Respirable PLGA microspheres containing rifampicin for the treatment of tuberculosis: screening in an infectious disease model.

PURPOSE: Targeted delivery of rifampicin loaded microspheres to the alveolar macrophage, the host cell for Mycobacterium tuberculosis (MTB), may be an effective targeted approach to pulmonary tuberculosis therapy. A guinea pig infection model has been adopted as a post-treatment screening method for antimicrobial effect. Insufflation and nebulization methods of drug delivery were evaluated. METHODS: Rifampicin alone (RIF, 1.03-1.72 mg/kg), within poly(lactide-co-glycolide) microspheres (R-PLGA, equivalent to 1.03-1.72 mg/kg) or polymer microparticles alone (PLGA) were administered by insufflation or nebulization, 24 h before bacterial aerosol exposure. Animals were infected with an aerosol containing a small number (2 x 10(5) cfu/mL) of virulent H37Rv strain of MTB. Lung and spleen tissue samples were collected 28 days after infection for quantitative bacteriology and histopathological analysis. RESULTS: There was a dose-effect relationship between insufflated R-PLGA and burden of bacteria in the lungs. In addition, guinea pigs treated with R-PLGA had a significantly smaller number of viable bacteria (P < 0.05), reduced inflammation and lung damage than lactose or saline control, PLGA or RIF treated animals. CONCLUSIONS: These studies indicate the potential of R-PLGA, delivered by insufflation or nebulization directly to the lungs, to affect the early development of pulmonary TB.

Airways delivery of rifampicin microparticles for the treatment of tuberculosis.

A Mycobacterium tuberculosis (H37Rv)-infected guinea pig model was used to screen for targeted delivery to the lungs by insufflation (with lactose excipient) or nebulization, of either rifampicin alone, rifampicin within poly(lactide-co-glycolide) microspheres (R-PLGA) or polymer microparticles alone (PLGA). Animals treated with single and double doses of R-PLGA microspheres exhibited significantly reduced numbers of viable bacteria, inflammation and lung damage compared with lactose-, PLGA- or rifampicin-treated animals 28 days post-infection (P < 0.05). Two doses of R-PLGA resulted in reduced splenic enlargement. These studies support the potential of R-PLGA delivered to the lung to treat pulmonary tuberculosis.

Isolation, maintenance, and characterization of human pancreatic islet tumor cells expressing vasoactive intestinal peptide.

Tissue from a vasoactive intestinal peptide (VIP)-secreting human tumor has been used to establish and characterize human neuroendocrine primary cell cultures from which permanent, clone-derived cell lines have been established. Viable cells were obtained by enzymatic and mechanical dissociation of freshly resected pancreatic islet tumor and hepatic metastatic tumor tissues. Aliquots of tumor cells were established ex vivo under culture conditions including porous substrata coated with type IV collagen and laminin and a low serum, hormonally defined culture medium. The small (<10 microm) rounded, grape-like cells had a very slow growth rate of doubling times estimated at several weeks or more. After several passages, morphologically uniform cells were derived that strongly expressed neuroendocrine markers of synaptophysin and synaptobrevin. Although chromogranin A and VIP had somewhat weaker expression, both demonstrated phorbol ester-stimulated secretion. The morphologic and secretory properties were maintained by the cells for nearly 2 years in culture. The establishment of this novel VIP-secreting human neuroendocrine cell line (HuNET) makes available a culture model with which to study a transformed version of this pancreatic islet cell type and offers approaches by which to establish islet tumor cell lines.

Reversal of ketamine/xylazine anesthesia in the rabbit with yohimbine.

Ketamine and xylazine used in combination have been shown to be effective, easily administered, cost efficient agents for surgical anesthesia in the rabbit. The effect of xylazine on the central nervous system has been shown to be mediated through alpha-2 adrenergic receptors. Yohimbine, an alpha-2 adrenergic antagonist has been shown to reverse xylazine induced depression and partially antagonize ketamine in other species. We evaluated the antagonistic effect of yohimbine on ketamine/xylazine anesthesia in the rabbit. Six New Zealand White rabbits were anesthetized with intramuscular ketamine (50 mg/kg) and xylazine (10 mg/kg) to establish baseline parameters including respiratory rate, heart rate, and palpebral, pedal and postural reflex activity. Fourteen days later each rabbit was subjected to the same anesthetic regimen followed 30 minutes later by the intravenous administration of yohimbine (0.2 mg/kg). The duration of anesthesia estimated by the time elapsed between the loss and return of the palpebral reflex was reduced in the yohimbine treated trial (means = 29.7 +/- 1.9 minutes) compared to the control trial (means = 67.0 +/- 13.5 minutes). The palpebral reflex returned within 5 minutes following yohimbine treatment. Our results indicated that yohimbine is an effective antagonist of ketamine/xylazine anesthesia in the rabbit. Yohimbine decreases anesthetic duration after intravenous administration and also may aid in the control of undesirable anesthetic effects and overdosage.

Rederivation of MHV and MEV antibody positive mice by cross-fostering and use of the microisolator caging system.

