Characterization of fecal bacterial populations in canines: effects of age, breed and dietary fiber.

The effects of age, breed, and diet on fecal chemistry, enzyme activity, and bacterial populations of dogs were studied. Eighteen dogs from two age groups (young: 2.5 +/- 0.5 years, old: 10.9 +/-0.7 years) and three different breeds (German shepherds, miniature schnauzers, and English setters) were rotated through a Latin Square design such that every dog was fed each of the diets. The test diets included a low-fiber (control) diet and a 10% fiber diet which contained 5% soybean hulls and 5% beet pulp. Inclusion of 10% fiber in the diet decreased the fecal concentration of ammonia, sulfide, and indole. Fiber inclusion significantly increased acetic, propionic, and butyric acid concentrations, while fecal pH decreased by 0.4 units. Fresh fecal samples were plated on selected aerobic and anaerobic culture media and DNA extracted for denaturing gradient gel electrophoresis (DGGE) analysis of PCR-amplified 16S ribosomal DNA fragments. Plate counts showed significant effects of breed (p < or = 0.05) and age (p < or = 0.01) on selected aerobic and anaerobic bacterial counts, while no significant effect of diet was found. Analysis of PCR-DGGE banding patterns showed there was a tendency for individual dogs to cluster together according to age (young or old dogs) and also for size (large or small dogs). However, the outstanding conclusion obtained from the DGGE analysis of fecal bacterial profiles was that individual dogs had their own characteristic banding pattern which was unique and stable. The relative stability and individuality of the patterns indicates that each individual harbored a characteristic fecal bacterial community which was independent of diet.

Comparative analysis of methods for collection and measurement of cytokines and immunoglobulins in cervical and vaginal secretions of HIV and HPV infected women.

The goal of these studies was to distinguish which of two techniques [cervicovaginal lavage (CVL) and cervical wick (SS)] is the optimal collection method for the measurement of the local immunological response in human papillomavirus (HPV) and HIV infected women. The following parameters were measured in 24 paired samples from 15 women (9 HIV+, 6 HIV-): total protein, immunoglobulin levels, HPV-specific antibodies, and Th1-Th2 cytokines. In addition, relative mRNA levels from CVL cell pellets were compared to protein levels from CVL supernatants. The total protein (2-fold) and IgG concentration (10-fold) are higher in the SS samples, were reproducible (%CV<3) and these levels correlated (P<0.0001) with their paired CVL sample. Type-specific HPV-L1 IgG and IgA antibodies were detected in CVL and SS (r>0.28, P<0.008) with excellent reproducibility (CV<3.0%). However, SS (%CV>18) failed to yield reproducible results for the cytokine assays as compared to the CVL (%CV<5.0). Furthermore, no correlations were found between relative mRNA levels from CVL cell pellet and cytokine protein levels in CVL supernatants. The CVL sample's superior reproducibility in the cytokine assays makes this the better collection method. In addition, cytokine protein level's failure to correlate with mRNA suggests tight regulation of cytokine genes or production from a different cell population.

X-ray photoelectron spectroscopy as a probe of intermolecular interactions in porphyrin polymer thin films.

High-resolution X-ray photoelectron spectroscopy (XPS) has been applied to a series of free-base and Zn-porphyrin polymers in which the macrocycles are separated by oligo(phenylene vinylene) units (OPV) of different, controllable lengths. Neat films of all the Zn-porphyrin polymers unexpectedly reveal two peaks in the N 1s XPS region ( approximately 400 eV). The peak areas vary with the length of the OPV bridge, suggesting an intermolecular interaction between the porphyrin and linker subunits. A series of XPS analyses were performed to identify the different interactions taking place in these thin films. To inhibit interpolymer interactions, one of the polymers was incorporated into a nonconjugated PMMA matrix, collapsing the N1s spectrum to a single peak at 398.0 eV, relative to the neat film signals at 397.8 and 400.1 eV. In a conjugated matrix of OPV, two peaks remain at 401.7 and 399.5 eV. Extensive vacuum drying of the neat film leads to a single peak at 398.3 eV, suggesting loss of trapped solvent molecules. Ultimately, we attribute the lower energy signal of the neat films to solvent ligation, and the higher energy peak is attributed to interactions between the porphyrins and conjugated bridges on nearby polymer chains. This interpretation is successfully applied to the N 1s XPS data from a previously reported Zn-porphyrin oligomer-based multilayer array.

