Candidal lung involvement
Candidal mycetomas have been reported rarely in the literature and are clearly not any more related to candidal pneumonia than aspergillomas are related to Aspergillus pneumonia. The recovery of Candida via protected brush or BAL specimens is common in patients receiving antimicrobial or corticosteroid therapy, as well as in diabetics, alcoholics, and HIV-infected patients. Therefore, the recovery of Candida from a bronchoscopy specimen, even with a protected tip, in a normal or compromised host with pulmonary infiltrates is not diagnostic of Candida pneumonia and should not be used as the basis for empiric anticandidal therapy. A Candida culture obtained via bronchial specimen should prompt the clinician to look for another explanation for the pulmonary infiltrates. Candidal lung involvement, even in systemic invasive disease, is rarely made antemortem. Therefore, if the diagnosis is made antemortem, the patient should be treated for systemic disease and not for an isolated pulmonary infection.
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Coccidioidomycosis
C immitiscauses the systemic illness coccidioidomycosis that is endemic in the southwestern United States. Approximately 60% of infected patients are asymptomatic while 40% will develop primary infection after an incubation period of 1 to 3 weeks. Most symptomatic patients with primary coccidioidomycosis develop a lower respiratory tract infection associated with a flu-like illness characterized by fever, chills, cough, malaise, anorexia, and night sweats. Erythema nodosum or erythema multiforme may occur. Chest radiographs are often abnormal with infiltrates, pleural effusions, or hilar adenopathy. Five percent of patients with primary disease have a residual pulmonary cavity or nodule, while 0.5% develop disseminated disease usually involving the meninges, bones, or joints.
In some patients, primary acute pneumonia may progress to chronic pulmonary coccidioidomycosis, a disease that closely mimics pulmonary tuberculosis. Most forms of acute pulmonary coccidioidomycosis are self-limited and require no therapy.
Reduce Your Anxiety! The Sensate Focus
The Sensate Focus Exercises
Okay, so what is Sensate Focus? These are relaxation exercises designed to reduce anxiety and help a couple discover and enjoy each other’s erogenous zones through non genital touching in non demanding situations. But the primary objective is to ease away tension and anxiety so that the husband can enjoy the moment of having sex eventually with his wife.
We have sensate focus exercises you can try below. There are each individual exercises that you have to do at least one time, and you move on to the next when you’re happy you’ve got as much out of the exercise as you can. Be sure you and your wife know and understand what’s going to happen before you embark on the exercises, both of you should be aware of the objective (although you don’t need to be that aware of the process, so that you can enjoy what you are doing in a spontaneous manner) and has a common understanding. As you can appreciate, it helps if you have a regular time each week to practice this work, when you will be free of stress, distractions, work and family worries.
Take note though, that most of the time, a single try cannot be enough. So never be anxious if things don’t go as what you expect it to be in the first try. If you can take the attitude that it’s an exciting and spontaneous process, you’re much more likely to find it sexy doing it and find that it’s successful in reducing the level of erection problems that you have – and your wife is more likely to like as well. The thing is, forget about everything, just be in the moment and enjoy the moment. Sure enough, whenever you feel pressured while you do the exercises, then you have to lighten up a bit and take it all less seriously.
Definitely, impotence official canadian health care mall website is a bothersome problem, but it can be solved and this treatment can be enjoyable if you just allow it to be. Take note, an erection is only one part of the sexual communication – and this series of exercise, this self-help treatment, will help improve much of the rest of your sex life, by reducing anxiety, getting you and your partner to be more intimate, and beginning to establish in your mind the understanding that you are not necessarily impotent for life.
It is vital that you maintain your focus on what’s in the moment, nothing more, nothing less. Never go off on some fantasy or other. Every time you begin to think about other instances, bring your attention gently back to the process. These distracters can be instances when you begin to think about what you look like, whether your partner is enjoying it, what state of erection your penis is in, whether this treatment for erectile dysfunction will work, or indeed anything irrelevant, including what you did at work that day and what you’ll be doing tomorrow! If you see yourself drifting off on thoughts or fantasies, just remind yourself that you are focusing on your body, and take a deep breath to relax any tension or anxiety that may have developed. Don’t give yourself a difficult time, and reject all negative thoughts: for example, if you think to yourself “don’t get anxious’’, guess what will probably happen?
