Bio-soliton model that predicts non-thermal electromagnetic frequency bands, that either stabilize or destabilize living cells.

Solitons, as self-reinforcing solitary waves, interact with complex biological phenomena such as cellular self-organization. A soliton model is able to describe a spectrum of electromagnetism modalities that can be applied to understand the physical principles of biological effects in living cells, as caused by endogenous and exogenous electromagnetic fields and is compatible with quantum coherence. A bio-soliton model is proposed, that enables to predict which eigen-frequencies of non-thermal electromagnetic waves are life-sustaining and which are, in contrast, detrimental for living cells. The particular effects are exerted by a range of electromagnetic wave eigen-frequencies of one-tenth of a Hertz till Peta Hertz that show a pattern of 12 bands, and can be positioned on an acoustic reference frequency scale. The model was substantiated by a meta-analysis of 240 published articles of biological electromagnetic experiments, in which a spectrum of non-thermal electromagnetic waves were exposed to living cells and intact organisms. These data support the concept of coherent quantized electromagnetic states in living organisms and the theories of Fröhlich, Davydov and Pang. It is envisioned that a rational control of shape by soliton-waves and related to a morphogenetic field and parametric resonance provides positional information and cues to regulate organism-wide systems properties like anatomy, control of reproduction and repair.

Liver slices as a model to study fibrogenesis and test the effects of anti-fibrotic drugs on fibrogenic cells in human liver.

Cell culture models have contributed significantly to the study of liver fibrosis, but cannot accurately incorporate in vivo cell-cell and cell-extracellular matrix interactions or account for the heterogeneity of the fibrogenic cell population involved in fibrosis development. Thus, there persists a need for an in vitro model that mimics the in vivo situation more closely, which may be provided by using precision-cut liver slices. In the present study we evaluated human liver slices as a tool to study fibrogenesis and test anti-fibrotic drugs. In this study we examined the responses of fibrogenic cells in human liver slices during control incubation and studied the effect of the anti-fibrotic compound pentoxifylline both during control incubation and after induction of early hepatic stellate cell (HSC) activation by carbon tetrachloride. After prolonged (>24 h) incubation, alphaSMA and pro-collagen 1a1 mRNA expression in human liver slices started to increase. Analysis of synaptophysin and fibulin-2 mRNA expression indicated that both activated HSC and other (myo)fibroblasts may be involved in this process. This response of fibrogenic cells to prolonged incubation of the liver slices was accompanied by an increased collagen protein content and could be inhibited by pentoxifylline. Early HSC activation, which was reflected by increased HSP47 and alphaB-crystallin mRNA expression, was not inhibited by pentoxifylline. Preparation and/or culturing of human liver slices induces fibrogenesis, which may be mediated by both activated HSC and resident liver (myo)fibroblasts and may represent a simple and rapid method to test the effects of potential anti-fibrotic drugs on fibrogenic cells in human liver.

Liver fibrosis in vitro: cell culture models and precision-cut liver slices.

Chronic liver injury of various etiologies can cause liver fibrosis, which is characterized by the progressive accumulation of connective tissue in the liver. As no effective treatment for liver fibrosis is available yet, extensive research is ongoing to further study the mechanisms underlying the development of disease- or toxicity-induced liver fibrosis and to identify potential pro- or anti-fibrotic properties of compounds. This review gives an overview of the in vitro methods that are currently available for this purpose. The first focus is on cell culture models, since the majority of in vitro research uses these systems. Both primary cells and cell lines as well as the use of different culture matrices and co-culture models are discussed. Second, the use of precision-cut liver slices, which recently came into attention as in vitro model for the study of fibrosis, is discussed. The overview clearly shows that continuous optimization and adaptation have extended the potential of in vitro models for liver fibrosis during the past years. By combining the use of the different cell and tissue culture models, the mechanisms underlying multicellular fibrosis development can be studied in vitro and potential pro- or anti-fibrotic properties of compounds can be identified both on single liver cell types and in human liver tissue.

Human liver slices as an in vitro model to study toxicity-induced hepatic stellate cell activation in a multicellular milieu.

