Proteolysis of vaginally administered bovine lactoferrin: clearance, inter-subject variability, and implications for clinical dosing.

This report describes proteolytic fragmentation and clearance of bovine lactoferrin (bLF) upon intravaginal administration in premenopausal women. Tablet formulations (MTbLF) containing 300 mg of bLF progressed through three phases: Pre-Dissolution, Dissolution, and Washout, over a 30-h time course. Tablets dissolved slowly, replenishing intact 80 kDa bLF in vaginal fluid (VF) as proteolysis occurred. bLF was initially cleaved approximately in half between its N- and C-lobes, then degraded into sub-fragments and small peptides. The extent of proteolysis was less than 10-20% across multiple subjects. Concentrations of both intact 80 kDa bLF and smaller fragments decreased in VF with a similar time course suggesting washout not proteolysis was the main clearance mechanism. Concentrations of intact and/or nicked 80 kDa bLF peaked between 4 and 8 h after administration and remained above 5 mg/mL for approximately 24 h. Experiments with protease inhibitors in ex vivo VF digests suggested an aspartyl protease was at least partially responsible for bLF cleavage. However, digestion with commercial pepsin or in vivo in the human stomach, demonstrated distinctly different patterns of fragments compared to vaginal proteolysis. Furthermore, the 3.1 kDa antimicrobial peptide lactoferricin B was not detected in VF. This suggests pepsin-like aspartyl proteases are not responsible for vaginal proteolysis of bLF.

Antimicrobial activity of bovine lactoferrin against Gardnerella species clinical isolates.

Gardnerella species play a key role in the development and recurrence of Bacterial Vaginosis (BV), a common imbalance of the vaginal microbiota. Because of the high rates of BV recurrence reported after treatment with standard of care antibiotics, as well as the emergence of antibiotic-resistant BV, the development of alternative treatment approaches is needed. Bovine lactoferrin, a well studied iron-binding glycoprotein with selective antimicrobial activity, may ameliorate vaginal dysbiosis either alone or in combination with antibiotics. The present study evaluated the antimicrobial resistance/susceptibility profile of seventy-one presumptive G. vaginalis clinical isolates to metronidazole and clindamycin. In addition, the in vitro antimicrobial activity of Metrodora Therapeutics bovine Lactoferrin (MTbLF) against the tested clinical isolates, both alone and in combination with metronidazole and clindamycin, was in depth evaluated using defined-iron culture conditions. All 71 presumptive G. vaginalis clinical isolates exhibited resistance to metronidazole, with MIC values greater than 256 µg/ml. Different susceptibility profiles were detected for clindamycin. In detail, the vast majority of the tested strains (45%), exhibiting MIC lower than 2 µg/ml, were considered sensitive; 18 strains (25%) with MIC higher or equal to 8 µg/ml, were classified as resistant, whereas the remaining 21 (30%) were classified as intermediate. MTbLF was tested in culture medium at different concentrations (32, 16, 8, 4, 2, 1, and 0.5 mg/ml) showing ability to inhibit the growth of the tested presumptive G. vaginalis clinical isolates, including those metronidazole-resistant, in a dose-dependent and not in a strain-dependent manner. MTbLF, at concentrations ranging from 32 to 8 mg/ml, exerted a statistically different antimicrobial activity compared with lower concentrations (4, 2, 1, and 0.5 mg/ml). A synergistic effect between MTbLF (8 and 4 mg/ml) and clindamycin was revealed for all the tested strains. When tested in the absence of other sources of iron, MTbLF did not support the growth of the tested presumptive G. vaginalis clinical isolates. Bovine lactoferrin may be a potential candidate to treat Gardnerella species infection.

Characterization of proteolytic degradation products of vaginally administered bovine lactoferrin.

When bovine lactoferrin (bLF) contacts human vaginal fluid (VF) it is subjected to proteolytic degradation. This report describes fragmentation patterns of bLF dosed vaginally in clinical trials or incubated ex vivo with VF. A consensus pattern of fragments was observed in samples from different women. The 80 kDa bLF molecule is initially cleaved between its homologous 40 kDa domains, the N-lobe and C-lobe, and then degraded into sub-fragments and mixtures of small peptides. We characterized this fragmentation process by polyacrylamide gel electrophoresis, western blotting, chromatographic separation, and mass spectral sequence analysis. Common to most VF fragmentation patterns were large amounts of an N-lobe 37 kDa fragment and a C-lobe 43 kDa fragment resulting from a single cleavage following tyrosine 324. Both fragments possessed full sets of iron-ligand amino acids and retained iron-binding ability. In some VF samples, alternative forms of large fragments were found, which like the 37+43 kDa pair, totaled 80 kDa. These included 58+22 kDa, 18+62 kDa, and 16+64 kDa forms. In general, the smaller component was from the N-lobe and the larger from the C-lobe. The 18+62 kDa pair was absent in some VF samples but highly abundant in others. This variability suggests multiple endopeptidases are involved, with the 18 kDa fragment's presence dependent upon the balance of enzymes. Further action of VF endopeptidases produced smaller peptide fragments, and we found evidence that exopeptidases trimmed their N- and C-termini. The 3.1 kDa antimicrobial peptide lactoferricin B was not detected. These studies were facilitated by a novel technique we developed: tricolor western blots, which enabled simultaneous visualization of N- and C-terminal epitopes.

Clinical surveillance and monitoring of arteriovenous access for hemodialysis.

Clinical surveillance and monitoring of arteriovenous access for hemodialysis can best ensure long term access function. The failing access can be identified and referred for intervention prior to complete access failure. This article reviews the basic science of access function and the various techniques for detecting the failing access. These techniques are utilized by the dialysis unit staff and by the physician, often the interventionalist, who takes primary care of the access. A combination of various techniques to detect dysfunction and trend analysis of various parameters is most likely to identify in a timely manner, those patients who need intervention.

Surgical versus percutaneous care of arteriovenous access.

Current clinical practice patterns have led to the performance of many percutaneous vascular procedures outside the traditional operating room and led to significant changes in management strategies for treatment of complications of arteriovenous access. In an effort to identify optimal treatment for failed or failing arteriovenous fistulas and grafts, percutaneous and surgical techniques are analyzed. Assessment of the functioning or failing access by physical examination is critical in determining proper treatment and in assessing outcomes of interventions. Percutaneous and surgical techniques are available for the treatment of AV access thrombosis and venous stenosis. Techniques for percutaneous thrombectomy and stent placement for venous stenosis and AV graft pseudoaneurysms are reviewed. Management of arterial inflow disease, steal syndrome and nonmaturing AV fistulas is also addressed. Overall, percutaneous techniques are assuming an increasingly important role in the treatment of failed or failing AV fistulas and grafts. Ongoing analysis of outcomes of both percutaneous and surgical interventions is necessary to continue to identify optimum treatment algorithms.