Operative Procedure: complications
Survival of implants ranges between 78 and 95% after 10 years. In a long-term study involving 2,384 patients who underwent penile prosthesis implantation, estimated 10-year revision-free survival was 68.5% and the 15-year revision-free implant survival was 59.7%. In 1992, the Mentor Alpha-1 (now the Coloplast Titan) device added pump reinforcement to fore-stall mechanical breakage which improved 10-year survival from 65.3% to 88.6%.
In January 2001, AMS CX added parylene coating to the cylinders that has increased 3-year mechanical survival from 88.4% to 97.9%.
Viagra does not only restore your erectile potential, it intensifies the quality of your erections and performance in general, making you last much longer. Your erections become much better controlled, since Viagra Australia shop decreases sensitivity of the penis – now you definitely will give your partner as many orgasms as she can take. Your woman will be beyond impressed; she will be astounded by the newly acquired power, hardness and girth of your member.
Since 1973, improvements have been made in penile prosthesis design. Notwithstanding these improvements, complications or adverse events can still occur, including infection, hematoma, urethral perforation, persistent pain, mechanical failure, malposition of components, and patient dissatisfaction. Extremely rare complications include erosion of reservoir into the bladder or bowel, penile gangrene, sepsis, glans necrosis, and hernia. The complication rate has decreased substantially from approximately 50% in the earliest models to 1–12% in the more recent devices. Regardless of the type of prosthesis used, meticulous surgical technique and surgeon’s experience are important factors in determining the final outcome.
A major postoperative concern for most implanting surgeons is the development of infection. Signs or symptoms of infection include a purulent exudate, increasing pain instead of gradual improvement, worsening erythema and induration, or low-grade fever. Most infections present within the first 3 months after surgery and the vast majority manifest within the first year, although delayed infections beyond 1 year occasionally occur. The literature lists several risk factors for infection, such as inadequate perioperative antibiotic prophylaxis prolonged hospitalization, concurrent urinary tract infection, prolonged operative time, repeat implantation procedures, and combined operations (hernia repair, circumcision, artificial urinary sphincter implanation) with penile prosthesis surgery. Some patients, such as diabetics, and patients with spinal cord injury may have a decreased host defense. Local factors which increase infection risk include capsule formation around a foreign body, which diminishes blood supply to the area, and biofilm production. These factors provide a protective cavity in which bacteria may remain in a low metabolic state with no systemic antibiotic contact. The severity of infections may range from simple superficial infections that can be managed by conservative measures and wound care, to penile gangrene and sepsis which may be life threatening. Penile gangrene is rare and may be due to gram-negative organisms with or without anaerobic superinfection.
Vive le Difference
His Sexuality and Her Sexuality
Men and women – when it comes to sex are we really so different that we come from different planets? Or are we more similar? In offering our perspective on gender and sex, we explore learnings from history; the biological and physiological makeup of men and women; what science tells us about men and women’s sexual attitudes, behaviors, and feelings; new information about the development of sex and love for men and women and how sexual drive and desires are similar yet different; the importance and value of men regulating their sex drive; thoughts about the “hot potato” issue between men and women – pornography; and finally how similar men and women are when it comes to the ultimate sexual goal – emotional and sexual satisfaction. These issues are important for sexual health because they help you develop realistic expectations about yourself as a sexual man, your relationship, and how to cooperate and share as an intimate team. Canadian health care mall online
Reflect on the sexual stereotypes about men and women. People believe that women are very complex in terms of sexuality, while men are very simple. Others (those who favor political correctness) believe that there are absolutely no differences between the sexes. Still others suggest that men and women engage in “the war between the sexes,” have for centuries, and always will.
To understand the sexuality of men and women, the optimal model is a multidimensional, integrated, biopsychosocial approach. Sexuality is a lifelong developmental process, grounded in the body, profoundly enriched through psychological growth, and culminating in interpersonal integration. The history of men and women’s sexuality in Western culture (as well as other cultures) demonstrates this developmental process. Traditionally, beliefs centered on understanding biological sexuality. Until about 1500 B.C., human beings did not understand that pregnancy was the result of sexual intercourse. The myths that culture designed to explain the meaning and significance of sex and fertility seem naïve and grandiose from our historical vantage point, but they were honest efforts to comprehend sexuality. The integration of the biological, psychological, and inter-personal aspects of sexuality in a scientific manner is new thinking in the last hundred years.
