Covalent Polymyxin B Conjugate with Human Immunoglobulin G as an Antiendotoxin Reagent

Polymyxin B (PMB) is a cyclic decapeptide antibiotic which also binds and neutralizes endotoxin. Unfortunately, PMB can be considerably nephrotoxic at clinically utilized doses, thereby limiting its utility as a therapeutic antiendotoxin reagent. We sought to change the pharmacokinetics and toxicity profile of PMB by covalently linking it to a human immunoglobulin G (IgG) carrier. Conjugates of PMB with IgG were prepared by EDAC [1-ethyl-3-(3-dimethylaminopropyl) carbodiimide]-mediated amide formation. Analysis by dot enzyme-linked immunosorbent assay with an anti-PMB monoclonal antibody showed that the purified conjugate contained bound PMB. The IgG-PMB conjugate reacted with lipid A and J5 lipopolysaccharide in Western blot assays in a manner comparable to that of whole antiserum with anti-lipid A reactivity; unconjugated IgG had no reactivity. The PMB bound in the conjugate retained its endotoxin-neutralizing activity compared to that of unbound PMB as evidenced by its dose-dependent inhibition of tumor necrosis factor release by endotoxin-stimulated human monocytes in vitro; unconjugated IgG had no activity. By this assay, the PMB-IgG conjugate was determined to have approximately 3.0 μg of bound functional PMB per 100 μg of total protein of conjugate (five molecules of PMB per IgG molecule). The PMB-IgG conjugate was also bactericidal against clinical strains of Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae relative to unconjugated IgG with MBCs of <4 μg of conjugate per ml for each of the tested strains. The conjugate appeared to be nontoxic at the highest doses deliverable and provided statistically significant protection from death to galactosamine-sensitized, lipopolysaccharide-challenged mice in a dose-dependent fashion when administered prophylactically 2 h before challenge. However, neither free PMB nor the PMB-IgG conjugate could protect mice challenged with endotoxin 2 h after administration. This suggests that these reagents can play a role in prophylaxis but not in therapy of sepsis. These experiments demonstrated that the PMB-IgG conjugate retains bound yet functional PMB as evidenced by its endotoxin-neutralizing activity both in vitro and in vivo. Further work is required to define the role that this or related conjugate compounds may play in the prophylaxis of endotoxin-mediated disease.     

Soluble and Membrane-bound Forms of Signaling Lymphocytic Activation Molecule (SLAM) Induce Proliferation and Ig Synthesis by Activated Human B Lymphocytes

In this study it is shown that both membrane-bound and soluble forms of signaling lymphocytic activation molecule (SLAM) induce proliferation and Ig synthesis by activated human B cells. Activated B cells express the membrane-bound form of SLAM (mSLAM), the soluble (s) and the cytoplasmic (c) isoforms of SLAM, and the expression levels of mSLAM on B cells are rapidly upregulated after activation in vitro. Importantly, recombinant sSLAM and L cells transfected with mSLAM efficiently enhance B cell proliferation induced by anti-μ mAbs, anti-CD40 mAbs or Staphylococcus aureus Cowan I (SAC) in the presence or absence of IL-2, IL-4, IL-10, IL-12, or IL-15. sSLAM strongly enhances proliferation of both freshly isolated B cells and B cells derived from long-term in vitro cultures, indicating that SLAM acts not only during the initial phase of B cell activation but also during the expansion of preactivated B cells. In addition, sSLAM enhances production of IgM, IgG, and IgA by B cells activated by antiCD40 mAbs. SLAM has recently been shown to be a high affinity self-ligand, and the present data suggest that signaling through homophilic SLAM–SLAM binding during B–B and B–T cell interactions enhances the expansion and differentiation of activated B cells.     

Voriconazole-induced photosensitivity

Voriconazole is a broad-spectrum triazole antifungal agent indicated for invasive aspergillosis, refractory Candida infections, and other emerging invasive fungal infections. Adverse cutaneous reactions associated with voriconazole therapy occur in fewer than 10% of treated patients and range from mild erythematous eruptions to life-threatening reactions such as the Stevens-Johnson syndrome and toxic epidermal necrolysis. Photosensitivity reactions are an uncommon but characteristic dermatitis in voriconazole recipients, particularly following chronic administration. We report a case of voriconazole-induced phototoxicity in a 50-year-old male with Candida parapsilosis endocarditis that reversed on discontinuation of the drug.     