Uniform thin films of poly-3,4-ethylenedioxythiophene (PEDOT) prepared by in-situ deposition.

In-situ deposited thin films of the conducting polymer poly-3,4-ethylenedioxythiophene (PEDOT) have been prepared on hydrophilic and hydrophobic substrates and characterized by UV-Vis spectroscopy, atomic force microscopy and resistivity measurements.

Polarized emission from a rhenium metal-ligand complex.

We report the first observation of polarized emission from a rhenium-phenanthroline complex, Re(CO)3(phen)Cl. Highly luminescent rhenium complexes are known, with quantum yields near 0.5 and lifetimes in excess of 10 µs. The detection of polarized emission suggests the use of rhenium complexes as probes of the hydrodynamics of large macromolecular complexes and for use in fluorescence polarization immunoassays with gated detection.

Immunomodulation by LFA3TIP, an LFA-3/IgG1 fusion protein: cell line dependent glycosylation effects on pharmacokinetics and pharmacodynamic markers.

LFA3TIP, a fusion protein comprised of the first extracellular domain of LFA-3 fused to the hinge, CH2, and CH3 domains of human IgG1 inhibits responses of human and non-human primate T cells in vitro. In seeking to optimize the expression efficiency to prepare large quantities of LFA3TIP for primate studies, the protein was produced in both the CHO (Chinese hamster ovary) and murine NS-0 myeloma cell lines. Although LFA3TIP derived from these cell lines performs identically in vitro in CD2 receptor binding and T cell assays, examination of a pharmacodynamic marker-the reduction in CD2+ lymphocyte numbers-following the administration of equal doses of NS-0 or CHO derived LFA3TIP to baboons, suggested that the effect of the NS-0 derived material was less sustained. Pharmacokinetic analysis of the materials in baboons and mice shows that LFA3TIP produced by NS-0 cells is rapidly cleared from circulation relative to the product derived from CHO cells. The disparate clearance profiles correlate with distinct glycosylation patterns, with LFA3TIP derived from NS-0 cells being less extensively sialylated than that from CHO cells due in part to alpha-galactosyl capping of selected lactosamine moieties in the N-linked glycans of NS-0 derived LFA3TIP. Moreover, enzymatic desialylation of CHO derived LFA3TIP results in a glycoprotein with an evanescent serum profile when administered to mice and baboons. These results correlate the extent of N-acetylneuraminic acid capping with the clearance rates of LFA3TIP derived from the two distinct cell lines, and underscore the importance of evaluating glycosylation dependent PK parameters when choosing production cell lines for recombinant immunotherapeutics.

The effects of an immunomodulatory LFA3-IgG1 fusion protein on nonhuman primates.

LFA3TIP, a fusion protein comprised of the first extracellular domain of LFA-3 fused to the hinge, CH2 and CH3 domains of human IgG1, inhibits proliferation of human T cells in vitro. LFA3TIP also inhibits responses of human CD2 transgenic mice by rapidly and totally depleting peripheral T cells. These effects require binding of the LFA-3 and CH2 domains of LFA3TIP to CD2+ T cells and Fc gamma R+ accessory cells, respectively. As CD2 is well conserved in primate species, we evaluated the effects of LFA3TIP in nonhuman primates. We report in vitro results leading to the selection of the baboon as a model for analysis of LFA3TIP, and in vivo effects of single and multidose regimens of LFA3TIP administration. This is the first report of the in vivo administration of an immunomodulatory fusion protein to primates. LFA3TIP is shown to mediate effects on primate T lymphocytes without apparent related toxicities or immunogenicity. Results are discussed in context of potential mechanisms of LFA3TIP immunotherapy.

Underdiagnosis of Chlamydia trachomatis infection. Diagnostic limitations in patients with low-level infection.

To determine limitations in commonly used methods for detection of Chlamydia trachomatis, 601 genitourinary specimens from patients in a sexually transmitted disease clinic were examined with quantitative cultures and by 2 different direct antigen tests, immunofluorescence (Micro Trak; Syva Company, Palo Alto, CA) and enzyme immunoassay (Chlamydiazyme; Abbott Laboratories, North Chicago, IL). Genital specimens were held no longer than 5 hours (at 4 degrees C) prior to inoculation for culture; 28% (168/601) were positive. To evaluate the effect of storage on culture efficacy, duplicate specimens were also stored at -70 degrees C and brought out subsequently for culture a second time. Only 32% (8/25) of specimens cultured within 5 hours and having less than 10 inclusions were positive on reculture, compared with 98% (49/50) positive for specimens with greater than or equal to 10 inclusions initially (P less than 0.001). Sensitivities of the two antigen tests were similar and taken together diminished significantly (P less than 0.001) as the number of organisms (inclusion forming units) in corresponding cultures decreased: 82% (51/62) sensitivity in cultures with greater than 100 inclusions; 50% (22/44) with 10-100 inclusions; and only 11% (6/53) with less than 10. Lack of urethral discharge in men with C. trachomatis infection (free of Neisseria gonorrhoeae) was associated with low numbers of inclusions (less than 10) and antigen tests failed in 68% (15/22) of these patients.

A study of factor XIIIa and MAC 387 immunolabeling in normal and pathological skin.

Immunolabeling with two new antibodies, Factor XIIIa and MAC 387, has been studied in routinely processed biopsy specimens of normal skin, subcutaneous tissues, lymph nodes, and a variety of pathological conditions. These presumptive cell markers of the monocyte-macrophage lineage appear to label totally different and possibly mutually exclusive subsets of cells. In normal skin, Factor XIIIa labeled fixed dermal connective tissue cells, emphasizing their dendritic morphological appearance. Factor XIIIa expression in fibroproliferative conditions and spindle cell tumors varied: in particular, scars, keloids, and dermatofibrosarcomas do not label, whereas histiocytomas, fibrous papule of the nose, and atypical fibroxanthomas were well labeled. In inflammatory conditions, increased numbers of Factor XIIIa cells were found, but most macrophages and epithelioid and multinucleate cells did not label. In contrast, normal dermal connective tissue cells did not label with MAC 387, nor did the constituent cells of various fibroproliferative disorders. In inflammatory conditions, variable numbers of MAC 387-positive cells were observed, corresponding to histiocytes and macrophages, but labeling of epithelioid cells and multinucleate foreign body giant cells was variable. Histiocytosis X cells do not express either label. The theoretical and practical implications of these results are explored.

Multinucleate cell angiohistiocytoma: an acquired vascular anomaly to be distinguished from Kaposi's sarcoma.

Multinucleate cell angiohistocytoma is a newly described benign vascular condition that usually arises on the extremities of women over the age of 40 as discrete grouped violaceous erythematous papules, often mimicking Kaposi's sarcoma. Of 10 patients, nine were women aged between 37-66 (average 51.5) years at the onset of their condition. The legs, and in particular the calves and thighs, were the commonest sites to be involved. However, in three patients the papules were confined to the back of the hands, and the male patient had lesions across the front of the chest. Bilateral lesions occurred in four patients. Histologically, the salient features were proliferation of capillaries and small venules at the level of the subpapillary plexus and the mid dermis, in association with prominent connective tissue cells and larger angulated multinucleate cells. Detailed histochemical and immunocytochemical studies have not elucidated the histogenesis of the multinucleate cells that seem to be a characteristic feature of this condition.

Some special skin tumours in the elderly.