Learnings From History and Canadian viagra online
Men’s sexual health has a fascinating history. Most of us have limited information about the variety and richness of sexuality in history and in cultures other than our own. We have a myopic (near-sighted) view about the powerful energy of sex in culture. In a macroview of history, human sexuality has experienced its own maturing process from a biological to a biopsychosocial understanding. People struggled for biological understanding sexual impulses, penis, erection, vagina, intercourse for centuries. Spiritual, theological, and religious perspectives and superstitions brought meaning in the absence of verifiable observations of science and medicine. Scientific objective, observable, empirical, tangible, and physical knowledge gradually emerged to help validate, integrate, and offer meaning to the realities of sex.
Therapeutic goals of t treatment: part 2
So, the goals of T therapy in adults with male hypogonadism are to:
- restore libido and improve erectile function;
- stimulate male hair growth;
- increase muscle mass and strength;
- increase BMD, potentially reducing the risk of fractures;
- improve energy, mood, and motivation;
- increase hematocrit into the normal adult male range.
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In most men with ED, T treatment alone is insufficient to restore complete erectile function and permit satisfactory intercourse. Because spermatogenesis requires high local T concentrations that cannot be achieved by exogenous androgen administration, T replacement therapy does not stimulate sperm production and testis size, nor does it restore fertility. Treatment of infertility in hypogonadal men is usually only possible in men with secondary hypogonadism and gonadotropin deficiency, using gonadotropin or gonadotropin-releasing hormone (GnRH) therapy.
The normal “physiological” range of serum T concentrations in adults is broad and usually established in healthy young men. In young hypogonadal men, T treatment produces beneficial clinical effects when serum T concentrations are increased into this normal range. With increasing age, serum T levels decline gradually and progressively, but the physiological significance of this decline is not clear. Initial studies in older hypogonadal men have also demonstrated some beneficial effects of T treatment that increase serum T levels into the normal range. Therefore, the goal of T treatment of male hypogonadism, irrespective of age, is to restore serum T concentrations to within the normal adult range.
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An important consideration in the clinical use of T therapy is the dose–response effect of T on different target organs. Recent studies suggest that some actions of T demonstrate continuous dose-response effects as T levels are increased from below normal to within and above the physiological range—e.g., muscle mass. In contrast, other T actions exhibit threshold effects—e.g., libido—that are stimulated near maximal levels at relatively low T concentrations. In some patients—e.g., elderly men with severe prostate disease—low-dose T supplementation may be more prudent than full T replacement. Although not demonstrated in clinical trials, low doses of T may be sufficient to induce some beneficial effects such as the stimulation of libido and, to a limited extent, anabolic actions on muscle and bone, while minimizing adverse stimulatory effects on prostate growth.
Therapeutic goals of t treatment
The therapeutic goals of T replacement therapy in male hypogonadism are to improve the clinical manifestations of androgen deficiency that vary with the stage of sexual development of the individual. Therefore, the specific goals of T treatment vary depending on whether the hypogonadal condition occurs in prepubertal boys or adults.
Males with prepubertal T deficiency usually present as adolescents or young adults with delayed puberty, manifesting varying degrees of eunuchoidism characterized by a small penis, a poorly developed scrotum, small testes (< 5 mL), and prostate, lack of male hair growth (facial, chest, axillary, pubic, perianal, and extremity hair), body habitus characterized by long arms and legs relative to height, poorly developed muscle mass, prepubertal fat distribution; reduced bone mass, high-pitched voice, poor libido (sexual interest and desire) and sexual function, reduced energy, mood alterations and lack of motivation, benign breast enlargement (gynecomastia), failure to produce an ejaculate (aspermia) and initiate spermatogenesis (azoospermia), and a relatively low hematocrit (in the female range).
Therefore, in boys with delayed puberty, the therapeutic goals of T treatment are to promote:
- the development of secondary sexual characteristics, including growth of the penis, the scrotum, and a male hair pattern;
- stimulate the acquisition of peak bone mass, long bone growth, and eventual closure of epiphyses [through the
- aromatization of T to estradiol (E2)] without compromising adult height;
- increase muscle mass and strength and reduce fat mass;
- induce laryngeal enlargement and deepening of the voice;
- stimulate libido and erections;
- improve energy, mood, and motivation;
- increase red-blood-cell production into the normal adult male range.
By stimulating accessory sex glands (seminal vesicles and prostate), T treatment stimulates seminal fluid production and an increase in ejaculate volume, but it does not stimulate sperm production sufficient for induction of fertility. Because the most common cause of delayed puberty is not pathological but rather constitutional, T therapy in boys with delayed puberty is usually intermittent and continued only until spontaneous puberty occurs.