INTRODUCTION: Hepatic stellate cell (HSC) activation is a key event in wound healing as well as in fibrosis development in the liver. Previously we developed a technique to induce HSC activation in slices from rat liver. Although this model provides a physiologic, multicellular milieu that is not present in current in vitro models it might still be of limited predictive value for the human situation due to species-differences. Therefore, we now aimed to evaluate the applicability of human liver slices for the study of HSC activation. METHOD: Liver slices (8 mm diameter, 250 microm thickness) were generated from human liver tissue and incubated for 3 or 16 h with 0-15 microl of carbon tetrachloride (CCl4) after which ATP-content and expression levels of HSC (activation) markers was determined. RESULTS: Human liver slices remained viable during incubation as shown by constant ATP levels. Incubation with CCl(4) caused a dose-dependent decrease in viability and an increase in mRNA expression of the early HSC activation markers HSP47 and alphaB-crystallin, but not the late markers for HSC activation, alphaSMA and pro-collagen 1a1. Synaptophysin mRNA expression remained constant during incubation with or without CCl4, indicating a constant number of HSC in the liver slices. CONCLUSION: We developed a technique to induce early toxicity-induced HSC activation in human liver slices. This in vitro model provides a multicellular, physiologic milieu to study mechanisms underlying toxicity-induced HSC activation in human liver tissue.

Preparation and characterization of conjugates of (modified) human serum albumin and liposomes: drug carriers with an intrinsic anti-HIV activity.

Human serum albumin (HSA) derivatized with cis-aconitic anhydride (Aco-HSA) that was earlier shown to inhibit replication of human immunodeficiency virus type 1 (HIV-1), was covalently coupled to conventional liposomes, consisting of phosphatidylcholine, cholesterol and maleimido-4-(p-phenylbutyryl)phosphatidylethanolamine, using the heterobifunctional reagent N-succinimidyl-S-acetylthioacetate (SATA). The amount of HSA that could be coupled to the liposomes depended on derivatization of the HSA and ranged from 64.2 +/- microgram HSA/micromol total lipid for native HSA to 29.5 +/- 2.7 microgram HSA/micromol total lipid for HSA in which 53 of the epsilon amino groups of lysine were derivatized with cis-aconitic anhydride (Aco53-HSA). Incorporation of 3.8 mol% of total lipid of a poly(ethylene glycol) derivative of phosphatidylethanolamine (PEG-PE) in the liposomes resulted in a lower coupling efficiency of Aco-HSA. The elimination and distribution of the liposomal conjugates in rats in vivo was largely dependent on the modification of the HSA coupled to the liposomes. With native HSA-liposomes, more than 70% of the conjugate was still found in the blood plasma 30 min after i.v. injection in rats, while at this time Aco-HSA-liposomes were completely cleared from the circulation. The rapid clearance of conventional Aco-HSA-liposomes was due to a rapid uptake into the liver and could be considerably decreased by incorporating PEG-PE in the liposomal bilayer. After 3 h 60% of Aco-HSA-PEG-liposome conjugates were found in the blood. In an in vitro anti-HIV-1 assay, the 50% inhibitory concentrations (IC50) for Aco39-HSA-liposomes and Aco53-HSA-liposomes expressed as protein weight, were 2.87 microgram/ml and 0.154 microgram/ml, respectively. When PEG-PE was incorporated, the Aco53-HSA-liposomes retained anti HIV-1 activity (IC50:3.13 microgram/ml). The possibility to modulate the residence time in the bloodstream of Aco-HSA-liposomes and the potent anti-HIV-1 activity of these conjugates, may allow the development of an intrinsically active drug carrier system. By incorporating anti HIV-1 drugs such as AZT into such liposomes a drug delivery system can be designed that might act simultaneously on the virus/cell binding by virtue of the coupled Aco-HSA and on the RNA/DNA transcription of the HIV-1 replication cycle through the nucleoside analogue.

Precision-cut organ slices as a tool to study toxicity and metabolism of xenobiotics with special reference to non-hepatic tissues.

