Prolactin exerts hematopoietic growth-promoting effects in vivo and partially counteracts myelosuppression by azidothymidine.

Prolactin (PRL) is a neuroendocrine hormone that influences immune and hematopoietic development. The mechanism of action of this hormone in vivo remains unclear; therefore, we assessed the effects of PRL on hematopoiesis in vivo and in vitro. Normal resting mice were treated with 0, 1, 10, or 100 microg of recombinant human prolactin (rhPRL) for 4 consecutive days and euthanized on the fifth day for analysis of myeloid and erythroid progenitors in the bone marrow and spleen. Both frequencies and absolute numbers of splenic colony-forming unit granulocyte-macrophage (CFU-GM) and burst-forming unit-erythroid (BFU-e) were significantly increased in mice receiving rhPRL compared to the controls that had received saline only. Bone marrow cellularities were not significantly affected by any dose of rhPRL, but the absolute numbers and frequencies of bone marrow CFU-GM and BFU-e were augmented by rhPRL. These results suggest that rhPRL can promote hematopoiesis in vivo. Because rhPRL augments myeloid development in vivo, we examined the potential of the hormone to reverse the anemia and myelosuppression induced by azidothymidine (AZT). Mice were given rhPRL injections concurrent with 2.5 mg/mL AZT in drinking water. rhPRL partially restored hematocrits in the animals after 2 weeks of treatment and increased CFU-GM and BFU-e in both spleens and bone marrow. The experiments with AZT and rhPRL support the conclusion that the hormone increases myeloid and erythroid progenitor numbers in vivo, and they suggest that the hormone is clinically useful in reversing myelosuppression induced by AZT or other myeloablative therapies.

Use of neuroendocrine hormones to promote reconstitution after bone marrow transplantation.

A survey of the previous literature and the data shown here indicate that neuroendocrine hormones such as growth hormone and prolactin may be of potential clinical use after bone marrow transplantation (BMT) to promote hematopoietic and immune recovery. The amounts of hormones used in our model do not promote weight gain suggesting that their lymphohematopoietic actions were independent of their anabolic effects. While the hormones may not produce the same extent of immune/hematopoietic effects when compared to conventional hematopoietic and immune stimulating cytokines (i.e. IL-2 or G-CSF), their pleiotropic effects and limited toxicity after systemic administration makes them attractive to test in the post-BMT setting. However, more work needs to be performed to understand the mechanism(s) of their action, particularly with regard to T-cell function and development.

Recombinant human growth hormone promotes hematopoietic reconstitution after syngeneic bone marrow transplantation in mice.

Recombinant human growth hormone (rhGH) was administered to mice after syngeneic bone marrow transplantation (BMT) to determine its effect on hematopoietic reconstitution. BALB/c mice were given 10 microg intraperitoneal injections of rhGH every other day for a total of 10 injections following syngeneic BMT. Mice that received rhGH exhibited significant increases in total hematopoietic progenitor cell content (colony-forming unit-culture) in both bone marrow and spleen. Erythroid cell progenitor content (burst-forming unit-erythroid) was also significantly increased after rhGH treatment. Analysis of peripheral blood indicated that administration of rhGH resulted in significant increases in the rate of white blood cell and platelet recovery. Granulocyte marker 8C5+ cells were also increased in the bone marrow and spleens of treated mice. Red blood cell, hematocrit, and hemoglobin levels were increased at all time points after rhGH treatment. No significant pathologic effects or weight gain were observed in mice receiving repeated injections of 10 microg rhGH. Thus, rhGH administration after syngeneic BMT promoted multilineage hematopoietic reconstitution and may be of clinical use for accelerating hematopoiesis after autologous BMT.

Differential immune responses to staphylococcal enterotoxin B mutations in a hydrophobic loop dominating the interface with major histocompatibility complex class II receptors.

Bacterial superantigens, such as staphylococcal enterotoxin B (SEB), can trigger acute pathologic effects in humans. A hydrophobic loop on the surface of SEB and other bacterial superantigens, centered around a conserved leucine (L45) residue, is essential for binding to class II major histocompatibility complex molecules. Single residue changes of wild type SEB, designated Q43P, F44P, or L45R, resulted in nonlethal proteins at a dose equivalent to 30 murine LD50 of SEB. Relative to SEB, the mutant proteins did not elevate serum concentrations of proinflammatory cytokines in mice and caused minimal proliferation of human lymphocytes. Anti-SEB titers of mice immunized with Q43P, F44P, L45R, or SEB were similar and protected 77%-100% of animals against a lethal SEB challenge. Levels of toxin-specific IgG1, IgG2a, IgG2b, and IgG3 in mice immunized with SEB, Q43P, or F44P were equivalent, but animals immunized with L45R had significantly elevated levels of IgG2a and IgG2b. Vaccines against staphylococcal superantigens should focus on this critical leucine residue.

Replication of coliphage Q beta as affected by host cell number, nutrition, competition from insusceptible cells and non-FRNA coliphages.

F-specific RNA (FRNA) coliphages, which infect Escherichia coli by attachment to F pili, might serve as indicators of human enteric viruses in groundwater, provided these phages do not replicate in groundwater and replicate only to a limited extent in wastewater. Several factors that could influence phage replication in either of these environments were examined. Q beta did not replicate when host cells were fewer than 10(4) cfu ml-1. Replication selected for insusceptible cells when Q beta was incubated with its E. coli host. Loss of Q beta, presumably by inactivation, occurred in autoclaved on-site and urban wastewater, autoclaved groundwater, and in filter-sterilized spent LB broth. Replication did not occur in LB broth diluted with sterile saline to 1% of its original strength, which indicates that replication of FRNA coliphages cannot occur in such nutrient-poor environments as wastewater and groundwater. Competition from non-FRNA coliphages and insusceptible cells tended to reduce Q beta replication, as predicted, but phage yields unexpectedly increased significantly when Enterococcus faecalis was added to cultures.

Staphylococcal enterotoxin B mutants (N23K and F44S): biological effects and vaccine potential in a mouse model.

Superantigens produced by Staphylococcus aureus can cause food poisoning and toxic shock syndrome. The biological activities and vaccine potential of mutant staphylococcal enterotoxin B (SEB) proteins, N23K and F44S, were studied in a lipopolysaccharide-potentiated mouse model. Although 10 micrograms of SEB per mouse is equivalent to 30 LD50, the same intraperitoneal dose of either mutant protein was nonlethal and did not elevate serum levels of tumor necrosis factors (TNF). N23K, F44S, and SEB were serologically identical in an enzyme-linked immunosorbent assay with polyclonal anti-SEB. Immunization with alum containing N23K, F44S, or SEB elicited an anti-SEB response that protected 80-87% of the mice against a 10 micrograms SEB challenge. Controls lacking an anti-SEB titer did not survive. Pooled sera from immunized mice effectively blocked SEB-induced T-cell proliferation in vitro. Naive mice survived a lethal SEB challenge when given pooled antisera 1, 2, or 4 h later, whereas the antisera failed to protect animals when administered 6 or 8 h after the toxin. Lethality at the later times was consistent with increased serum levels of TNF observed 6 h after SEB injection. These studies suggest that the N23K and F44S mutant proteins of SEB are less biologically active than the wild-type toxin, yet retain epitopes useful for eliciting a protective antibody response.

Effects of temperature and host cell growth phase on replication of F-specific RNA coliphage Q beta.

Human enteric viruses have been found in groundwater in the absence of fecal coliforms. Because detection of human enteric viruses is costly, time-consuming, and lacking in sensitivity, F-specific RNA (FRNA) coliphages, which infect Escherichia coli by attachment to F pili, are being examined for suitability as indicators of human enteric viruses in groundwater. Temperatures and host cell growth conditions that constrain F-pilus expression will limit FRNA coliphage replication in groundwater and wastewater, as is desirable in an indicator. Below 25 degrees C F-pilus synthesis ceases; FRNA coliphage Qbeta did not replicate below this temperature in batch cultures. One-step replication studies indicated that the replicative cycle is prolonged and that fewer progeny are released as the temperature decreases. The decreases in phage replication observed in the one-step replication studies were a consequence of fewer cells infected as the temperature was lowered or as host cells entered stationary phase. The numbers of phage particles released from infected cells did not change. The minimum temperature for replication of Qbeta, 25 degrees C, is not maintained in wastewater and does not occur in Wisconsin groundwater. On the basis of temperature and host cell growth phase, we have concluded that extensive replication of FRNA coliphages does not occur in wastewater and groundwater in Wisconsin and areas with similar cool climates.

Modification of carboxyl groups in botulinum neurotoxin types A and E.

Effects of chemical modification of carboxyl groups of botulinum neurotoxin serotypes A and E were studied by using a water soluble carbodiimide-nucleophile reaction that is highly specific for modifying carboxyl groups of proteins. In both types A and E, increasing levels of the reagents, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and norleucine methyl ester or glycine methyl ester, at pH 4.8 caused increased loss of toxicity. More glycine could be incorporated than norleucine. Amino acid analysis did not reveal modification of any amino acid residue other than carboxyl groups (possible reaction of sulfhydryl groups was not studied). Loss of one carboxyl group did not severely affect toxicity, but modification of three carboxyl groups caused greater than 95% detoxification in both types. Complete detoxification could not be achieved with any amount of the reagents. Modification of three to five carboxyl groups did not affect serological activity.

Fast protein liquid chromatography of botulinum neurotoxin types A, B and E.

Three antigenically different botulinum neurotoxins (NTs, relative molecular mass approximately 150,000), classically distinguished only by specific antisera, were for the first time chromatographically resolved. Mixed NTs eluted from a Mono-Q column in order of types E, A and B, and from Mono-S as B, E and A. Type A and B NTs were successfully chromatographed on the cation-exchange Mono-S column above their isoelectric points. Purification of type A and B NTs by automated liquid chromatography was also accomplished for the first time. Type A, B and E NTs were purified by application on an anion-exchange Mono-Q column, followed by use of a cation-exchange Mono-S column.

Amino acid composition of Clostridium botulinum type B neurotoxin.

To characterize type B botulinum neurotoxin based on reliable data on the amino acid composition, three batches of the neurotoxin were analyzed. Each batch was isolated from a separate neurotoxin producing bacterial culture (strain Okra). Two batches were purified by the same method and one was purified by a different method. The toxin preparations were comparable in purity (judged by polyacrylamide gel--sodium dodecyl sulfate electrophoresis) and similar in amino acid composition. The best estimate of the number of amino acid residues per toxin molecule (mol. wt 152,000) was: Asp212,Thr54,Ser83,Glu130,Pro46,Gly61++ +,Ala44,Val54,CyS11,Met23,Ile144,Leu107 , Tyr81,Phe77,Lys118,His7,Arg39,Trp18.