Unless severe, T deficiency in adult males is usually more difficult to diagnose because the clinical manifestations are often subtle and attributable to other causes. T-deficient men usually present with poor sexual performance manifested by reduced libido and erectile dysfunction – viagra in canada (ED) as their major complaints, although T deficiency is not the primary etiology in the majority of men with ED.
- They may also manifest gynecomastia;
- infertility due to impaired sperm production;
- diminished chest, axillary, and pubic hair;
- decreased muscle bulk and strength;
- low BMD that may result in osteopenia or osteoporosis;
- low energy and motivation;
- irritability and a depressed mood;
- a mild hypoproliferative anemia in the normal female range – female viagra australian.
Testis size is usually normal but may be small (< 15 mL) in men with profound reductions in sperm production. Hot flushes may occur in men with a rapid onset of severe androgen deficiency.
V3 loop peptides from HIV-1 strain MN have different polyanion binding specificities than the DBP subdomain 1 HBM peptide
The heparin affinities of hbs-wt peptide, the cyclic V3 loop peptide of HIV-1 strain MN, and RANTES were the same when tested in heparin-Sepharose columns, and X4 strains of HIV, PvRII and hbs-wt can be inhibited by the same polyanions, but not chondroitin sulfate. To see if there are differences in the specificity by which the V3 peptides and the hbs-wt peptide bind to heparin, an ELISA based on RANTES binding to BSA-heparin was developed much like the hbs-wt ELISA. The only difference to the hbs-wt ELISA is that RANTES is substituted for the hbs peptide, and detection is through a biotinylated mouse anti-RANTES monoclonal antibody followed by horseradish peroxidase-conjugated streptavidin. The V3 loop peptides and hbs peptides were added at different concentrations to compete with RANTES at a fixed 5 nM. The linear and cyclic V3 loop peptides of strain MN that had significant binding on heparin-Sepharose were able to compete with RANTES binding to BSA-heparin in a dose-dependent manner, but the hbs-wt peptide was not.
The subdomain 1 HBM has a conserved role in the DBP protein family for binding to diverse receptors, but only members of the family that bind to DARC are inhibited by polyanions
Studies by Ranjan and Chitnis have identified a site in PvRII in the C-terminal flanking region to the DBP V3-like peptide, between C4 and C7, that contain residues necessary for DARC binding [27]. This study also showed that the C1-C4 region of the P. knowlesi beta protein, a member of the DBP family that does not bind to DARC, was capable of substituting for the P. vivax C1-C4. Upon closer inspection, the consensus heparin binding motif is well conserved in the DBP family, with great similarity between proteins that bind different receptors. The P. knowlesi alpha and gamma proteins have an identical consensus heparin binding site, but only alpha binds to DARC. To see if the consensus heparin binding motif may play a similar role in the binding proteins of other members of the DBP family, the same three alanine substitutions found in pv22KARA were introduced in our previous report by site directed mutagenesis into the plasmids pHKADR22, pHKBDR22, and pHKGDR22. This yielded the constructs pkalphaKARA, pkbetaKARA, and pkgammaKARA, which contain the K221, R224, and R227 alanine substitution in the P. knowlesi alpha, beta, and gamma genes, respectively. All three of these mutants failed to bind rhesus erythrocytes when expressed in COS-7 cells, whereas the parent vectors bind very well. When binding of rhesus erythrocytes to the wild type plasmids in COS-7 cells was measured in the presence of polyanions, only the DARC binding alpha protein was inhibited in a dose-dependent manner.
Heparitinase digestion. Part 2
The hbs-wt, hbs-kara, an assortment of V3 loop peptides, and RANTES were bound to a heparin-Sepharose column and eluted with 0.01, 0.15, 0.5, 1.0 or 2.0 M NaCl. The NaCl concentration required to elute the peptides provides a relative value for the affinity between the peptide and heparin, and is directly proportional to the Kd value. The 0.15 M NaCl concentration is physiologically relevant but reflects weak binding, 0.5 M indicates moderate binding, and 1.0 M and above represents strong binding (reviewed in chapter 6 of “Heparin-Binding Proteins”. Most of the peptides could be detected in the fractions by their absorbance of light at 280 nm on a spectrophotometer, with the exception of the linear V3 loop peptide of strain IIIB that lacks aromatic side chains. The Pierce BCA Protein Assay was used to detect this peptide.