Metabolism of xenobiotics is often seen as an exclusive function of the liver, but some current findings support the notion that the lungs, kidneys and intestine may contribute considerably. After the establishment of the use of liver slices as a useful in vitro model to study metabolism and toxicity of xenobiotics, the same concept is currently being used for slices from lung, kidney and intestine. It is the aim of this review to discuss the use of organ slices in biotransformation research. The basic idea behind the use of tissue slices in biomedical research is the assumption that the cells under study will function optimally in vitro if they are cultivated in an environment that is most alike to their natural in vivo embedding, which is the case in tissue slices. Advantages in the use of organ slices are the relatively easy preparation as well as the potential standardization of both the preparation and use. Moreover, a direct interspecies comparison can be made between liver, lungs, kidneys and intestines, for example with respect to their metabolic capacity and their sensitivity for toxicants. Of major importance is that organ slices can be made with a similar procedure from organs/tissues originating from different species, including man. This latter aspect is useful in drug development in general but also for a better insight in the metabolic fate of compounds in man. Importantly the use of slices may largely contribute to a reduction in the use of experimental animals.

Drug uptake systems in liver and kidney.

The hepatobiliary system and the kidneys are the main routes by which drugs and their metabolites leave the body. Compounds that are mainly excreted into bile in general have relatively high molecular weights, are amphipathic and highly bound to plasma proteins. In contrast, compounds that are predominantly excreted into urine have relatively low molecular weights, are more hydrophilic and generally less protein bound. The first step in drug elimination in liver and kidney is uptake into hepatocytes or into proximal tubular cells. The substrate specificity and affinity of the uptake carriers expressed at the basolateral membranes of hepatocytes and proximal tubular cells could therefore play an important role for the determination of the main elimination route of a compound. This review discusses the tissue distribution, substrate specificity, transport mechanism, and regulation of the members of the organic anion transporting polypeptide (Oatp/OATP) superfamily (solute carrier family SLC21A) and the SLC22A family containing transporters for organic cations (OCTs) and organic anions (OATs). The Oatps/OATPs are mainly important for the hepatic uptake of large amphipathic organic anions, organic cations and uncharged substrates, whereas OCTs and OATs mediate uptake of predominantly small organic cations and anions in liver and kidney.

Selective activity of several cholic acid derivatives against human immunodeficiency virus replication in vitro.

Several cholic acid derivatives such as taurolithocholic acid, lithocholic acid 3-sulfate, taurolithocholic acid 3-sulfate, and glycolithocholic acid 3-sulfate were shown to inhibit selectively the replication of human immunodeficiency virus type 1 (HIV-1) in vitro. These compounds completely protected MT-4 cells against HIV-1-induced cytopathogenicity at a concentration of 100 micrograms/ml, whereas no toxicity for the host cells was observed at 200 micrograms/ml. They also inhibited HIV-1 antigen expression in HIV-1-infected CEM cells. The bile acids (cholic acid, deoxycholic acid, chenodeoxycholic acid, and lithocholic acid) did not show any inhibitory effect on HIV-1 replication at concentrations that were not toxic to the host (MT-4) cells. From a structure-function analysis of a number of cholic acid derivatives, the presence of either a sulfonate (as in the tauro conjugates) or a sulfate group as well as the "litho" configuration appeared to be necessary for the expression of anti-HIV-1 activity. The active cholic acid derivatives did not directly inactivate the virus particles at the concentrations that were not toxic to the host cells. Lithocholic acid 3-sulfate, taurolithocholic acid 3-sulfate, and glycolithocholic acid 3-sulfate, but not taurolithocholic acid, partially inhibited virus adsorption to MT-4 cells. These three compounds were also inhibitory to the reverse transcriptase activity associated with HIV-1.

Optimization of the reaction conditions for the synthesis of neoglycoprotein-AZT-monophosphate conjugates.

