Inhibition of intimal hyperplasia by the tetracycline derived mmp inhibitor doxycycline in vein graft disease in vitro and in vivo.

AIMS: Intimal hyperplasia, characterised by smooth muscle cell migration and proliferation, requires extracellular matrix degradation which is mediated by matrix metalloproteinases (MMPs). In this study, the effect of tetracycline derived doxycycline, a specific MMP inhibitor of both activity and synthesis, on intimal hyperplasia in vitro and in vivo was assessed. METHODS AND RESULTS: Segments of human saphenous veins were cultured for 4 weeks in absence or presence of doxycycline (10microg/ml) (n=6). A 81% inhibition in intimal hyperplasia was observed in the doxycycline treated segments compared to controls. To assess the effect of doxycycline on intimal hyperplasia in vivo, perivascular cuffs were placed around femoral arteries in mice with or without doxycycline in the drinking water (3 mg/ml). In this in vivo model for intimal hyperplasia doxycycline significantly reduced (68%, n=6) intimal hyperplasia when compared to controls. In addition the effect of doxycycline on vein graft thickening was assessed in a murine venous interposition model. In this in vivo model vein graft thickening was reduced by 35 % in the doxycycline treated mice (3 mg/ml in drinking water). Furthermore, a reduction in vascular MMP expression was observed in these mice. CONCLUSION: Treatment with tetracycline derived doxycycline results in significant inhibition of intimal hyperplasia in vitro and in vivo and may be an effective strategy to prevent post interventional restenosis and vein graft disease.

Plasminogen activators in multiple sclerosis lesions: implications for the inflammatory response and axonal damage.

Components of the plasminogen activator (PA) and matrix metalloprotease (MMP) cascade have been characterized in multiple sclerosis lesions by immunohistochemistry, enzyme-linked immunosorbent assay and enzyme activity assays in order to establish a functional role for the enzyme sequence in lesion development. Highly significant quantitative increases in urokinase PA (uPA), urokinase receptor (uPAR) and plasminogen activator inhibitor-1 were detected in acute multiple sclerosis lesions (P < 0.0001) and in uPAR in normal-appearing white matter (P < 0.0001) compared with control tissue. All three proteins were immunolocalized to mononuclear cells in perivascular cuffs and to macrophages in the lesion parenchyma. MMP-9 and the tissue inhibitor of metalloprotease-1 also increased during lesion development but the enzyme was present largely in the inactive pro-form. In contrast to uPA, the concentration and activity of tissue PA (tPA), the most abundant plasminogen activator in normal control brain, were reduced in multiple sclerosis specimens. In acute lesions tPA co-localized with fibrin(ogen) on large diameter axons also stained with SMI-32, an immunohistochemical marker of axonal damage. The uPA-uPAR complex, concentrated on inflammatory cells in the perivascular zone of the evolving lesion, may facilitate cellular infiltration into the CNS which is amplified by MMP- mediated degradation of blood vessel matrix. tPA localization on injured axons may be a marker of axonal damage or represent a protective mechanism aimed at removal of fibrin deposits and restoration of axonal function.

MMP-9 activity in urine from patients with various tumors, as measured by a novel MMP activity assay using modified urokinase as a substrate.

Matrix metalloproteinases (MMPs) play an important role in many pathologic processes, but their activities are difficult to determine since no simple specific and/or chromogenic substrate exists. We have developed a novel MMP activity assay using a modified urokinase as a substrate. Protein engineering enabled the plasmin activation site in this urokinase to be substituted by a specific activation site recognized by MMPs. In this way the MMP activity can be monitored via urokinase activity as measured by a simple chromogenic assay. The assay was made specific for MMP-9 by a capture step using MMP-9-specific antibodies that do not interfere with MMP-activity. This assay monitors the amount of active enzyme as well as the latent, but potentially active proform. Using this assay the levels of MMP-9 were investigated in urine from patients with various kinds of carcinoma. High levels of both active and latent MMP-9 were detected in urine from patients with carcinoma of the bladder, whereas hardly any activity was observed in urine from healthy controls. MMP-9 in urine was present in its intact form. Surprisingly, MMP-9 was also increased in the urine of patients with nonurogenital carcinoma. Therefore, measurement of urinary MMP-9 activity levels may be a convenient diagnostic tool for various types of carcinoma.

Infectious cellular load in human immunodeficiency virus type 1 (HIV-1)-infected individuals and susceptibility of peripheral blood mononuclear cells from their exposed partners to non-syncytium-inducing HIV-1 as major determinants for HIV-1 transmission in homosexual couples.