Hemodynamic effects after conversion of arrhythmias

Systemic and coronary hemodynamic parameters were determined during an arrhythmia and immediately after a direct current transthoracic shock given in an attempt to convert the arrhythmia to a sinus mechanism. No anesthesia or drugs were administered between the two studies. 16 patients with atrial fibrillation converted to sinus rhythm and five did not. In two patients with atrial flutter and one with supraventricular tachycardia, the arrhythmia was corrected. The arrhythmia persisted in a single patient with ventricular tachycardia. Utilizing each patient as his own control, we compared statistically various hemodynamic parameters before and after the shock. In addition, the group of patients whose atrial fibrillation terminated was compared to the group treated in the same manner but in which the atrial fibrillation persisted. Pressures in the right side of the heart decreased in both groups so that the changes appeared to be caused by factors associated with the transthoracic direct current shock or the catheterization procedure. The differences between those with atrial fibrillation who converted to sinus rhythm as compared to those who did not were a decrease in heart rate, an increase in stroke volume, and an increase in cardiac efficiency. There was no immediate effect on the cardiac output or coronary blood flow.     

Myc confers androgen-independent prostate cancer cell growth

Prostate cancer is one of the most diagnosed and mortal cancers in western countries. A major clinical problem is the development of androgen-independent prostate cancer (AIPC) during antihormonal treatment. The molecular mechanisms underlying the change from androgen dependence to independence of these tumors are poorly understood and represent a challenge to develop new therapies. Based on genetic data showing amplification of the c-myc gene in AIPC, we studied the ability of c-myc to confer AIPC cell growth. Human androgen-dependent prostate cancer cells overexpressing c-myc grew independently of androgens and presented tumorigenic properties in androgen-depleted conditions. Analysis of signalling pathways by pharmacological inhibitors of the androgen receptor (AR) or by RNA interference directed against AR or c-myc showed that c-myc acted downstream of AR through multiple growth effectors. Thus c-myc is required for androgen-dependent growth and following ectopic expression can induce androgen-independent growth. Moreover, RNA interference directed against c-myc showed that growth of human AIPC cells, AR-positive or -negative, required c-myc expression. Furthermore, we showed that c-myc-overexpressing cells retain a functional p53 pathway and thus respond to etoposide.     

Fatality after deliberate ingestion of the pesticide rotenone: a case report

Rotenone is a pesticide derived from the roots of plants from the Leguminosae family. Poisoning following deliberate ingestion of these plant roots has commonly been reported in Papua New Guinea. However, poisoning with commercially available rotenone in humans has been reported only once previously following accidental ingestion in a 3.5-year-old child. Therefore, the optimal management of rotenone poisoning is not known. After deliberate ingestion of up to 200 ml of a commercially available 0.8% rotenone solution, a 47-year-old female on regular metformin presented with a reduced level of consciousness, metabolic acidosis and respiratory compromise. Metformin was not detected in premortem blood samples obtained. Despite intensive supportive management, admission to an intensive care unit, and empirical use of N-acetylcysteine and antioxidant therapy, she did not survive. Poisoning with rotenone is uncommon but is potentially fatal because this agent inhibits the mitochondrial respiratory chain. In vitro cell studies have shown that rotenone-induced toxicity is reduced by the use of N-acetylcysteine, antioxidants and potassium channel openers. However, no animal studies have been reported that confirm these findings, and there are no previous reports of attempted use of these agents in patients with acute rotenone-induced toxicity.     

Serum bactericidal activities of high-dose daptomycin with and without coadministration of gentamicin against isolates of Staphylococcus aureus and Enterococcus species

The purpose of this experiment was to evaluate the pharmacokinetics and serum bactericidal titers (SBTs) of daptomycin alone and in combination with gentamicin against strains of Staphylococcus aureus and enterococci to determine if there might be any benefit to the addition of the aminoglycoside. A multiple-dose, randomized crossover study was performed in 11 healthy volunteers to evaluate the steady-state pharmacokinetic profile of 6 mg/kg of body weight daptomycin once daily with or without 1 mg/kg gentamicin every 8 h. SBTs were determined against clinical isolates of nosocomial (MRSA 494) and community-acquired (CA-MRSA 44) methicillin-resistant S. aureus, vancomycin-susceptible Enterococcus faecalis (VSEF 49452), vancomycin-resistant Enterococcus faecium (VREF 80), and quality control strains of methicillin-susceptible S. aureus (ATCC 29213) and vancomycin-susceptible E. faecalis (ATCC 29212). Enhancement of bactericidal activity was evaluated by calculating and comparing the areas under the bactericidal curve (AUBC) for each dosing regimen against each isolate. The area under the concentration-time curve from 0 to 24 h and clearance for daptomycin alone were 645 +/- 91 microg.h/ml and 9.47 +/- 1.4 mg/h/kg, respectively, compared with 642 +/- 69 microg.h/ml and 9.45 +/- 1.0 mg/h/kg for daptomycin plus gentamicin. Daptomycin alone displayed sustained bactericidal activity against five of the six isolates over the entire 24-h dosing interval; bactericidal activity was maintained for 8 h against VREF 80. Mean AUBCs for daptomycin alone ranged from 935 to 1,263 and 36 to 238 against staphylococcal and enterococcal isolates, respectively, compared with 902 to 972 and 34 to 213 against staphylococci and enterococci when coadministered with gentamicin. The results of this study suggest that the addition of gentamicin does not alter the pharmacokinetic profile or enhance the bactericidal activity of daptomycin against staphylococcal or enterococcal isolates.     