The coupling of the monophosphate derivative of 3-azido-2,3-dideoxythymidine (AZTMP) to glycoproteins by water soluble carbodiimide (1-ethyl-3-[3-(dimethylamino)propyl]-3-ethylcarbodiimide) was greatly improved, relative to a recently reported method, by using also N-hydroxysulfosuccinimide (NHS) in the conjugation reaction. The hydrolysis of the activated AZTMP intermediate, responsible for the low degree of conjugation in the earlier method, could be delayed considerably if the activated phosphate group was converted into an activated ester by addition of NHS. In order to minimize the use of compounds needed for the preparation of AZTMP-protein conjugates, the present study was undertaken to determine if the reaction conditions could be optimized such that a conjugate with 2 AZTMP molecules/mol of neoglycoprotein would result. In addition a low proportion of cross-linked conjugates was desired. Optimization was achieved studying the shape of three-dimensional response surfaces, in which the degree of AZTMP coupling and the percentage of monomeric conjugates were regarded as the relevant responses. It appeared that the optimal conditions for coupling 1-2 mol of AZTMP to 1 mol of glycoprotein were an incubation time of 30 h, an AZTMP amount of 4 mg, an NHS amount between 8 and 15 mg, and a glycoprotein amount of 50 mg.

Neoglycoproteins as carriers for antiviral drugs: synthesis and analysis of protein-drug conjugates.

In order to investigate whether neoglycoproteins can potentially act as carriers for targeting of antiviral drugs to certain cell types in the body, various neoglycoproteins were synthesized using thiophosgene-activated p-aminophenyl sugar derivatives. These neoglycoproteins were conjugated with the 5'-monophosphate form of the antiviral drug AZT. For a proper characterization of these preparations, both protein and drug content have to be determined. Comparison of the Lowry and the Bio-Rad protein assays revealed that for both the neoglycoprotein carriers themselves and the AZTMP conjugates, the Lowry assay yielded the most reliable and reproducible results. It was demonstrated that both the reagent used for drug conjugation (ECDI) as well as the introduction of phenyl-sugar groups in the protein interfered with the analysis of bound nucleotide as based on spectral differences between protein and protein-drug conjugate. Therefore, we developed a rapid HPLC system for determination of the drug-protein coupling ratio through acid hydrolysis of the covalently bound nucleotide. With the ECDI-mediated conjugation of 5'-monophosphate drug derivatives to neoglycoproteins, products with molar ratios of drug to protein ranging from 1.2 to 5.6 were obtained. The drug-neoglycoprotein conjugates appeared to be fairly stable during storage, in lyophilized form, at -20 degrees C. The anti-HIV-1 activity of the neoglycoprotein-drug conjugates, as determined in vitro in MT-4 cells, was shown to be dependent on glycosylation of the albumin and also on the kind of sugar present in the neoglycoprotein. The anti-HIV-1 activity of the AZTMP-mannose-albumin conjugate exceeded that of the parent drug by more than 4 times.

Low molecular weight proteins as carriers for renal drug targeting. Preparation of drug-protein conjugates and drug-spacer derivatives and their catabolism in renal cortex homogenates and lysosomal lysates.