A review of uncommon skin tumours, all of which characteristically arise in elderly patients, is presented. Angiosarcoma of the face and scalp is a highly progressive tumour with a poor prognosis, but initially it may be misdiagnosed as a simple bruise or cellulitis. In well-differentiated tumours a characteristic feature is interlacing endothelial cell-lined channels showing considerable nuclear atypia. Merkel cell tumours, which develop as deep-seated nodules in the skin, occur most frequently in photodamaged areas, especially on the head and neck. The prognosis of Merkel cell tumours is generally poor; histological evidence suggests that some of these tumours may be a special type of poorly differentiated squamous cell carcinoma. Atypical fibroxanthoma, which is normally benign, occurs most frequently in elderly, fair-skinned males, especially around the face. It is typically a polypoid lesion with a vascular appearance. Acantholytic squamous cell carcinomas, also known as adenoid squamous cell carcinomas, are most common in fair-skinned men with a history of keratoses. They also occur in the female genital region and the oral mucosa. Histologically, these tumours differ from squamous cell carcinomas because of their tendency to form duct-like areas in tumour lobules.

Antifungal activity of novel 5-carbonyl derivatives of 3-phenyl-3-(1H-imidazol-1-ylmethyl)-2-methylisoxazolidines.

The synthesis and antifungal activity of a series of novel 5-carbonyl derivatives of 3-phenyl-3-(1H-imidazol-1-ylmethyl)-2-methylisoxazolidines (4) are discussed. The preparation of the title compounds involved a 1,3-dipolar cycloaddition reaction of alpha-substituted ketonitrones with either acrylic esters, acrylamide or methyl vinyl ketone to furnish cis/trans-diastereomeric mixtures of the desired 5-carbonyl isoxazolidines 4. The anifungal activity was evaluated in vitro in solid agar cultures. Some of the compounds tested exerted moderate to potent activity against a wide variety of dermatophytes and yeast and systemic fungi.

The mechanism of action of phenformin in starved rats.

The ability of phenformin to lower the blood glucose concentration after an intraperitoneal glucose load, with a concomitant increase in blood lactate concentration, indicated that the drug was increasing the rate of anaerobic glycolysis. The results of experiments in which glucose and gluconeogenic precursors were given to starved rats were explained by a hypothesis for the mechanism of action of phenformin involving inhibition of certain NAD+-dependent dehydrogenases. Substrates with NAD+-linked oxidations could be discriminated from those, like succinate, with FAD-linked oxidations, and succinate may be of use in the treatment of clinical lacticacidosis caused by biguanide drugs.

Human toxocariasis in a rural community.

A questionnaire and serologic survey of a Pennsylvania community revealed that 23 (54%) of 43 study participants had antibody titers to Toxocara canis (enzyme-linked immunosorbent assay test [ELISA]) in the range defined as positive (greater than or equal to 1:32). Age-specific seropositivity rates were highest among adults (greater than or equal to 18 years). Positive titers ranged from 1:32 to 1:512. Seropositivity did not cluster by family, and seropositivity rates were significantly higher than those of a control group. Physical examination and laboratory tests designed to detect clinical signs of toxocariasis were performed on individuals with positive ELISA titers. Nine of 23 had a history of pica, seizures, or chronic pulmonary symptoms; however, physical examination revealed no abnormalities. Although medical examination and laboratory tests revealed a few abnormalities, neither the visceral larva migrans syndrome nor the ocular lesions characteristic of those produced by Toxocara sp were observed in any individual. We conclude that although certain populations may have high seropositivity rates to Toxocara antigens, indicating previous infection, clinical diseases may be uncommon.

Niclosamide as a treatment for Hymenolepis diminuta and Dipylidium caninum infection in man.

In the 5-year period 1973-1977, 43 patients infected with Dipylidium caninum and 43 patients infected with Hymenolepis diminuta were treated with Yomesan (niclosamide) in the dosages recommended by the Parasitic Disease Drug Service, Center for Disease Control. The first post-treatment stool specimen and 1-week and 3-month specimens were examined in 13 patients with D. caninum and 19 patients with H. diminuta. One hundred percent (13/13) of those with D. caninum and 89% (17/19) of those with H. diminuta had negative examinations at 3 months and were considered cured. Two patients with persistent H. diminuta were cured with a second course of drug without changing the dosage or time schedule. Four of these cases had been unresponsive to an initial course of quinacrine hydrochloride. Thus, niclosamide seems to be an effective, relatively nontoxic drug for the initial therapy of these cestode infections.