were stained about 2-fold more intensely by anti-AWN than by preimmune immunoglobulin, indicating the presence of small amounts of AWN. Immunocytochemistry showed the live cells to be faintly stained all over their surface, whereas staining of the dead cells was largely localized to the acrosomal region. This latter staining was non-specific, preimmune immmunoglobulin resulting in as much bound fluorescence as anti-AWN. Attempts to block non-specific staining with appropriate pretreatment with chicken or goat serum (as compared with routine use of BSA) met with variable and incomplete success, and did not increase staining by anti-AWN relative to preimmune serum in either live or dead cells. It is concluded that limited amounts of spermadhesin AWN bind tightly over the whole surface of live ejaculated boar sperm. However, the acrosomal region of disrupted sperm has an alarming tendency to bind fluoro-conjugates of immunoglobulins non-specifically.

Decreased Adherence of EnterohemorrhagicEscherichia coli to HEp-2 Cells in the Presence of Antibodies That Recognize the C-Terminal Region of Intimin

ABSTRACT Antiserum raised against intimin from enterohemorrhagicEscherichia coli (EHEC) O157:H7 strain 86-24 has been shown previously by our laboratory to inhibit adherence of this strain to HEp-2 cells. In the present study, we sought to identify the region(s) of intimin important for the effect of anti-intimin antisera on EHEC adherence and to determine whether antisera raised against intimin from an O157:H7 strain could reduce adherence of other strains. Compared to preimmune serum controls, polyclonal sera raised against the histidine-tagged intimin protein RIHisEae (intiminO157) or against His-tagged C-terminal fragments of intimin from strain 86-24 reduced adherence of this strain. Furthermore, an antibody fraction purified from the anti-RIHisEae serum that contained antibodies to the C-terminal third of intimin, the putative receptor-binding domain, also reduced adherence of strain 86-24, but a purified fraction containing antibodies to the N-terminal two-thirds of intimin did not inhibit adherence. The polyclonal anti-intiminO157 serum raised against RIHisEae inhibited, to different degrees, the adherence of another O157:H7 strain, an EHEC O55:H7 strain, one of two independent EHEC O111:NM isolates tested, and one of two EHEC O26:H11 strains tested. Adherence of the other O26:H11 and O111:NM strains and an EPEC O127:H6 strain was not reduced. Finally, immunoblot analysis indicated a correlation between the antigenic divergence in the C-terminal third of intimins from different strains and the capacity of anti-intiminO157 antiserum to reduce adherence of heterologous strains. Taken together, these data suggest that intiminO157 could be used as an immunogen to elicit adherence-blocking antibodies against O157:H7 strains and closely-related EHEC.

Colocalization of H-ATPase and H,K-ATPase immunoreactivity in the rat kidney.

Urinary acidification in the collecting duct (CD) is via V-type H-ATPase and P-type H,K-ATPase. The localization and polar distribution of H-ATPase in intercalated cells (IC) have been well studied. The localization of H,K-ATPase to IC has been reported, but its intracellular distribution has not been defined. To colocalize these pumps, a murine monoclonal antibody (E11) to the 31-kd subunit of the H-ATPase and a rabbit antiserum (HK alpha N2) to a synthetic peptide based on the N terminus of the hog gastric H,K-ATPase alpha-subunit were used. In immunocytochemical staining of rat kidney, H,K-ATPase was present only in the IC of the CD with the same polar distribution as H-ATPase. The preabsorption of HK alpha N2 with affinity-purified bovine H-ATPase did not affect the H,K-ATPase staining, whereas preabsorption with the immunizing synthetic peptide eliminated immunoreactivity. HK alpha N2 stained only parietal cells in the rat gastric mucosa, whereas preimmune serum and E11 showed no immunoreactivity. On immunoblots of rat gastric mucosal microsomes, HK alpha N2 labeled a single 94-kd band, and this staining disappeared after preabsorption with the immunizing synthetic peptide. HK alpha N2 labeled no bands on immunoblots of affinity-purified bovine H-ATPase. All immunochemistry was negative with preimmune serum.(ABSTRACT TRUNCATED AT 250 WORDS)

Dynamic changes in the distribution of cytoplasmic myosin during Drosophila embryogenesis

Dramatic changes in the localization of conventional non-muscle myosin characterize early embryogenesis in Drosophila melanogaster. During cellularization, myosin is concentrated around the furrow canals that form the leading margin of the plasma membrane as it plunges inward to package each somatic nucleus into a columnar epithelial cell. During gastrulation, there is specific anti-myosin staining at the apical ends of those cells that change shape in regions of invagination. Both of these localizations appear to result from a redistribution of a cortical store of maternal myosin. In the preblastoderm embryo, myosin is localized to the egg cortex, sub-cortical arrays of inclusions, and, diffusely, the yolk-free periplasm. At the syncytial blastoderm stage, myosin is found within cytoskeletal caps associated with the somatic nuclei at the embryonic surface. Following the final syncytial division, these myosin caps give rise to the myosin rings observed during cellularization. These distributions are observed with both whole immune serum and affinity-purified antibodies directed against Drosophila non-muscle myosin heavy chain. They are not detected in embryos stained with anti-Drosophila muscle myosin antiserum or with preimmune serum. Although immunolocalization can only suggest possible function, these myosin localizations and the coincident changes in cell morphology are consistent with a key role for non-muscle myosin in powering cellularization and gastrulation during embryogenesis.

Immunoelectron microscopy on lysis of the oocyte vitelline coat induced by a protein from spermatozoa in a marine mollusk, Tegula pfeifferi

A protein responsible for lysis of the oocyte vitelline coat (VC), that is, a VC lysin, has been isolated from spermatozoa of the mollusk, Tegula pfeifferi. Biochemical studies using purified VC lysin and isolated VCs have revealed that the action of this VC lysin is stoichiometric rather than enzymatic. The present immunoelectron microscopic study was undertaken to obtain morphological evidence for this uncommon lytic mechanism using antibodies against the lysin and the soluble product (SP) released from the VC upon lysin treatment.Oocytes isolated from dissected ovaries were treated with the lysin (1 mg/ml) at 25°C for 10 min until their VCs were elevated high from the oocyte surface. The lysin-treated oocytes were prefixed with either 1% glutaraldehyde (GA) for cytochemical study or 2.5% GA plus tannic acid (TA) for morphological study. To localize the lysin and the SP in the VCs, prefixed oocytes were treated with ovalbumin followed by one of the antisera or preimmune serum (control).