Low molecular weight proteins (LMWPs) are known to be reabsorbed and catabolized primarily by the proximal tubular cells of the kidneys. As such, LMWPs might serve as drug carriers that release drugs site-specifically in the kidney. We tested this concept in vitro by coupling different drugs to the LMWP lysozyme both directly (amide bond) and via different spacers: oligopeptides (amide bond), (poly-)alpha-hydroxy acids (ester bond), and a pH sensitive cis-aconityl spacer (amide bond). The capability of the kidney to release the parent drug from such drug-spacer derivatives and drug-LMWP conjugates by enzymatic or chemical hydrolysis of the bond was tested by incubation experiments in renal cortex homogenates and lysosomal lysates. Directly coupled conjugates of terminal carboxyl group containing drugs and lysozyme were catabolized to single amino acids, but did not result in release of the parent drug. The amide bond between the drug and the final amino acid (lysine) appeared to be stable in the incubation milieu. Different oligopeptide spacers coupled to the drugs showed similar results: the oligopeptide itself was cleaved but the amide bond between the drug and different single amino acids remained untouched. Only amide bonds of derivatives of carboxylic drugs with peptide structures themselves were cleaved. Some of the directly coupled conjugates of terminal amino drugs and oligopeptides showed clear release of the parent drug whereas others were stable. Terminal amino drugs were rapidly released from an acid-sensitive cis-aconityl spacer. Terminal carboxyl group containing drugs were enzymatically released from their glycolic and lactic ester spacers at different rates. These kinds of drugs were also released as parent drug from LMWP conjugates with ester spacers like L-lactic acid. Increasing spacer length by intercalating a tetra(L-lactic acid) molecular between the drug and the protein further increased the extent and rate of drug release, indicating increased accessibility of the bond to the enzymes. Terminal amino group containing drugs were rapidly generated as parent drug from LMWP conjugates using an acid-sensitive spacer. In addition the conjugates were found to be adequately stable in plasma, considering their rapid clearance from the bloodstream. It is concluded that LMWPs may indeed be of use as carriers for specific renal delivery of drugs, since renal cortex homogenates and lysosomal lysates are able to catabolize the protein and generate the parent drug from drug-LMWP conjugates bearing suitable spacers. The option of enzymatic release is limited by the narrow specificity of the lysosomal enzymes.(ABSTRACT TRUNCATED AT 400 WORDS)

Value of the in vitro or in vivo monoethylglycinexylidide test for predicting liver graft function.

BACKGROUND: An adequate function test for donor livers is still lacking. The monoethylglycinexylidide (MEGX) test, performed in vivo in the donor to measure the metabolic rate of lidocaine conversion to MEGX, has been proposed as a function test for donor livers to predict postoperative organ function. METHODS: In the present study, we investigated whether the MEGX formation rate measured in needle biopsy specimens in vitro correlates with the rate of MEGX formation in vivo. The in vivo MEGX test was performed in the donors and in the recipients on days 1 and 2. The in vivo and in vitro MEGX tests were compared with posttransplant liver function in the recipients in order to investigate their possible relevance as predictors of graft function. RESULTS: The MEGX formation rate in needle biopsy specimens in vitro showed a significant correlation with the MEGX serum concentration found in the donor. A low rate of MEGX formation in the biopsy specimens tended to predict initial poor function of the grafts. In the donor, the MEGX test did not correlate with general liver function after transplantation. Only the MEGX serum concentration in the recipients on day 2 gave an indication of graft function. CONCLUSIONS: MEGX formation in liver biopsy specimens in vitro properly reflects metabolic function of the particular liver. Therefore, liver biopsies may be a valuable tool to help predict liver function in vivo. However, the MEGX test alone is not sufficient to provide the gold standard to determine liver function in donor and transplantation livers.

Hepatobiliary and intestinal clearance of amphiphilic cationic drugs in mice in which both mdr1a and mdr1b genes have been disrupted.

1. We have used mice with homozygously disrupted mdr1a and mdr1b genes (mdr1a/1b (-/-) mice) to study the role of the mdr1-type P-glycoprotein (P-gp) in the elimination of cationic amphiphilic compounds from the body. These mice lack drug-transporting P-gps, but show no physiological abnormalities under laboratory conditions and have normal bile flow. 2. 3H-labelled cationic drugs were administered intravenously (i.v.) to mice as a single bolus dose and the disposition of the studied cationic drugs was investigated by focusing on drug secretion into bile, intestinal lumen and urine. 3. Hepatobiliary secretion of the investigated cationic drugs was profoundly reduced in mice devoid of the mdr1-type P-gps. In fact, the cumulative biliary output, measured during 1 h, of the small type 1 compounds tri-butylmethyl ammonium (TBuMA) and azidoprocainamide methoiodide (APM), as well as of the more bulky type 2 cationic drug vecuronium, was reduced by at least 70% in the mdrla/lb (-/-) mice compared to wild-type. 4. The intestinal secretion of TBuMA, APM and vecuronium was also profoundly reduced in mdrla/lb (-/-) mice compared to wild-type mice. The absence of the mdrl-type P-gp resulted in virtual elimination of intestinal secretion of TBuMA and APM (>90% reduced as compared to wild-type (P=0.0001 and 0.0022, respectively)). The intestinal secretion of the type 2 cation drug vecuronium was reduced by 58% (P=0.0004) compared to the wild-type mice. 5. Increased renal clearances of both the type 1 compounds TBuMA and APM and also of the type 2 cationic compound vecuronium in the mdrla/lb (-/-) mice were observed. Furthermore, the balance between hepatic, intestinal and renal clearances of small type 1 organic cations clearly shifted towards a predominant role for renal clearance. Increased renal clearance may be explained by (over)expression of additional mechanisms for renal organic cation secretion, alternatively they may also point to an as yet undefined role of P-glycoprotein in kidney physiology and renal secretory function. 6. We conclude that the elimination from the body of a broad spectrum of cationic amphiphilic drugs via liver and intestine, is largely dictated by the activity of mdrl-type P-glycoproteins.