Characterization of Ileal Vitamin B12 Binding Using Homogeneous Human and Hog Intrinsic Factors

Elucidation of the mechanism of intrinsic factor (IF)-mediated vitamin B(12) (B(12)) binding to ileal binding sites has been hampered by the use of crude or only partially purified preparations of IF in previous studies. We have used homogeneous human IF and hog IF isolated by affinity chromatography to study [(57)Co]B(12) binding to ileal mucosal homogenates. The following observations were made: (a) Human IF-B(12) and hog IF-B(12) were bound to human, monkey, hog, dog, rabbit, mouse, hamster, and guinea pig ileal, but not jejunal, homogenates in amounts significantly greater than free B(12) or B(12) bound to five other homogeneous B(12)-binding proteins; (b) only IF-mediated B(12) binding was localized to ileal homogenates and was inhibited by EDTA; (c) values for the association constant (K(a)) for the various ileal homogenates mentioned above and human IF-B(12) and hog IF-B(12) ranged from 0.3 x 10(9) M(-1) to 13.0 x 10(9) M(-1). Apparent differences in the K(a) for human IF-B(12) and hog IF-B(12) existed in most species; (d) the number of ileal IF-B(12) binding sites per gram (wet weight) of ileal mucosa ranged from 0.3 x 10(12) to 4.9 x 10(12). The same value was always obtained with human IF-B(12) and hog IF-B(12) for any given homogenate preparation; (c) 100-fold excesses of free B(12) or human IF and hog IF devoid of B(12) did not significantly inhibit human IF-B(12) and hog IF-B(12) binding to human and hog ileal homogenates.THESE EXPERIMENTS PERFORMED WITH HOMOGENEOUS IF INDICATE THAT: (a) gastric factors other than IF are not required for B(12) binding to ileal IF-B(12)-binding sites: (b) the mechanism of ileal IF-B(12) binding is different from that of free B(12) or of B(12) bound to non-IF-B(12)-binding proteins; (c) human IF and hog IF have different structures; (d) human IF-B(12) and hog IF-B(12) bind to the same ileal binding sites; and (c) human and hog ileal IF-B(12) binding sites bind free B(12) and human and hog IF devoid of B(12) poorly, if at all.     

Effect of Glycine-Conjugated Bile Acids with and without Lecithin on Water and Glucose Absorption in Perfused Human Jejunum

Perfusion studies were performed in healthy volunteers to test whether the secretory effect of conjugated bile acids, previously shown for the colon, was also present in the jejunum. A perfusion system with a proximal occlusive balloon (and continuous aspiration of duodenal secretions) was used; isotonic test solutions contained glycine-conjugated bile acids with or without lecithin. Fluid movement was measured by changes in the concentration of polyethylene glycol (PEG, mol wt 4,000). Conjugated dihydroxy bile acids inhibited electrolyte and fluid absorption and, at higher concentrations, evoked secretion of an isotonic fluid. Glucose absorption continued, despite fluid secretion, but its rate decreased. The secretory effects of bile acids were abolished by the addition of lecithin to the bile acid solutions. A trihydroxy bile acid (cholylglycine) had no effect on jejunal absorption. Small amounts (6-9%) of conjugated bile acids were absorbed in the jejunum; lecithin was well absorbed (72-90%). The results indicate that dihydroxy bile acids influence salt and water transport in the human jejunum but that this effect may be abolished when a polar lipid such as lecithin is present. We speculate that this effect of bile acids may modify fluid movement in the small intestine postprandially after fat absorption has occurred.