The hbs-wt peptide eluted at 0.5 M NaCl (Table 1). The hbs-kara peptide did not bind to the column at all, eluting with the wash buffer. In a subsequent experiment, without the wash step, it eluted with 0.01 M NaCl. The cyclic V3 loop peptide of X4 HIV strain MN also eluted at 0.5 M as did the linear peptide of X4 strain IIIB. Of the linear peptides that are overlapping subunits of the MN cyclic peptide, the peptide containing the consensus heparin binding motif eluted at 1.0 M. This is a stronger interaction than the cyclic full-length V3 peptide. Other V3 peptides based on consensus sequences of subtypes B and EA, or specific strains RF and SF2, eluted at 0.15 M. RANTES eluted at 0.5 M. There was no direct relationship between net charge of the peptides and affinity to heparin, as the cyclic MN peptide with a charge of +7 eluted at a lower NaCl concentration than the linear peptide at a charge of +6. The most neutral net charge in the tested group of peptides was 0 for the hbs-wt, and it eluted at 0.5 M NaCl.
An ELISA based on coating plates with BSA-heparin and determining the binding of the hbs-wt peptide, or hbs-kara as a control, was developed. This assay showed high sensitivity for detecting bound hbs-wt peptide with low background as demonstrated by low signal produced by the hbs-kara control (data not shown). The binding was inhibited at the same NaCl concentrations that eluted the hbs-wt peptide in the heparin-Sepharose column at 0.5 M and above (data not shown). When sulfated polysaccharides were added to the ELISA, they inhibited the binding of the hbs-wt peptide in a dose-dependent manner. The same sulfated polysaccharides found to be inhibitory in the erythrocyte invasion assay and the PvRII region binding assay where inhibitory in the ELISA, with no inhibition from chondroitin sulfate C.
Heparitinase digestion
Red blood cells were washed 3 times in PBS and resuspended at a hematocrit of 10%. For the PvRII erythrocyte binding assay, 1 ml of 10% hct blood was used. To each 1 ml of red blood cells, 0, 0.001, 0.002 or 0.01 International Units (which correspond to 0.6, 1.2 and 6 Sigma Units, respectively) of Heparitinase (EC 4.2.2.8, Seikagaku America, Falmouth, MA) was added. The cells were incubated at 43°C for 90 min. with intermittent agitation. An aliquot of 30 μl of the cells was taken for analysis by flow cytometry (data not shown). The remaining cells were then centrifuged at 3000 RPM for 5 min., and resuspended in 1 ml of complete for use in the PvRII erythrocyte binding assay.
Region II of the P. Vivax DBP is blocked from binding to DARC by the same polyanions that inhibit X4 HIV strains
Within the DBP V3-like peptide is a site that conforms to the consensus heparin binding sequences BBXB and BBBXXB, where B represents a basic amino acid and X represents any amino acid including basic amino acids. Some strains of HIV, such as MN, contain a consensus heparin binding motif in the V3 loop, and many X4 strains can be inhibited from infecting target cells by polyanions which may bind to the V3 loop. Polyanions that have been shown to inhibit HIV infection include pentosan polysulfate, heparin, and the algal-derived sulfated polysaccharides Na-spirulan, Ca-spirulan and Na-hornan (Na-HOR). These polyanions also inhibited the binding of DARC+ erythrocytes to PvRII in a dose-dependent manner. They also block P. knowlesi invasion of DARC+ erythrocytes. Chondroitin sulfate C represents a polyanion with similar charge to heparin, but differs in the conformational placement of those charges. Chondroitin sulfate C does not block PvRII binding to DARC, or P. knowlesi invasion of DARC+ erythrocytes, suggesting that the interaction between PvRII and polyanions is related to conformation as well as charge. The same is true for inhibition of the V3 loop by polyanions.
A peptide based on the consensus heparin binding motif in the DBP V3-like peptide binds to heparin with the same affinity as V3 loop peptides of X4 HIV strains and recapitulates polyanion inhibition of PvRII binding to DARC
A peptide based on the consensus heparin binding motif in the DBP V3-like peptide was designed to test the affinity of this site for heparin and compare it to V3 loop peptides and RANTES. Based on results with an alanine substitution mutant of the consensus heparin binding motif in the DBP V3-like peptide, two peptides were designed; one contains the wild type heparin binding site (hbs-wt), and the other contains the same alanine substitutions as the pv22KARA construct (hbs-kara) found in our previous work to abrogate binding to DARC [16]. The peptides are FITC conjugated at the N-terminus and terminate at the carboxyl end with the DYKDDDDK “flag epitope” sequence for fluorescence or antigenic detection, respectively. They are identical with the exception of the alanine substitutions.