Interactions between P-glycoprotein substrates and other cationic drugs at the hepatic excretory level.

1. In the present study it was tested whether known P-glycoprotein (P-gp) substrates/MDR reversal agents interact with small (type 1) and bulky (type 2) cationic drugs at the level of biliary excretion in the rat isolated perfused liver model (IPRL). The studies were performed with model compounds tri-n-butylmethylammonium (TBuMA) (a relatively small type 1 organic cation), rocuronium (Roc) (a bulky type 2 organic cation) and the classical P-gp substrate doxorubicin (Dox). 2. Inhibitors were given in a 4 fold molar excess to the substrate studied. To minimize an interaction of the substrates at the hepatic uptake level, the competing compounds were added when over 55% to 85% of the administered dose of the model compounds had been removed from the perfusate and taken up by the liver. 3. We found a mutual interaction between TBuMA and procainamidethobromide (PAEB), both type 1 cationic compounds during biliary excretion. Interestingly, type 2 compounds, such as rocuronium, clearly inhibited type 1 cationic drugs as well as Dox secretion into bile, whereas type 1 compounds did not significantly inhibit type 2 drug excretion into bile. The type 1 cations PAEB and TBuMA only moderately inhibited Dox biliary excretion. Dox did not inhibit the biliary excretion of the type 2 agent rocuronium whereas rocuronium reduced Dox biliary excretion by 50% compared to controls. 4. MDR substrates/reversal agents like verapamil, quinine, quinidine and vinblastine strongly reduced both type 1 and type 2 organic cation excretion into bile. Dox secretion into bile was also profoundly reduced by these drugs, vinblastine being the most potent inhibitor in general. 5. The lack of mutual inhibition observed in some combinations of substrates may indicate that major differences in affinity of the substrates for a single excretory system exist. Alternatively, multiple organic cation transport systems with separate substrate specificities may be involved in the biliary excretion of amphiphilic drugs. Furthermore, the present study revealed a clear positive correlation between the lipophilicity of the potential inhibitors studied and their respective inhibitory activity on the biliary excretion of the model drugs investigated. 6. Our data are compatible with a potential involvement of P-glycoprotein in the hepatobiliary excretion of doxorubicin as well as of some type 1 and type 2 organic cations. Furthermore we postulate that the hydrophobic properties of the amphiphilic cationic drugs studied play a crucial role in the accommodation of these agents by P-glycoprotein and/or other potential cationic drug carrier proteins in the canalicular membrane.

Substantial excretion of digoxin via the intestinal mucosa and prevention of long-term digoxin accumulation in the brain by the mdr 1a P-glycoprotein.

