Bruce K. Patterson

Viral burden in genital secretions determines male-to-female sexual transmission of HIV-1: a probabilistic empiric model.

ObjectiveTo develop a model to predict transmission of HIV-1 from men to women. DesignHIV-1 in seminal plasma, and endocervical CCR5 receptors were correlated with epidemiological studies of HIV-1 transmission to develop a probabilistic model. SettingsSemen samples were collected from patient subjects in Seattle Washington, Chapel Hill, North Carolina, and St. Gallen, Switzerland. Endocervical biopsy specimens were obtained from women in Chicago, Illinois. ParticipantsEighty-six men (not receiving antireroviral therapy) in whom CD4 cell count and semen volume were available, and 24 women in whom the number of endocervical CCR5 receptors were determined. Main outcome measuresPrediction of transmission of HIV-1 from men to women per episode of vaginal intercourse based on the absolute burden of HIV (volume × HIV RNA copies/ml seminal plasma). ResultsThe model suggests efficient heterosexual transmission of HIV-1 when semen viral burden is high. When semen contains 100 000 copies of non-syncytium-inducing (NSI) HIV RNA the probability of HIV-1 transmission is 1 per 100 episodes of intercourse; conversely, with 1000 copies NSI HIV RNA in semen, transmission probability is 3 per 10 000 episodes of intercourse. ConclusionsThis model links biological and epidemiological data related to heterosexual HIV-1 transmission. The model can be used to estimate transmission of HIV from men with high semen viral burden from inflammation, or reduced burden after antiretroviral therapy. The results offer a biological explanation for the magnitude of the HIV epidemic in places where earlier studies have shown men have high semen viral burden, such as in sub-Saharan Africa. The model can be used to develop and test HIV-1 prevention strategies.

Repertoire of Chemokine Receptor Expression in the Female Genital Tract : Implications for Human Immunodeficiency Virus Transmission

Sexually transmitted diseases, genital ulcer disease, and progesterone therapy increase susceptibility to lentivirus transmission. Infection of cells by human immunodeficiency virus (HIV) is dependent on expression of specific chemokine receptors known to function as HIV co-receptors. Quantitative kinetic reverse transcription-polymerase chain reaction was developed to determine the in vivo expression levels of CCR5, CXCR4, CCR3, CCR2b, and the cytomegalovirus-encoded US28 in peripheral blood mononuclear cells and cervical biopsies from 12 women with and without sexually transmitted diseases, genital ulcer disease, and progesterone-predominant conditions. Our data indicate that CCR5 is the major HIV co-receptor expressed in the female genital tract, and CXCR4 is the predominantly expressed HIV co-receptor in peripheral blood. CCR5 mRNA expression in the ectocervix was 10-fold greater than CXCR4, 20-fold greater than CCR2b, and 100-fold greater than CCR3. In peripheral blood, CXCR4 expression was 1.5-fold greater than CCR5, 10-fold greater than CCR2b, and 15-fold greater than CCR3. US28 was not expressed in cervical tissue despite expression in peripheral blood mononuclear cells from five individuals. CCR5 was significantly increased (p < 0.02) in biopsies from women with sexually transmitted diseases and others who were progesterone predominant. In vitro studies demonstrate that progesterone increases CCR5, CXCR4, and CCR3 expression and decreases CCR2b expression in lymphocytes and monocytes/macrophages. Characterization of chemokine receptors at the tissue level provides important information in identifying host determinants of HIV-1 transmission.

Development of an in vitro organ culture model to study transmission of HIV-1 in the female genital tract

Development of an in vitro organ culture model to study transmission of HIV-1 in the female genital tract

In Situ Localization and Quantification of mRNA for 92-kD Type IV Collagenase and Its Inhibitor in Aneurysmal, Occlusive, and Normal Aorta

