The influence of a colonic microbiota on HPMA copolymer lectin conjugates binding in rodent intestine.

Germ-free (GF) animals lack a colonic microflora like that seen in conventional (CV) animals. Bacterial presence plays a role in the development of glycoproteins in the gastrointestinal (GI) tract; the absence of a microbiota has been seen to suppress the production of certain glycoproteins and glycolipids. Binding patterns of lectins are modified when glycoprotein structures are altered (e.g., during development or disease). Little information on lectin binding patterns in mature GF animals is available. We examined the binding of free and N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-conjugated fluorescein isothiocyanate (FITC)-labeled wheat germ agglutinin (WGA) [P(HPMA)-(WGA-FITC)] and FITC-labeled peanut agglutinin (PNA) [P(HPMA)-(PNA-FITC)] in CV and GF mouse colon with and without neuraminidase pretreatment. Anti-Thomsen-Friedenreich (TF) antigen (a development and disease-related glycoprotein) antibody binding was also examined in these tissues. Subtle differences were seen in the binding patterns between CV and GF animals. CV animals showed strong P(HPMA)-(WGA-FITC) binding in goblet cells, but minimal P(HPMA)-(PNA-FITC) binding was visible. In GF animals, luminal surface binding of P(HPMA)-(WGA-FITC) was visible, and goblet cell binding of P(HPMA)-(PNA-FITC) was seen. These subtle changes suggest that altered glycoprotein expression occurred under GF conditions.

Potential of lectin-N-(2-hydroxypropyl)methacrylamide copolymer-drug conjugates for the treatment of pre-cancerous conditions.

N-(2-Hydroxypropyl)methacrylamide (HPMA)-lectin (wheat germ agglutinin (WGA), peanut agglutinin (PNA)) drug conjugates for treatment of the pre-cancerous conditions ulcerative colitis and Barrett's esophagus are being developed. Cell-surface glycoproteins that are altered in disease and development bind lectins. PNA binds alpha-lactose and the Thomsen-Friedenreich (TF) antigen, a disease- and development-associated glycoprotein. PNA incorporation in conjugates may allow for preferential delivery to diseased over healthy tissues. Conjugates were prepared by attaching lectins to HPMA copolymers via an amide linkage. Frontal affinity chromatography was used to measure dissociation constants (K(d)) of free and conjugated lectins. Animal models of colitis (DSS, TNBS/EtOH) were developed. Human biopsy specimens were obtained. Free and HPMA copolymer-conjugated FITC-labeled lectin and anti-TF antigen antibody binding patterns were examined in normal neonatal, adult and diseased rodent tissues and normal and diseased human tissues. K(d) values of free and conjugated lectins were similar ( approximately 10(-5) M(-1)). Free and conjugated lectins had comparable binding patterns. In health, strong WGA binding was seen in goblet cells; PNA binding was minimal, occurring only in the supranuclear goblet cell region. In disease, WGA binding was not altered, but PNA binding was increased in both human and rodent tissues; entire goblets bound the lectin. Anti-TF antigen antibody binding was minimal, but did overlap with PNA binding patterns both in normal and diseased tissues. Conjugation of lectins to HPMA copolymers does not affect binding affinity. Alterations in glycoprotein structures in development and disease resulted in modified lectin binding patterns. In development and disease, the PNA binding seen was to the TF antigen and other lactose-containing glycoproteins. The results suggest that site-specific delivery of therapeutic agents such as cyclosporin A (CsA) for ulcerative colitis and mesochlorin e(6) for Barrett's esophagus may be achieved. P(HPMA)-lectin-CsA conjugates have been prepared and preliminary in vivo studies are underway.

Biorecognition of HPMA copolymer-lectin conjugates as an indicator of differentiation of cell-surface glycoproteins in development, maturation, and diseases of human and rodent gastrointestinal tissues.

Lectins are proteins that bind glycoproteins; binding patterns are altered with changes in glycoprotein expression accompanying maturation or disease. Binding of two lectins, wheat germ agglutinin (WGA) and peanut agglutinin (PNA), in human and rodent colon were previously examined. Normal tissue showed intense WGA binding; PNA binding was minimal. Diseased tissues showed increased PNA binding. We hypothesized that N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-lectin-drug conjugates could deliver therapeutic agents to diseased tissues by targeting colonic glycoproteins. We examined biorecognition of free and HPMA copolymer-conjugated WGA and PNA and anti-Thomsen-Friedenreich (TF) antigen antibody binding in normal neonatal, adult, and diseased rodent tissues, human specimens of inflammation, and Barrett's esophagus. Neonatal WGA binding was comparable to the adult, with additional luminal columnar cell binding. PNA binding was more prevalent; luminal columnar cell binding existed during the first 2.5 weeks of life. WGA binding was strong in both normal and diseased adult tissues; a slight decrease was noted in disease. PNA binding was minimal in normal tissues; increases were seen in disease. Anti-TF antigen antibody studies showed that PNA did not bind to the antigen. The results suggest that HPMA copolymer-lectin-drug conjugates may provide site-specific treatment of conditions such as colitis and Barrett's esophagus.

Velar-pharyngeal status in cleft palate patients with expected adenoidal involution.

This study was designed to provide information about whether cleft palate patients with hypertrophied adenoids maintain velar-pharyngeal contact during the time of expected adenoidal atrophy. Thirty-nine subjects were selected from a large longitudinal study on the basis of availability of lateral still x-ray films taken in series from 5 to 16 years of age. Ratings of velar-pharyngeal contact and ratings of adenoid size were obtained from the films. The obtained data indicated the expected decrease in adenoid size but also, for the group, maintenance of velar-pharyngeal contact. However, three of the 39 subjects were judged to show loss of such contact during the period of study, and an additional four had surgery for velopharyngeal incompetence after the completion of the study. All seven appeared to show significant deterioration of velopharyngeal status in middle or late adolescence. Implications of these findings are discussed.

Biological time-related changes in tolerance of male rats to hypoxia--I. Survival rate and carbohydrate metabolism.

Investigations were carried out on male Wistar rats, synchronized in standard conditions to a light-dark regiment (LD 12:12 with L from 0600 to 1800). Rats exposed to hypoxia equivalent to 10,500m at a clock-hour of 1000 had a survival time twice as long as that of animals exposed at 2200. Data from this study indicate the ability to mobilize energy stores through the conversion of liver glycogen to glucose along with circadian differences in hormonal response (e.g. corticosterone and insulin) contributes to the tolerance to hypoxia being greater during diurnal rest than nocturnal activity in rats.

Biological time-related changes in tolerance of male mice to hypoxia--II. Circadian rhythm of lysosomal susceptibility to hypoxia.

Circadian variations of mouse liver, brain and heart lysosomal susceptibility to hypoxia were investigated. Lysosomal disruption during hypoxia was estimated on the basis of the following measurements: changes in percentage free activity of beta-galactosidase and acid phosphatase, tissue loss of both lysosomal enzymes and accumulation of serum beta-galactosidase. When exposure to hypoxia took place at the end of the rest phase or at the beginning of the active phase, it was accompanied by maximum increase of percent free activity. This, presumably represents a diffusion of enzymes from lysosomes due to altered membrane permeability. However, hypoxia when occurring during the second part of the active phase and first part of the rest phase resulted in tissues loss of lysosomal enzymes and accumulation of serum lysosomal enzymes. This is believed to represent the release of lysosomal enzymes in bulk from damaged or ruptured lysosomal membranes.