1. We have used mice with a disrupted mdr 1a P-glycoprotein gene (mdr 1a (-/-) mice) to study the role of P-glycoprotein in the pharmacokinetics of digoxin, a model P-glycoprotein substrate. 2. [3H]-digoxin at a dose of 0.2 mg kg-1 was administered as a single i.v. or oral bolus injection. We focussed on intestinal mucosa and brain endothelial cells, two major pharmacological barriers, as the mdr 1a P-glycoprotein is the only P-glycoprotein normally present in these tissues. 3. Predominant faecal excretion of [3H]-digoxin in wild-type mice shifted towards predominantly urinary excretion in mdr 1a (-/-) mice. 4. After interruption of the biliary excretion into the intestine, we found a substantial excretion of [3H]-digoxin via the gut mucosa in wild-type mice (16% of administered dose over 90 min). This was only 2% in mdr 1a (-/-) mice. Biliary excretion of [3H]-digoxin was not dramatically decreased (24% in wild-type mice versus 16% in mdr 1a (-/-) mice). 5. After a single bolus injection, brain levels of [3H]-digoxin in wild-type mice remained very low, whereas in mdr 1a (-/-) mice these levels continuously increased over a period of 3 days, resulting in a approximately 200 fold higher concentration than in wild-type mice. 6. These data demonstrate the in vivo contribution of intestinal P-glycoprotein to direct elimination of [3H]-digoxin from the systemic circulation and to the pattern of [3H]-digoxin disposition, and they underline the importance of P-glycoprotein for the blood-brain barrier.

Plasma lactoferrin levels are decreased in end-stage AIDS patients.

The antimicrobial protein lactoferrin (Lf) is present in plasma and in mucosal secretions. Using ELISA we analysed plasma and saliva of HIV-infected patients, patients with AIDS, and healthy controls for the presence of secreted Lf. The plasma Lf levels of AIDS patients (classification C3) were significantly lower (p < 0.001) as compared to asymptomatic and symptomatic HIV infected patients, or controls. In addition, plasma Lf levels closely correlated with neutrophilic granulocyte counts in the HIV-infected patients. Thus, basal plasma Lf levels are likely the result of Lf release by neutrophilic granulocytes. The Candida titres present in the oral cavity were determined in a part of the HIV-infected patient group. As it appeared, the presence of this opportunistic pathogen always coincided with low levels of salivary Lf levels. We conclude that Lf, as part of the nonspecific immune system, might play an important role in the first line of defense against opportunistic microbial infections in AIDS patients.

Potent inhibition of replication of primary HIV type 1 isolates in peripheral blood lymphocytes by negatively charged human serum albumins.

We previously reported the antiviral capacity of human serum albumin (HSA), which was modified by the introduction of a single (Suc-HSA) or two carboxylic groups (Aco-HSA) per lysine residue, yielding strongly negatively charged polypeptides. Here we report the antiviral effect of these modified HSAs on replication of primary HIV-1 isolates that differed with respect to syncytium-inducing (SI) capacity and cell tropism. Both Suc-HSA and Aco-HSA potently inhibited replication of primary HIV-1 variants, independent of the SI capacity of the HIV-1 variant, with IC50 values in the range of 50 to 187 microg/ml. The inhibition of the formation of syncytia and the absence of proviral DNA products in cells inoculated with HIV-1 in the presence of Suc-HSA or Aco-HSA pointed to interference at an early level in the virus replication cycle. The inhibitory capacity of Suc-HSA and Aco-HSA on primary HIV-1 variants suggests that these agents are potential candidates for use in antiviral therapy in HIV-infected individuals.

The in vitro anti-HIV efficacy of negatively charged human serum albumin is antagonized by heparin.

Succinylated human serum albumin (Suc-HSA) was synthesized by treating human serum albumin with succinic anhydride. Among similar proteins and neo(glyco)proteins tested, Suc-HSA exhibits a pronounced net negative charge, a feature that largely contributes to its efficacy against replication of human immunodeficiency virus type 1 (HIV-1). To assess further the antiviral effect of Suc-HSA, the effect on HIV-1 replication was studied in the presence of whole human plasma. Pretreatment of MT2 cells with Suc-HSA was more efficacious than direct Suc-HSA treatment of HIV prior to addition to the cells. No changes in the antiviral effect of Suc-HSA were observed in tissue culture medium, 30% plasma, or whole plasma when CPDA-1 (citrate-phosphate-dextrose-adenine 1) was used as the anticoagulant. However, a dramatic decrease (greater than 99%) in the antiviral activity was observed when these experiments were performed in plasma prepared from blood using heparin as anticoagulant. The antagonistic effect by heparin was observed both in the case that heparin was added prior to or after addition of Suc-HSA to the test system. In the present study we demonstrate that heparin largely reduces Suc-HSA activity on HIV replication in the same concentration in which if affects binding of Suc-HSA to the envelope protein gp120 and in particular its V3 domain. In the same concentration range, heparin reduced binding of Suc-HSA to MT4 cells, another HTLV-I-transformed cell line. It is concluded that heparin can displace Suc-HSA from its binding sites on hybrid lymphoid cells as well as on HIV-1 particles. Therefore, we conclude that both the binding to cells and to virus contribute to the potent anti-HIV-1 effect. The fact that heparin and heparin degradation products antagonize Suc-HSA without having a significant anti-HIV-1 effect indicates that the anticoagulant acts as a relatively weak partial inhibitor.