Ninety-two-kilodalton type IV collagenase (MMP-9) is present in aortic aneurysms and may be important to the pathogenesis of this disease. Alteration in expression of MMP-9 or its inhibitor, the tissue inhibitor of metalloproteinase type 1 (TIMP-1), could increase degradation of extracellular matrix and lead to aneurysm formation. The purpose of this study was (1) to measure tissue levels of MMP-9 and TIMP-1 mRNA in aneurysmal (AAA), atherosclerotic occlusive (AOD), and normal (NL) human infrarenal aorta; (2) to test for their expression by cultured AAA and NL vascular smooth muscle cells (VSMCs); and (3) to locate in situ the cells responsible for mRNA production within AAA, AOD, and NL aortic wall. Total RNA extracted from AAA (n = 8), AOD (n = 8), and NL (n = 7) tissue was subjected to Northern analysis. Signals for MMP-9 and TIMP-1 were normalized to alpha-tubulin. Mean values +/- SEM were compared by ANOVA. NL and AAA VSMCs were cultured, passaged, and grown to confluence before RNA extraction and Northern analysis. In situ hybridization with digoxigenin-labeled RNA probes localized cells responsible for MMP-9 and TIMP-1 mRNA expression within sections of AAA (n = 5), AOD (n = 2), and NL (n = 2) aorta. MMP-9 mRNA levels were significantly greater in AAA (0.855 +/- 0.180) than NL (0.046 +/- 0.23) (P < .02), but differences between AOD (0.406 +/- 0.196) and AAA or AOD and NL were not significant.(ABSTRACT TRUNCATED AT 250 WORDS)

Early reduction of immune activation in lymphoid tissue following highly active HIV therapy.

Objective:To evaluate immune reconstitution within HIV-infected lymphoid tissue during highly active antiretroviral therapy (HAART). Design and methods:In situ cellular responses were studied in sequential tonsillar biopsies in three asymptomatic HIV-infected (CD4 cells greater than 400 × 106/l) antiretroviral treatment-naive volunteers enrolled in a clinical trial to determine the early effect of HAART. Computerized image analysis was used to study immunohistochemically stained sequential tonsil sections for the patterns of local cytokine production, chemokine receptor expression and cellular distribution. Replicate quantitative assessments of samples before and after 4 weeks of therapy were used for the evaluation of drug effects and compared with four uninfected controls. Tonsillar HIV proviral-DNA was determined by fluorescent in situ 5′-nuclease assay. Results:HIV-infected tonsil tissue was characterized by extensive pro-inflammatory and type 1 cytokine expression. A five- to 15-fold elevation of interleukin (IL)-1α, IL-12, IL-2 and interferon (IFN)-γ protein expression was found compared with controls, and each encompassed a mean of at least 4.5% of the tissue compartment. This was reduced by 20–90% in all individuals after 4 weeks of HAART. In contrast, type 2 cytokine expression (IL-4, IL-10), plus tumour necrosis factor (TNF)-α, remained low throughout the study. HAART reduced, by 40%, the expression of HIV co-receptors, CCR5 and CXCR4, which initially were elevated four to six times over the control values. In addition, the myelomonocytic inflammatory proteins, CD68 and calprotectin, diminished by 26–83% after therapy. The HIV RNA was reduced to undetectable levels in plasma by HAART. However, a large pool of tonsil cells (2–7%), remained HIV DNA positive after 4 weeks of therapy. Conclusions:Although immune activation may be the direct consequence of HIV replication, HAART-associated reconstitution begins with a reduction in inflammatory cytokine production which precedes the elimination of local proviral reservoirs.

Human Immunodeficiency Virus Type 1 Shedding Pattern in Semen Correlates with the Compartmentalization of Viral Quasi Species between Blood and Semen

High levels of human immunodeficiency virus (HIV) type 1 have been detected in semen at all stages of disease. However, it is not clear whether HIV-1 is shed in semen continuously or intermittently. In a prospective longitudinal study, viral RNA was measured weekly for 10 weeks in semen and blood of HIV-seropositive subjects. Results showed three different patterns of HIV-1 shedding in semen: none (28%), continuous (28%), and intermittent (44%). In contrast, there was no change in blood plasma virus load during the study period. Phylogenetic analysis of the envelope sequences of HIV-1 RNA in semen and blood revealed distinct virus populations in semen and blood of intermittent shedders but similar virus populations in the semen and blood of continuous shedder. These results indicate for the first time that HIV-1 is shed primarily in an intermittent manner and that shedding patterns of HIV-1 in semen are related to compartmentalization of HIV-1 between semen and blood.