This study established the feasibility of rederiving numerous mouse hepatitis virus (MHV) and mouse encephalomyelitis virus (MEV) antibody positive strains of mice using cross fostering techniques and a new caging system, thus permitting introduction of virus antibody free mice into a barrier facility. Serologic status of dams within the nucleus breeding colony was determined, and all mice within the breeding colony were housed in individual Microisolator cages. Specific pathogen free (SPF) foster mothers purchased from a commercial source were determined to have no detectable serum antibody to 11 murine viruses including MEV and MHV. Pups delivered naturally from time pregnant dams were cross fostered onto the SPF foster dams. The procedure of cross fostering was conducted within a positive flow, HEPA-filtered, mass air displacement unit within 24 hours of parturition. The virus status of pups from 49 litters was monitored serologically at weaning and again at 6 weeks of age. All cross fostered litters were serologically negative for antibody to mouse hepatitis virus. Seven of 29 litters were negative for MEV antibody titer using this cross fostering technique. Those litters negative serologically to both MHV and MEV (at 3 and 6 weeks) were transferred to a barrier facility and held in isolation. All rederived mice transferred to the barrier facility remained negative for MHV and MEV when sampled at 12 weeks of age.

The pathogenicity of Salmonella mbandaka in specific pathogen free rabbits.

Salmonella mbandaka was isolated from the lungs of three rabbits that developed a peracute disease and died three to eight weeks following extensive experimental manipulation. To examine the pathogenicity of this rare serotype, clinically normal, S. mbandaka-carrier rabbits were subjected to either corticosteroid immunosuppression alone or corticosteroid immunosuppression with a minor surgical procedure. Acute or peracute salmonellosis developed in one of two rabbits immunosuppressed only, and in three of four rabbits immunosuppressed and surgically manipulated. Stressful experimental procedures may precipitate episodes of clinical salmonellosis in rabbits harboring this organism. Salmonella mbandaka, should be considered a zoonotic agent in the laboratory environment.

Labeling hen's egg with 74Se for use in human metabolic experiments.

Experiments were conducted with laying hens to explore quantitative aspects of incorporation of 75Se ( radioselenium ) at various dosing levels and for different chemical forms of an orally administered tracer. Quantitative distribution of the incorporated isotope in egg white and egg yolk was strongly influenced by both the chemical form of the label and the dosing level. The ratio of egg yolk:egg white selenium decreased with increased level of administered dose of selenite. In addition, the rate of incorporation and the amount of selenium in whole egg were higher when [75Se]selenomethionine was given as compared to [75Se]selenite. Characterization of the chemical form of selenium in egg white and egg yolk labeled biologically by giving hens radioactive selenite or selenomethionine was performed by classification as: selenite, selenoprotein and fat-bound selenium. Studies were then undertaken to achieve intrinsic labeling of egg white and egg yolk with stable isotope 74Se for purposes of exploring selenium bioavailability in humans. Enrichments of 74Se in egg white and egg yolk of hens given high dose selenite (54.4 micrograms 74Se ) were 20- and 28-fold, whereas in egg white and egg yolk of hens given low dose (10.9 micrograms 74Se ) they were 4- and 10-fold the level of natural abundance, respectively. The stable isotope-labeling studies indicated that a 7-day sequential dosing protocol with 20-100 micrograms Se per dose permitted sufficient enrichment of egg white (only high dose) and of yolk with the stable isotope 74Se for use in human metabolic studies.

Seborrheic dermatitis in a rhesus monkey (Macaca mulatta).

Seborrheic dermatitis was diagnosed in an adult, rhesus monkey presenting with erythematous, exfoliative and focally exudative skin lesions distributed over the perioral, perirhinal and brow regions of the face. Similar areas of involved skin also were observed on the extremities. Lesions appeared to be exacerbated during increased experimental utilization. Histopathologic changes in skin biopsy specimens of affected skin included acanthosis, parakeratosis, spongiotic edema, vascular ectasia, and mild lymphohistiocytic infiltrates.

Mastitis caused by hemolytic Escherichia coli in the ferret.

Hemolytic Escherichia coli was isolated from the mammary glands of 8 ferrets with gangrenous mastitis. Clinical signs included firm swelling of one or more mammary glands and discoloration of the overlying skin. Peracute disease and acute septicemia were observed, and in some cases the animals rapidly became moribund. Antibiotic therapy alone did not alter the course of the disease. Wide surgical resection of the involved glands in combination with systemic antibiotic therapy (ampicillin 10 mg/kg, BID, and gentamicin 5 mg/kg, SID) was the most successful treatment. Histopathologic changes included extensive edema, hemorrhage, and necrosis, with a mixed leukocytic infiltrate and large numbers of bacteria. The agent of this disease was isolated from rectal swab specimens from clinically normal ferrets as well as ferrets that had mastitis.

A technique for saliva collection in dogs.