Antiviral effects of milk proteins: acylation results in polyanionic compounds with potent activity against human immunodeficiency virus types 1 and 2 in vitro.

A number of native and modified milk proteins from bovine or human sources were analyzed for their inhibitory effects on human immunodeficiency virus type 1 (HIV-1) and HIV-2 in vitro in an MT4 cell test system. The proteins investigated were lactoferrin, alpha-lactalbumin, beta-lactoglobulin A, and beta-lactoglobulin B. By acylation of the amino function of the lysine residues in the proteins, using anhydrides of succinic acid or cis-aconitic acid, protein derivatives were obtained that all showed a strong antiviral activity against human immunodeficiency virus type 1 and/or 2. The in vitro IC50 values of the aconitylated proteins were in the concentration range of 0.3 to 3 nM. Succinylation or aconitylation of alpha-lactalbumin and beta-lactoglobulin A/B also produced strong anti-HIV-2 activity with IC50 values on the order 500 to 3000 nM. All compounds showed virtually no cytotoxicity at the concentration used. Peptide-scanning studies indicated that the native lactoferrin as well as the charged modified proteins strongly bind to the V3 loop of the gp120 envelope protein, with Kd values in the same concentration range as the above-mentioned IC50. Therefore, shielding of this domain, resulting in inhibition of virus-cell fusion and entry of the virus into MT4 cells, may be the likely underlying mechanism of antiviral action.

Resistance of the human immunodeficiency virus to the inhibitory action of negatively charged albumins on virus binding to CD4.

Negatively charged albumins (NCAs) have been identified as potent inhibitors of HIV-1 replication in vitro. Time of addition studies suggest that succinylated and aconitylated human serum albumin (Suc-HSA and Aco-HSA) act at an early stage of the virus life cycle, and surface plasmon resonance (BIAcore) experiments have confirmed a direct interaction of NCAs with HIV-1 gp120. Resistance to Suc-HSA and Aco-HSA was analyzed by characterizing HIV-1 variants that were selected in cell culture after serial passage of the NL4-3 strain in the presence of the compounds. After 24 passages (126 days) we isolated variants that were resistant to Suc-HSA (>27-fold) and Aco-HSA (37-fold), as compared with the wild-type NL4-3 virus. The binding of the NCA-resistant HIV strains to CD4+ MT-4 cells could no longer be inhibited by either Suc- or Aco-HSA. The emergence of mutations in the envelope gp120 of the resistant virus paralleled the emergence of the resistant phenotype. The Suc-HSA-resistant strain was 100-fold cross-resistant to the G quartet-containing oligonucleotide AR177 (Zintevir, an HIV-binding inhibitor), and partially cross-resistant to dextran sulfate, but remained sensitive to the bicyclam AMD3100 and the chemokine SDF-1alpha, which block HIV replication by interaction with the chemokine receptor CXCR4. Furthermore, neither Suc-HSA nor Aco-HSA inhibited the binding of monoclonal antibodies 12G5 and 2D7 (directed to CXCR4 and CCR5, respectively) in SUPT-1 cells or THP-1 cells. These results confirm that NCAs bind primarily to gp120 and do not interact directly with the HIV chemokine receptor but block the binding of the virus particles (through gp120) with CD4+ cells.