In situ localization and quantification of seventy-two – kilodalton type IV collagenase in aneurysmal, occlusive, and normal aorta

PURPOSE Seventy-two-kilodalton type IV collagenase (MMP-2), a potent collagenase and elastase, is present in inflammatory disease states and may be important in the pathogenesis of aortic aneurysms. Alteration in expression of MMP-2 or its inhibitor, the tissue inhibitor of metalloproteinases type two (TIMP-2), could increase extracellular matrix degradation and lead to aneurysm formation. The purpose of this study is (1) to measure relative tissue levels of MMP-2 and TIMP-2 mRNA in aneurysmal, occlusive, and normal human infrarenal aorta; (2) to test for expression by cultured aneurysmal and normal vascular smooth muscle cells (VSMCs); and (3) to identify, in situ, the cells responsible for mRNA production within aneurysmal, occlusive, and normal aortic wall. METHODS Total RNA extracted from aneurysmal (n = 8), occlusive (n = 9), and normal (n = 7) tissue was subjected to Northern analysis. Signals for MMP-2 and TIMP-2 were normalized to alpha-tubulin. Mean values +/- SE were compared by use of analysis of variance. Aneurysmal and normal VSMCs were cultured, passaged, and grown to confluence before RNA extraction and Northern analysis. In situ hybridization with digoxigenin RNA probes localized cells responsible for MMP-2 and TIMP-2 mRNA production in histologic sections of aneurysmal (n = 7), occlusive (n = 4), and normal (n = 3) aorta. RESULTS Tissue MMP-2 mRNA levels were significantly greater in aneurysmal aorta (1.032 +/- 0.164, n = 5) than in either occlusive (0.553 +/- 0.027, n = 4, p < 0.02) or normal aorta (0.230 +/- 0.038, n = 3, p < 0.002). Differences in TIMP-2 mRNA levels were not significant (aneurysmal aorta 0.207 +/- 0.042, n = 3; occlusive aorta 0.413 +/- 0.164, n = 3; normal aorta 0.260 +/- 0.079, n = 4; p = 0.34), although numbers were small. Cultured aneurysmal and normal VSMCs constitutively expressed both MMP-2 and TIMP-2. In situ studies colocalized tissue MMP-2 and TIMP-2 expression to VSMCs and macrophages surrounding inflammation in aneurysmal adventita, but to atherosclerotic plaque in occlusive aorta. CONCLUSIONS MMP-2 and TIMP-2 are expressed in aneurysmal, occlusive, and normal aorta. MMP-2 expression is significantly greater in aneurysmal than in either occlusive or normal aorta. Cultured aneurysmal VSMCs constitutively express both MMP-2 and TIMP-2. Differential patterns of expression seen in situ and elevated tissue MMP-2 mRNA levels in aneurysmal versus occlusive aorta suggest that MMP-2 may be responsible for localized plaque remodeling in occlusive disease and for diffuse adventitial collagen and elastin destruction in aneurysms.

Perforin is not co-expressed with granzyme A within cytotoxic granules in Cd8 T lymphocytes present in lymphoid tissue during chronic Hiv infection

BACKGROUND Residual HIV-1-infected cells are poorly eliminated from lymphoid tissue (LT) reservoirs by effector cytotoxic T lymphocytes (eCTL) despite antiretroviral therapy. Perforin and granzyme A (grA) constitute major effector molecules within eCTL granules that induce apoptosis and lysis of virally infected cells. OBJECTIVE Expression of perforin and grA was studied at the single cell level in LT and blood from 16 patients infected with HIV-1 (stage A1-C) who were not taking antiretroviral therapy. METHOD Immunohistochemical analysis by in situ imaging of cells from blood and LT. RESULTS Quantitative in situ imaging showed that perforin-expressing CD8 T cells comprised 0.3-1.5% of total cells within the LT from recent HIV-1 seroconverters, while grA was found in 2.1-7.2% of total cells. However, despite high-level grA upregulation (1.5-4.5% of total cells) compared with that in non-infected individuals (0.4-0.9%), perforin expression remained low (< 0.1% of total cells) (P < 0.02) in LT from patients with chronic HIV-1 infection (stage A2-C). This contrasted with findings in peripheral blood mononuclear cells (PBMC) from the same HIV-1 infected cohort where perforin was detected in 13-31% of all PBMC, which was 10- to 100-fold higher than in lymphoid tissue (P < 0.001); grA was found in 14-32% of total PBMC. Two-colour staining showed that granular expression of perforin and grA was restricted to CD8 T cells in over 90% of total cells in both LT and blood. CONCLUSIONS These findings indicate that cytotoxic perforin expression is impaired at local sites of HIV replication within lymphoid tissue. Since perforin is required together with grA for granule-mediated cytolysis, the low perforin expression in the LT may limit the ability of eCTL to eliminate HIV-1 infected cells in lymphoid tissue.