A plastic two-chambered vacuum-collection device was developed to collect saliva produced by the parotid gland in dogs. Repeated application of the device on the parotid gland papilla was atraumatic. Dogs were restrained during the collection period with a continuous intravenous infusion of ketamine hydrochloride and diazepam in lactated Ringer's solution. The anesthetic regime did not interfere with salivation.

Isolation of Chlamydia psittaci from naturally infected African clawed frogs (Xenopus laevis).

An inclusion-forming agent was isolated from the livers of commercially raised African clawed frogs (Xenopus laevis) involved in an epizootic of high morbidity and mortality. Original isolation was made in McCoy cells. This agent was identified as Chlamydia psittaci based on the formation of typical intracytoplasmic inclusions which developed within 48 h, were not stained by iodine, and were resistant to sulfadiazine. The isolate from one particular frog (designated as strain 178) was further studied and found to be lethal for 7-day-old embryonated chicken eggs after intra-yolk sac inoculation. This strain was demonstrated not to be pathogenic for mice when inoculated intraperitoneally. The cell culture isolate of C. psittaci was transmitted to uninfected X. laevis, causing disease and death.

Ferret as a potential reservoir for human campylobacteriosis.

A survey was conducted to determine the frequency of Campylobacter fetus subsp jejuni in feces of ferrets purchased for use in biomedical research. Over a 12-month period, 168 ferrets from 2 commercial breeders were examined by bacteriologic cultural procedures for Campylobacter spp; 61% were culturally positive for C fetus subsp jejuni. In a therapeutic trial with 16 ferrets shedding C fetus subsp jejuni in feces, erythromycin given orally failed to eliminate intestinal carriage of the organism in 15 of the animals.

Spontaneous and experimental infections of Xenopus laevis with Chlamydia psittaci.

Chlamydial infection was suspected when widespread pyogranulomatous inflammation and large basophilic intracytoplasmic inclusion bodies were evident histopathologically in African clawed frogs (Xenopus laevis) dying of a spontaneous disease of high morbidity and mortality. Organism morphology was determined by electron microscopy of infected hepatic sinusoidal lining cells, and it was characteristic of the unique developmental cycle of a chlamydial agent. Isolation and speciation of the organism was achieved in a McCoy cell culture system. The infected cells were inoculated into disease-free frogs reproducing the disease.

Acute gastric dilatation in nonhuman primates: review and case studies.

Acute gastric dilatation occurs sporadically in laboratory-housed nonhuman primates. Clinical histories often include chronic drug administration, food restriction, accidental overfeeding, and prior anesthesia. Monkeys may be found dead or may have clinical signs of colic, abdominal distention, and dyspnea. Death in untreated cases is due to impaired venous return and cardiopulmonary failure. Gastric distention with fermented gaseous ingesta and congestion of the abdominal viscera are the predominant lesions. The cause of acute gastric dilatation is unknown, but it probably is multifactorial. Two principal factors seem to be intragastric fermentation associated with Clostridium perfringens, and abnormal gastric function.

Acute gastric dilatation in nonhuman primates: review and case studies.

Acute gastric dilatation occurs sporadically in laboratory-housed nonhuman primates. Clinical histories often include chronic drug administration, food restriction, accidental overfeeding, and prior anesthesia. Monkeys may be found dead or may have clinical signs of colic, abdominal distention, and dyspnea. Death in untreated cases is due to impaired venous return and cardiopulmonary failure. Gastric distention with fermented gaseous ingesta and congestion of the abdominal viscera are the predominant lesions. The cause of acute gastric dilatation is unknown, but it probably is multifactorial. Two principal factors seem to be intragastric fermentation associated with Clostridium perfringens, and abnormal gastric function.

Enzyme-linked immunosorbent assay of serum protein SAA in rhesus monkeys with secondary amyloidosis.

Tissue deposits of amyloid protein AA and a concomitant elevation of serum protein SAA have been demonstrated previously in mice and humans with secondary amyloidosis, but not in rhesus monkeys (Macaca mulatta). In this study, protein SAA was quantitated in normal and amyloidotic rhesus monkeys using an enzyme-linked immunosorbent assay. Protein AA was isolated from the liver of a rhesus monkey with secondary amyloidosis by a combination of water extraction and gel filtration chromatography. The purified material had rigid, nonbranching fibrillar structures typical of amyloid on electron microscopy and a molecular weight of 9000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The amino acid content and partial sequence were comparable to those reported previously for protein AA of rhesus monkeys. As measured by enzyme-linked immunosorbent assay, normal rhesus monkeys had SAA levels of 40 to 64 ng. of AA equivalents per ml., whereas amyloidotic rhesus monkeys had SAA levels of 1700 to 95,000 ng. of AA equivalents per ml. An elevation of protein SAA was also detected in an amyloidotic pigtailed macaque (Macaca nemestrina) using rabbit antirhesus protein AA. Rabbit antisera against human and mouse protein AA reacted strongly with rhesus protein AA and with amyloidotic rhesus serum, but only slightly with normal rhesus serum, indicating that rhesus proteins AA and SAA have antigenic determinants cross-reactive with protein AA of xenotypic species.