To study risk factors for homosexual transmission of human immunodeficiency virus type 1 (HIV-1), we compared 10 monogamous homosexual couples between whom transmission of HIV-1 had occurred with 10 monogamous homosexual couples between whom HIV-1 transmission had not occurred despite high-risk sexual behavior. In the group of individuals who did not transmit virus, peripheral cellular infectious load was lower and the CD4+ T-cell counts were higher than in the group of transmitters. HIV-1 RNA levels in serum did not differ between transmitters and nontransmitters. Compared with peripheral blood mononuclear cells (PBMC) from normal healthy blood donors, 8 of 10 nonrecipients and only 3 of 8 recipients had PBMC with reduced susceptibility to in vitro infection with non-syncytium-inducing (NSI) HIV-1 variants isolated from either their respective partners or an unrelated individual. No difference in susceptibility was observed for infection with a syncytium-inducing variant. Among the individuals who had PBMC with reduced susceptibility, five nonrecipients and one recipient had PBMC that were equally or even less susceptible to NSI variants than PBMC that had low susceptibility and that were derived from healthy blood donors that were heterozygous for a 32-bp deletion in the CCR5 gene (CCR5 delta32). Three of these individuals (all nonrecipients) had a CCR5 delta32 heterozygous genotype themselves, confirming an association between low susceptibility to NSI variants and CCR5 delta32 heterozygosity. All three recipients with less susceptible PBMC had partners with a high infectious cellular load; inversely, both nonrecipients with normally susceptible PBMC had partners with a very low infectious cellular load. These results suggest that a combination of susceptibility of target cells and inoculum size upon homosexual exposure largely determines whether HIV-1 infection is established.

Diagnosis of Chlamydia pneumoniae infection in patients with chronic obstructive pulmonary disease by micro-immunofluorescence and ELISA.

The incidence of Chlamydia pneumoniae infection was determined in patients with chronic obstructive pulmonary diseases (COPD) by prospective serial serology. Chlamydia-specific IgG, IgM and IgA antibodies were detected with a recombinant DNA lipopolysaccharide (LPS) ELISA as well as with a micro-immunofluorescence (MIF) assay with C. pneumoniae elementary bodies. From 271 consecutive COPD patients who visited the outpatient clinic of the department of pulmonary diseases (211 males, 60 females, age range 34-88 years, mean age 66 SD 10 years), blood samples (n = 1058) were taken every 2-7 months; the observation period ranged from 3 to 19 months (mean 15 SD 4). The prevalence of chlamydial IgG was 72% with the MIF and 53% with the rDNA LPS ELISA. More than 90% of the COPD patients had no significant changes in their chlamydia-specific IgG, IgA and IgM titres in either test during the observation period. Seven (3%) patients had MIF results indicating acute C. pneumoniae infection during their surveillance period, of whom five were confirmed by rDNA LPS ELISA. Eleven (4%) additional patients were infected during observation, as determined by rDNA LPS ELISA only. These patients had significantly elevated C. pneumoniae-specific IgG and IgA MIF titres, as compared with the patients without infection. All 18 patients with serological evidence of acute infection during their surveillance period were re-tested in a commercial MIF test that can distinguish between C. pneumoniae, C. trachomatis and C. psittaci LPS-specific antibodies, but no evidence of C. trachomatis or C. psittaci infection was found. The incidence of chlamydial infection was 2.2 and 5.3/100 person-years, when diagnosed by MIF and rDNA LPS ELISA, respectively. It is concluded that the rDNA LPS chlamydia assay may currently be the most sensitive serological tool for diagnosing recent respiratory chlamydia infections and that C. pneumoniae infection occurs frequently in COPD patients.

Macrophage-tropic variants initiate human immunodeficiency virus type 1 infection after sexual, parenteral, and vertical transmission.

Macrophage-tropic, non-syncytium-inducing, HIV-1 variants predominate in the asymptomatic phase of infection and may be responsible for establishing infection in an individual exposed to the mixture of HIV-1 variants. Here, genotypical and phenotypical characteristics of virus populations, present in sexual, parenteral, or vertical donor-recipient pairs, were studied. Sequence analysis of the V3 domain confirmed the presence of a homogeneous virus population in recently infected individuals. Biological HIV-1 clones were further characterized for syncytium inducing capacity on the MT2 cell line and for macrophage tropism as defined by the appearance of proviral DNA upon inoculation of monocyte-derived macrophages. Both sexual and parenteral transmission cases revealed a selective outgrowth in the recipient of the most macrophage-tropic variant(s) present in the donor. In three out of five vertical transmission cases, more than one highly macrophage-tropic virus variant was present in the child shortly after birth, suggestive of transmission of multiple variants. In three primary infection cases, homogeneous virus populations of macrophage-tropic, non-syncytium-inducing variants were present prior to seroconversion, thus excluding humoral immunity as the selective pressure in favour of macrophage-tropic variants. These observations may have important implications for vaccine development.

