Two-photon imaging of cancer cell extravasation in live mice

Abstract MDA-MB-231 breast cancer cells were engineered to express cytoplasmic paxillin-GFP and nuclear H2B-mCherry. In order to image extravasation, the cancer cells were injected in the blood stream of nude mice. Using 2-photon excitation microscopy we can simultaneously excite the two probes and also visualize the autofluorescence of tissues. A skin flap was opened to visualize blood vessels and recognize the position of the cancer cells. Two-photon imaging showed that after an initial phase in which the cells are non-adherent, some cells spread on the internal surface of the capillaries. Days later some cells started to appear on the external side of the capillary. The extravasated cells extend very long protrusions into the tissue. The goal was to determine if at the end of the long protrusion, if it is possible to observe the formation of focal adhesions by imaging paxillin-GFP. Preliminary results show that when cells start to adhere to the blood vessel wall they form focal adhesions as determined by the characteristic elongated features observed in the paxillin-GFP channel. New approaches will allow the tracking of the tip of the protrusion to determine if focal adhesions are forming there as the cells extravasate. This is important in establishing the mechanism of cell extravasation and migration in tissues. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1412. doi:10.1158/1538-7445.AM2011-141

Real-time imaging of 3-dimensional cancer cell movement in tissues

Abstract Our knowledge of how cells move in 3D in tissues is limited due to the lack of imaging methods that can produce 3D images fast enough and with sufficient resolution. Cancer cells migrate in 3D by forming adhesion points at the end of very long cellular protrusions. These protrusions are very thin and it is difficult to visualize adhesions along the protrusion surface. Conventional 3D stack reconstruction has relatively low resolution unless it is done using many frames. This results in a very slow acquisition in 3D confocal microscopy. Faster methods of 3D data acquisition (spinning disk microscopy) cannot be easily implemented since there is significant amount of scatter in tissues. A major obstacle in imaging adhesions is to find and track them so that they will not go out of focus. We are developing a new method which is based on orbiting imaging around cellular protrusions to visualize protein dynamics during extravasation. A feedback mechanism controls the center of the orbit to be at the center of the fluorescence distribution. A program reconstructs the shape of the protrusions in 3D. The fluorescence intensity in one or more channels is also simultaneously measured. The fluorescence intensity of one channel is used to paint the protrusion shape, which results in the 3D reconstruction of the protrusion. During the orbit, the second channel of the microscope measures the second harmonic generation (SHG) signal. We then correlated the appearance of bright fluorescence spots on the protrusion surface with the points of contact of the protrusion. This method will enable imaging of cancer cell invasion in 3-dimentions in live mice in real time. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4750. doi:10.1158/1538-7445.AM2011-475

Raster-image-correlation spectroscopy of paxillin-GFP-expressing breast cancer cell in vitro and in vivo

Abstract Raster-image-correlation spectroscopy (RICS) is a noninvasive technique to detect and quantify events in the living cell, including concentrations of molecules and their diffusion coefficients. Any cell containing a fluorophore that can be imaged with a laser scanning microscope can be analyzed with RICS. We obtained RICS images with an Olympus FluoView FV1000 confocal microscope using Olympus FluoView software to acquire data and SimFCS software to perform RICS analysis. Paxillin is involved in the assembly of focal adhesions, which was linked to green fluorescent protein (GFP) for the current study. In this study, we describe RICS of paxillin-GFP expression in breast cancer cells (MDA-MB-231) in vitro and in vivo. Slow-moving membrane-bound paxillin proteins were measured in live breast cancer cells in vitro. