Therapy of Staphylococcal Infections in Monkeys. IV. Further Comparison of Triacetyloleandomycin and Erythromycins

Intravenous inoculation of a penicillin-resistant, phage type 80/81 staphylococcus caused lethal infection in 8 of 15 untreated monkeys. Daily intragastric administration of 50 mg/kg of triacetyloleandomycin, erythromycin estolate, and erythromycin ethylsuccinate was followed by mortalities of 0 of 16, 3 of 16, and 3 of 10, respectively. At dose levels of 25 and 12.5 mg/kg, none of 7 and 4 of 7 receiving triacetyloleandomycin and erythromycin estolate, respectively, died, as compared to 3 of 4 deaths in controls. In vitro sensitivity data and serum antibacterial levels would suggest that triacetyloleandomycin would be the least effective therapeutically. However, this prediction was not fulfilled in these studies of experimental infections in monkeys wherein triacetyloleandomycin was a very effective antimicrobial agent.     

Therapy of Staphylococcal Infections in Monkeys: VI. Comparison of Clindamycin, Erythromycin, and Methicillin

Intravenous inoculation of a penicillin-resistant, phage type 80/81 staphylococcus caused lethal infection in six of eight untreated monkeys. Daily intragastric administration of clindamycin hydrochloride and erythromycin stearate and intramuscular inoculation of clindamycin-2-phosphate and methicillin, all at a dose level of 50 mg/kg, was followed by mortalities of one of eight, one of eight, none of eight, and one of eight monkeys, respectively. Duration of obvious acute illness in surviving monkeys and time required for complete recovery were not significantly different in the four therapy groups with the exception that duration of acute illness in monkeys treated with clindamycin-2-phosphate (mean, 4.1 days) was significantly shorter than in monkeys given erythromycin stearate (mean, 7.1 days). In vitro sensitivity data and serum antibacterial levels would suggest that methicillin would be the least effective therapeutically, followed by erythromycin stearate and the two clindamycin preparations in that order. However, this prediction was not fulfilled in these studies in experimentally infected monkeys.     

Enhancing Effect of Alkalinization of the Medium on the Activity of Erythromycin Against Gram-negative Bacteria

The antibacterial activity of erythromycin was markedly enhanced by alkalinization of the culture medium or urine within the clinical range (pH 6.0 to 8.2). This effect was demonstrated against recent isolates of Escherichia coli, Klebsiella pneumoniae, Enterobacter sp., and Pseudomonas aeruginosa, as well as against Staphylococcus aureus and Streptococcus faecalis. The urine of normal volunteers was made alkaline by ingestion of sodium bicarbonate or acetazolamide (Diamox) during administration of 1.0 g of erythromycin every 8 hr; such urine was capable of inhibiting E. coli and K. pneumoniae even when diluted up to (in one instance) 128 times with broth of the same pH as the urine. Undiluted urine of the same subjects, without alkalinization, was seldom capable of inhibiting these organisms. The range of pH (6.6 to 8.6) over which the antibacterial effect was enhanced coincided with that over which there was decreasing ionization of a basic group.     

Microbiological and Pharmacological Behavior of 7-Chlorolincomycin

Replacement of the 7-(R) hydroxyl group of lincomycin by a 7-chloro-substituent produced a compound with greater in vitro activity than the parent. Laboratory studies of this compound showed it to be highly active against all of the following strains of gram-positive organisms examined, including penicillinase- and nonpenicillinase-producing staphylococci, Diplococcus pneumoniae, Streptococcus viridans and Streptococcus pyogenes. The enterococci, as well as all the gram-negative organisms tested, with the exception of some strains of Haemophilus, were uniformly insensitive to this agent. The activity of 7-chlorolincomycin was not affected by serum or inoculum size. Resistance developed in a slow stepwise pattern. Peak levels of approximately 2 mug/ml were achieved in the serum of volunteers after ingestion of 150 mg either in the fasting state or after a meal. No untoward effects were noted. The antibiotic appears to be of potential value in the treatment of infections due to gram-positive organisms, with the exception of enterococcus.     

Active immunization of cynomolgus monkeys (Macaca fascicularis) with porcine zonae pellucidae

Cynomolgus monkeys (Macaca fascicularis) were immunized with porcine zonae pellucidae to assess the possible antifertility effects of the zona antibodies. Serum antibody titers were evaluated utilizing a rapid solid-phase radioimmunoassay. Six of twelve monkeys conceived 6 to 10 wk after vaccination. All monkeys reached maximal antiserum titers by the time of conception, although the six animals that did not conceive had considerably lower antibody titers. Further pregnancies did not occur until antibody level had declined markedly, 8 mo after last immunization. The menses of all but one of the remaining six monkeys were interrupted intermittently. Also, the usual midcycle elevated estradiol levels were absent for several cycles. Both menses and midcycle estradiol peaks were reestablished in all but one monkey 3 to 5 mo after the last booster was given. Two monkeys conceived when serum antibody levels dropped to one fourth of maximal, but both had a still birth. Histological observations showed accumulation of luteal tissue and massive atresia of small follicles at the end of the study (18 mo). We conclude that through heteroimmunization with porcine zona pellucida monkeys can become infertile and that this condition is reversible. Because the zona preparation used in this study appeared to contain traces of nonzona material, it was not possible to determine whether the menstrual irregularities and oocyte atresia that we observed were owing to immunological effects on the zona itself or to the production of antibodies against other ovarian components.     

Reduced immunogenicity and improved pharmacokinetics of humanized anti-Tac in cynomolgus monkeys

The anti-Tac mAb has been shown to bind to the p55 chain of the IL-2R, block IL-2 binding and inhibit T cell proliferation. A humanized form of anti-Tac (HAT) has been constructed that retains the binding properties of murine anti-Tac (MAT). These two mAb were evaluated in cynomolgus monkeys to compare relative immunogenicity and pharmacokinetic properties. Monkeys treated with HAT daily for 14 days exhibited anti-HAT antibody titers which were 5- to 10-fold lower than their MAT-treated counterparts and these antibodies developed later than in the MAT-treated monkeys. Two of four monkeys receiving a single injection of MAT developed anti-MAT antibodies, whereas none of four monkeys developed antibodies after a single treatment with HAT. In monkeys injected with either HAT or MAT daily for 14 days, the anti-antibody titers induced were inversely related to the amount of anti-Tac administered. Antibodies that developed against MAT were both anti-isotypic and anti-idiotypic, whereas those developed against HAT appeared to be predominantly anti-idiotypic. The pharmacokinetic properties, that is the half-life and area under the curve values, of HAT were also significantly different from those of MAT. The area under the curve values for HAT in naive monkeys were approximately twofold more than those for MAT, and the mean serum half-life of HAT was 214 h, approximately four- to fivefold more than MAT. These pharmacokinetic values were reduced in monkeys previously sensitized with HAT or MAT suggesting that the presence of anti-antibodies altered these parameters.     

The use of freeze-preserved treponemes in the Treponema pallidum immobilization test

Treponema pallidum extracted from rabbit testes and quick frozen in liquid nitrogen were vigorously motile upon thawing, when cryoprotected with dimethyl sulfoxide (DMSO). However, this chemical was toxic to unfrozen treponemes unless normal serum, 10–20% final concentration, was added to suspensions before freezing, or immediately after thawing. Such organisms survived the in vitro incubation of the T. pallidum immobilization (TPI) test, but DMSO partially inhibited immune immobilization. This inhibition was concentration dependent and was not observed in treponeme preparations diluted fourfold with fresh medium after 10% DMSO storage. Frozen treponeme suspensions used in this fashion yielded TPI test results comparable to those of fresh suspensions, and were still satisfactory after 12 months storage.     

Prevention of the toxicity of erythromycin estolate in the perfused rat liver by endotoxin

The possibility that endotoxin pretreatment could prevent the hepatotoxic effects of erythromycin estolate (EE) was investigated using the isolated perfused rat liver. The addition of E. coli endotoxin (25 micrograms/ml) to the perfusate, 30 min prior to EE administration at 150 or 200 microM, significantly ameliorated the decreases in bile and perfusate flow caused by either concentrations of the drug in control liver preparations. This phenomenon was also studied using liver isolated from rats pretreated in vivo with endotoxin for three days. In these preparations, EE at both concentrations did not alter bile flow and caused reductions of perfusate flow which were far less than those observed in untreated control livers. Furthermore, in livers from endotoxin-treated rats EE induced less reduction of bile acid excretion and, at 150 microM, it did not increase the bile to perfusate ratio of sucrose seen in control preparations after the drug, which may be an expression of altered hepatocytic membrane permeability. Since it is known that both endotoxin and EE interact with membranes, it is suggested that the "protective" effects of endotoxin may occur at the membrane level.     

Effects of Certain O-Acyl Derivatives of Oleandomycin and Erythromycin on Chloroplasts of Euglena gracilis

SYNOPSIS. A series of mono-, di- and triesters of oleandomycin and monoesters of erythromycin was tested for bleaching activity on the chloroplast system of Euglena gracilis. Many esters were more active than the original antibiotic base. The most active substances were those with esterification at the neutral sugar oleandrose. The effect of triacetyloleandomycin relative to oleandomycin on Euglena cells correlates directly with its relative antibacterial activity in vivo , while the effect correlates inversely with relative antibacterial activity in vitro . Like the macroorganism, E. gracilis probably accommodates the triester in a manner affording a net, relatively higher concentration of substances to which the chloroplasts are more sensitive.     

Synthetic peptides containing T and B cell epitopes from human immunodeficiency virus envelope gp120 induce anti-HIV proliferative responses and high titers of neutralizing antibodies in rhesus monkeys.

We have previously described a synthetic peptide (T1-SP10) derived from two noncontiguous regions of HTLVIIIB envelope gp120 (T1, amino acids 428-443; SP10, amino acids 303-321) that induced type-specific anti-HIV neutralizing antibodies and T cell proliferative responses against native HIV gp120 when used as a carrier-free immunogen in goats. In this study, HTLVIIIB T1-SP10 synthetic peptides were used to immunize rhesus monkeys to determine if the peptides were capable of eliciting HIV-specific neutralizing antibody and proliferative responses in primates. Four compounds (alum, polyA:polyU, threonyl-muramyldipeptide (MDP) and IFA) were also compared for efficacy as adjuvants in this system. Rhesus monkeys immunized with T1-SP10 peptides generated high titers of antibodies against the immunogens and also against HTLVIIIB gp120. Sera from all four animals given T1-SP10 in IFA or threonyl-MDP neutralized infection by HTLVIIIB and blocked virus-dependent cell fusion events. A peak neutralization titer of 1:940 was seen in one animal given IFA (19600) and a titer of 1:900 was seen in one of the monkeys (17371) given threonyl-MDP. Proliferative responses of immune rhesus PBMC to T1-SP10 appeared after the first injection. After eight immunizations, two of eight monkeys (one injected with peptides in threonyl-MDP and one given peptides in IFA) had PBMC proliferative responses to native HTLVIIIB gp120. These data demonstrate that the carrier-free T1-SP10 synthetic peptide construct can induce high titers of neutralizing anti-HIV antibody responses and PBMC proliferative responses to HIV in primates.     

Antibodies from Chicken Eggs as Probes for Antigens from Pasteuria penetrans Endospores

The bacteria Pasteuria spp. have been identified as among the most promising of several microbial organisms currently under investigation as biological control agents of plant-parasitic nematodes. As part of our goal to develop methods to discriminate isolates of Pasteuria penetrans with different host preferences, we investigated the potential of developing antibody probes to identify endospores of different isolates of P. penetrans. Polyclonal IgY antibodies were raised in chickens against endospores of P. penetrans isolates P20 and P100. Hens were injected with P20 or P100 endospore suspensions and boosted at 14 days. Anti-spore titers were determined with ELISA on yolk extracts of individual eggs as a function of time. The highest titers were found in eggs produced at 22 to 35 days after initial injections. Yolk extracts showing the highest titers were combined and processed to provide partially purified IgY preparations. SDS-PAGE and immunoblot analyses identified protein antigens with Mr values of 23-24, 46, and 57-59 KDa common to both P20 and P100 endospores. One protein antigen with an Mr value of 62 KDa was unique to the PI00 endospores. The IgY antibodies reduced the attachment of Pasteuria endospores to their nematode hosts, indicating antibody interaction with antigens on the endospore surface that are involved in the recognition and attachment processes.     

Large scale isolation of bovine and pig zonae pellucidae: Chemical,immunological, and receptor properties

A procedure is described for isolating milligram quantities of bovine and porcine zonae pellucidae, uncontaminated by follicle cells or their processes. On SDS-polyacrylamide gel electrophoresis the isolated bovine zona material formed one major glycoprotein band with an estimated molecular weight of approximately 100,000 daltons and two minor lower molecular weight components. The isolated pig zonae formed only one glycoprotein band with a molecular weight of approximately 62,000 daltons. Rabbit antisera raised against the isolated zonae were zona-specific and formed only a single precipitin line against the heat-solubilized zonae on immunoelectrophoresis. An adjuvant was not required for high-antibody titers. High titers were also obtained by injection of the dog and rhesus monkey. Anti-zona antibody was detected by immunofluorescence, zona-coating, double-immunodiffusion, and the inhibition of spermbinding to eggs, including those of human origin. Antigenic and sperm receptor properties were stable at 100°C for five minutes, but some activity was lost after longer exposure. The serum antibody produced by rabbits immunized with pig zonae was predominantly IgG and cross-reacted with the zonae of a variety of other species, including primates. Pregnancy was inhibited in female rabbits immunized with pig zona preparations.     

Acute suppression of FSH secretion by oestradiol in the ovariectomized rhesus monkey

Using long-term ovariectomized rhesus monkeys, we examined the ability of oestradiol to decrease circulating FSH concentrations in the absence of other ovarian factors. Daily blood samples were obtained from untreated monkeys for 8 days, followed by insertion of oestradiol capsules after the Day-8 sample was taken. Samples were then taken on Days 9-15, the capsules were removed after the Day-15 sample, and samples were obtained on Days 16-19. Serum was assayed for concentration of oestradiol, FSH and LH by RIA. The concentration of FSH (ng/ml) in serum did not change during the first 8 days before oestradiol treatment (overall mean = 356 +/- 55) but decreased from the Day-8 value of 320 +/- 8 to 190 +/- 42 on Day 9 and by Day 15, after 7 days of oestradiol treatment, had reached a nadir of 20 +/- 5. By Day 17, i.e. 2 days after removal of the oestradiol capsules, serum FSH had increased (P less than 0.05) to 92 +/- 23 with a further increase (P less than 0.05) on Day 19 (171 +/- 16). This study demonstrates that, unlike in rats, mice, and sheep, administration of oestradiol alone to ovariectomized rhesus monkeys reduces immunoreactive serum FSH to concentrations measured in intact animals.     

An enzyme-linked immunosorbent assay for detection of chronic subclinical Pasteurella pneumotropica infection in mice.

Serum samples from seventy-five, 3- to 12-week-old and 16 retired breeder male Swiss mice from a conventional colony with enzootic chronic subclinical Pasteurella pneumotropica infection were tested by enzyme-linked immunosorbent assay (ELISA) and Western blots for IgG antibodies to whole cell (WC) and lipooligosaccharide (LOS) antigens of P. pneumotropica. In 3- to 12-week-old mice, serum antibody levels to LOS exceeded those to the WC preparation. Western blots of sera from mice in this age group substantiated that a major component of the early IgG antibody response was directed against LOS antigens. Higher antibody levels to both antigen preparations in 3-week-old mice compared to mice 4 and 6 weeks old were interpreted as reflecting a decline in antibodies acquired from the dam. Active immunity indicative of infection was first detected at 8 weeks of age. Serum samples from retired breeder mice (28 weeks of age) also had substantial antibody titers to LOS but, in contrast to sera from mice in the younger age groups, retired breeders had significantly greater IgG reactivity to WC preparations than to LOS antigens. The superior specificity of the LOS antigen compared to the WC preparation in the ELISA was demonstrated by testing serum samples from retired breeder mice against WC and LOS antigens from P. ureae, P. multocida, and P. hemolytica. The reactivity of IgG against LOS antigens from these organisms was negligible, whereas substantial titers were evident to WC antigens. This ELISA, using LOS preparations as antigen, is a useful serologic assay for the detection of subclinical P. pneumotropica infection in mice.     

Experimental American leishmaniasis and Chagas'' disease in the Brazilian squirrel monkey: cross immunity and electrocardiographic studies of monkeys infected with Leishmania braziliensis and Trypanosoma cruzi

, and 1988. Experimental American leishmaniasis and Chagas' disease in the Brazilian squirrel monkey: cross immunity and electrocardiographic studies of monkeys infected with Leishmania braziliensis and Trypanosoma cruzi. International Journal for Parasitology 18: 1053–1059. Adult, laboratory-bred squirrel monkeys (Saimiri sciureus) previously infected with either Leishmania braziliensis braziliensis or L. b. panamensis were challenge infected with blood-form trypomastigotes of Trypanosoma cruzi (Brazil strain). Monkeys previously infected with T. cruzi were challenged with stationary phase promastigote forms of L. b. braziliensis. Monkeys were examined during the course of challenge for evidence of infection, electrocardiographic alterations and parasite-specific antibody responses. T. cruzi epimastigotes were cultured from the blood of monkeys up to 3 months after challenge with this parasite. Unulcerated cutaneous lesions appeared and persisted in monkeys challenged with L. b. braziliensis. The formation of satellite lesions was observed in one monkey. Increased QRS intervals were not observed in T. cruzi challenged monkeys without prior cardiac irregularities and QRS left axis shifts were observed in only two of these monkeys. Elevated titers of parasite binding IgM and IgG specific for both T. cruzi and L. braziliensis were observed in all monkeys following challenge. These results indicate that prior infection with T. cruzi or L. braziliensis does not protect against heterologous challenge infection with these organisms. However, prior infection with Leishmania parasites may provide some protection against chagasic cardiopathies.     

Nitrate reductase system in Staphylococcus aureus wild type and mutants.

Respiratory nitrate reductase with lactate as a hydrogen donor has been studied in cells and spheroplast preparations of wild type and heme-deficienct mutants of Staphylococcus aureus. The activity is rapidly induced when suspensions of aerobically grown cells are incubated without aeration in a complete medium with nitrate. In ruptured spheroplast preparations, the activity with lactate as the donor is located in the membrane fraction, whereas at least 50% of the activity assayed with reduced benzyl viologen is in the cytoplasm. The reductase is inhibited by azide and cyanide, and the lactate-linked system is also sensitive to oxamate, 2-heptyl-4-hydroxyquinoline-N-oxide, dicoumarol, and p-chloromercuribenzoate. An inactive form of the reductase is apparently made during induction with tungstate; this can be activated by subsequent incubation with molybdate in the presence of chloramphenicol. Nitrate reductase activity with reduced benzyl viologen as the donor is induced in suspensions of heme-deficient mutants in the presence or absence of heme. The proportion of cytoplasmic activity is increased in the absence of heme. The staphylococcal nitrate reductase has many of the characteristics commonly associated with the respiratory enzyme in other organisms, but the apparent predominance of cytoplasmic activity is unusual.     

Isolation of Campylobacter jejuni from experimental dogs and monkeys in Japan

Isolation of Campylobacter jejuni (C. jejuni) from experimental dogs and monkeys was undertaken. C. jejuni was detected from 14.7% of the fecal samples obtained from beagles in a production colony, whereas 32% of newly imported beagles harbored the organisms. C. jejuni was isolated from 25% of the young and 3.9% of adult dogs in an animal center. The organisms were isolated from newly imported cynomolgus and rhesus monkeys at high frequencies (49.2% and 38.8%, respectively). Almost all of the strains isolated were highly sensitive to erythromycin, chloramphenicol and gentamicin.     

Longitudinal analysis of early HIV-1-specific neutralizing activity in an elite neutralizer and in five patients who developed cross-reactive neutralizing activity

We previously established that at 3 years postseroconversion, ~30% of HIV-infected individuals have cross-reactive neutralizing activity (CrNA) in their sera. Here we studied the kinetics with which CrNA develops and how these relate to the development of autologous neutralizing activity as well as viral escape and diversification. For this purpose, sera from five individuals with CrNA and one elite neutralizer that were obtained at three monthly intervals in the first year after seroconversion and at multiple intervals over the disease course were tested for neutralizing activity against an established multiclade panel of six viruses. The same serum samples, as well as sera from three individuals who lacked CrNA, were tested for their neutralizing activities against autologous clonal HIV-1 variants from multiple time points covering the disease course from seroconversion onward. The elite neutralizer already had CrNA at 9.8 months postseroconversion, in contrast with the findings for the other five patients, in whom CrNA was first detected at 20 to 35 months postseroconversion and peaked around 35 months postseroconversion. In all patients, CrNA coincided with neutralizing activity against autologous viruses that were isolated <12 months postseroconversion, while viruses from later time points had already escaped autologous neutralizing activity. Also, the peak in gp160 sequence diversity coincided with the peak of CrNA titers. Individuals who lacked CrNA had lower peak autologous neutralizing titers, viral escape, and sequence diversity than individuals with CrNA. A better understanding of the underlying factors that determine the presence of CrNA or even an elite neutralizer phenotype may aid in the design of an HIV-1 vaccine.     

Rapid engineering of polyketide overproduction by gene transfer to industrially optimized strains

Development of natural products for therapeutic use is often hindered by limited availability of material from producing organisms. The speed at which current technologies enable the cloning, sequencing, and manipulation of secondary metabolite genes for production of novel compounds has made it impractical to optimize each new organism by conventional strain improvement procedures. We have exploited the overproduction properties of two industrial organisms—Saccharopolyspora erythraea and Streptomyces fradiae, previously improved for erythromycin and tylosin production, respectively—to enhance titers of polyketides produced by genetically modified polyketide synthases (PKSs). An efficient method for delivering large PKS expression vectors into S. erythraea was achieved by insertion of a chromosomal attachment site (attB) for φC31-based integrating vectors. For both strains, it was discovered that only the native PKS-associated promoter was capable of sustaining high polyketide titers in that strain. Expression of PKS genes cloned from wild-type organisms in the overproduction strains resulted in high polyketide titers whereas expression of the PKS gene from the S. erythraea overproducer in heterologous hosts resulted in only normal titers. This demonstrated that the overproduction characteristics are primarily due to mutations in non-PKS genes and should therefore operate on other PKSs. Expression of genetically engineered erythromycin PKS genes resulted in production of erythromycin analogs in greatly superior quantity than obtained from previously used hosts. Further development of these hosts could bypass tedious mutagenesis and screening approaches to strain improvement and expedite development of compounds from this valuable class of natural products.