Epidemiology of human immunodeficiency virus type 1 infection among homosexual men participating in hepatitis B vaccine trials in Amsterdam, New York City, and San Francisco, 1978-1990.

Homosexual/bisexual men from Amsterdam, The Netherlands, New York, New York, and San Francisco, California, were entered into trials of the efficacy of hepatitis B vaccine shortly before the acquired immunodeficiency syndrome (AIDS) epidemic was recognized (1978-1980). The authors analyzed data, including serial blood samples tested for antibody to human immunodeficiency virus type 1 (HIV-1) as well as demographic and behavioral information, to characterize the spread of HIV-1 infection within the cohorts. By the end of 1982, the cumulative incidence of HIV-1 infection within the cohorts. By the end of 1982, the cumulative incidence of HIV-1 infection was 7.5% in Amsterdam, 26.8% in New York City, and 42.6% in San Francisco. Covariate analysis showed that differences in sexual activity (number of male sexual partners) and correlates of sexual activity (age and hepatitis B incidence) accounted for the differences in incidence of HIV-1 infection between the New York City and San Francisco cohorts. These covariates did not explain the lower incidence in the Amsterdam cohort. In conclusion, significant differences were found in the spread of HIV-1 in cohorts of homosexual men in Amsterdam, New York City, and San Francisco. These dissimilarities were probably due to a combination of differences in sexual activity at the time the epidemic began and a later introduction of HIV-1 in Amsterdam.

Orogenital sex and the transmission of HIV among homosexual men.

OBJECTIVE: To investigate the possibility of orogenital transmission of HIV. DESIGN: Cohort study on HIV infection among homosexual men. SETTING: The Municipal Health Service, Amsterdam, The Netherlands. PATIENTS, PARTICIPANTS: Homosexual men for whom the date of HIV seroconversion was known (n = 102) were included in our study. MAIN OUTCOME MEASURES: Data on the sexual behaviour of our subjects in the 6-9 months preceding HIV seroconversion were collected. In order to identify those men who consistently denied practising receptive anogenital intercourse, information was retrieved from written questionnaires and from face-to-face interviews. RESULTS: Receptive anogenital intercourse in the 6-9 months before seroconversion was denied by 20 seroconverters in their written questionnaires. However, in face-to-face interviews, 11 men later reported this sexual practice. CONCLUSIONS: Orogenital transmission of HIV appears to occur, but its frequency may be overestimated because of reluctance to report the practice of receptive anogenital intercourse.

Spread of human T-cell leukemia virus (HTLV-I) in the Dutch homosexual community.

Sequential sera of 697 homosexual men, participating in a prospective study (1984-1986) of the risk to acquire human immunodeficiency virus (HIV) or AIDS, were tested for antibodies to human T-cell leukaemia virus (HTLV-I) by particle agglutination and immunoblotting. No intravenous drug users were included in this trial. Three men (0.4%) were HTLV-I antibody positive at intake and an additional 2 at the end of the observation period, resulting in an attack rate of approximately 0.3%. One of the 3 men with HTLV-I antibodies at intake was a Brazilian. One man had an acute HTLV-I infection after sexual intercourse with a Brazilian during holiday in Brazil. No serological cross-reactivity with HIV was observed nor a relationship with other sexually transmissible viral or bacterial infections. In contrast to HIV no relationship with anogenital intercourse was noted; both primary HTLV-I infected men practiced only orogenital intercourse. This suggests that HTLV-I was imported in the Dutch homosexual community after HIV was introduced in the Netherlands. HTLV-I appears to spread slower within the homosexual community than HIV and possibly by other routes.

Hepatitis B among homosexual men in The Netherlands.

In 1980-1981 a study for the presence of markers of hepatitis B virus was done among 2, 946 homosexual men living in The Netherlands, mainly in Amsterdam. Serologic evidence of a past or present hepatitis B infection was found in 60.3% of this group, and the prevalence differed significantly among the different age groups. Of the total population studied, 4.8% were positive for HBsAg. This prevalence is very high in comparison to that in the general Dutch population, among whom the prevalence of hepatitis B is low, as is shown by the fact that the prevalence of HBsAg was 0.22% among a large group of Dutch first-time blood donors. Another blood sample was collected two to 20 months after the first sample from 316 male homosexuals who were initially negative for hepatitis B markers. It was found that 36 persons had seroconverted, most of them without clinical manifestations of hepatitis. The annual attack rate of hepatitis B in this group was calculated as 27.6% and differed significantly among the different age groups.