PvRII erythrocyte binding assay
COS-7 cells were transfected by Lipofectamine with 1-2 μg of pHVDR22 DNA, a plasmid kindly provided by L. Miller which expresses region II of the DBP of P. vivax on the cell surface as a chimera with the HSV gD protein. Duffy Fy (a-b+) erythrocytes were washed three times in RPMI 1640, resuspended to a hematocrit of 1% in 1 ml of complete DMEM with polyanions at concentrations of 0, 1, 10, 100, and 1000 μg/ml. This suspension was swirled over aspirated COS-7 cells 40-60 h after transfection and allowed to settle over 2 h at 37°C. The COS-7 cells were then washed three times with 2 ml of PBS to remove nonadherent erythrocytes. The number of adherent erythrocyte rosettes was scored in 20 randomly chosen fields at a magnification of 40 using an inverted microscope. Percentage inhibition of binding was determined by dividing the number of rosettes at each polyanion concentration by the percentage of rosettes at 0 μg/ml of the polyanion, multiplying by 100 and subtracting this value from 100.
PvDBP peptide BSA-heparin ELISA
Polyvinyl chloride 96-well microtiter plates were coated with 5 μg/ml heparin-albumin (Sigma) in a volume of 100 μl per well in 50 mM Tris-HCl pH 7.5 wash buffer overnight at room temperature. Plates were washed three times with wash buffer and blocked for 2 h at room temperature with 1% BSA in wash buffer at 400 ul per well. For polyanion blocking experiments, WT or mutant DBP polyanion binding site peptides were diluted to 10 μg/ml in a final concentration of polyanions at 0, 0.1, 1, 10, 100, or 1,000 μg/ml in wash buffer and added at 100 μl per well for 2 h. For controls, the DBP peptides were added at 10 μg/ml in 0.01, 0.15, 0.5, 1.0, and 2.0 M NaCl 50 mM Tris HCl pH 7.5 (data not shown). Plates were washed three times with wash buffer. Chicken anti-DYKDDDDK epitope antibody (Aves Labs, OR) at 1:5000 in 1% BSA wash buffer was added at 100 μl per well for 1 h at room temperature. Rabbit anti-chicken horseradish peroxidase antibody (Jackson ImmunoResearch) was added at 1:5000 in 1% BSA wash buffer at 100 μl per well for 1 h at room temperature. Plates were washed three times with wash buffer. The reaction was developed with 100 μl per well of 2% 3,3′,5,5′-tetramethylbenzidine 0.1 M NaOAc containing 0.001% hydrogen peroxide for about 5 min. The reaction was stopped with 100 μl per well of 1 M phosphoric acid. Absorbance measurements were made at 450 nm on a Biotek 133 microtiter plate reader. Percentage inhibition of binding was determined by dividing the absorbance at each polyanion concentration by the absorbance at 0 μg/ml of the polyanion, multiplying by 100 and subtracting this value from 100.
RANTES BSA-heparin ELISA
The same ELISA format used for the PvDBP Peptide-BSA-heparin ELISA described above was used for a competitive ELISA to detect RANTES binding to heparin, and competitors to this binding. Wells were coated with BSA-heparin, blocked with BSA and washed. A volume of 100 μl of 5 nM RANTES in wash buffer supplemented with 0, 0.5, 5, 50, 500 or 5000 nM peptide was added to triplicate wells. After incubation for 1.5 h at room temperature, the plates were washed in wash buffer and 100 μl of biotinylated anti-RANTES monoclonal antibody (R&D Systems) was added at 1:500 in 0.1% BSA wash buffer. After 1 h at room temperature, the plates were washed and 100 μl of streptavidin-horseradish peroxidase (Jackson ImmunoResearch) was added at 1:2000 in 0.1% BSA wash buffer. After 1 h at room temperature, the plates were washed. The reaction was developed as above. Percentage inhibition of binding was determined by dividing the absorbance at each peptide concentration by the absorbance at 0 nM of the peptide, multiplying by 100 and subtracting this value from 100.