Reducing cellular autofluorescence in flow cytometry: An in situ method

Cellular autofluorescence affects the sensitivity of flow cytometric assays by interfering with detection of low level specific fluorescence. These detection limits increase with use of protocols, such as thermocycling and fluorescent in-situ hybridization (FISH), that can increase intrinsic cellular fluorescence to 5,000-20,000 fluorescein isothiocyanate (FITC) equivalents. In order to improve signal to noise ratios when using FITC labeled probes in these procedures, we employed a method using the polyanionic azo dye, trypan blue, to reduce intracellular autofluorescence. Dyes such as these are commonly used in immunofluorescent microscopy to reduce background fluorescence. By using this method, we realized an approximately 5-fold increase in signal to noise ratio (S/N) in the direct detection of RNA target probes using flow cytometry. Trypan blue aided in the resolution of dim surface antibodies, internal markers and probes, and functions to reduce background autofluorescence after thermocycling and hybridization. This technique is rapid and easily applicable for reducing intracellular autofluorescence, and can be used in single and dual color applications.

p16INK4A immunohistochemistry is superior to HPV in situ hybridization for the detection of high-risk HPV in atypical squamous metaplasia.

In situ hybridization (ISH) assays for high-risk human papillomavirus (HR-HPV) and immunohistochemical (IHC) assays for surrogate markers such as p16INK4A can be useful in detecting HR-HPV in cervical dysplasia, but the use of these markers in problematic cervical biopsies has not been well-established. We evaluated 3 chromogenic ISH assays (Ventana INFORM HPVII and HPVIII and DakoCytomation GenPoint) in conjunction with p16INK4A IHC and HPV polymerase chain reaction in a study set consisting of 12 low-grade squamous intraepithelial lesions, 16 high-grade squamous intraepithelial lesions, and 30 benign cervix samples. A test set of 28 cases of atypical squamous metaplasia were also evaluated withVentana HPVIII ISH and p16INK4A IHC. In the study set, the sensitivity of the DakoCytomation ISH assay (which detects HPV subtypes 16, 18, 31, 33, 35, 39, 45, 52, 56, 58, 59, and 68) was similar to the Ventana HPVII assay but less than that of the Ventana HPVIII ISH assay (both of which detect HPV subtypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 66) and less than p16INK4A IHC (55.6% vs. 53.6 vs. 69.2% vs. 82.1%). All HPV ISH assays exhibited 100% specificity. p16INK4A reactivity consisted of 2 patterns: focal strong and diffuse strong. Because focal strong p16INK4A reactivity was identified in benign squamous epithelium (6.7% cases) and dysplastic epithelium, it was considered an equivocal result and only diffuse strong reactivity was considered to be specific for the presence of HR-HPV. In the squamous intraepithelial lesions study set, the difference in sensitivity between Ventana HPVIII ISH and p16INK4A was not statistically significant. However, in the atypical squamous metaplasia test set cases, p16INK4A reactivity (focal strong and diffuse strong) was significantly more sensitive than Ventana HPVIII ISH in correlating with the presence of human papillomavirus as detected by polymerase chain reaction (83.3% vs. 33.3% P=0.004). Because focal strong p16INK4A reactivity is less specific, cases with this staining pattern are considered atypical and require further evaluation by other means. Overall, p16INK4A IHC is considered the best candidate for the initial assessment of cervical biopsies that are histologically indeterminate for dysplasia given its wide availability, comparative ease of interpretation, and high sensitivity and specificity.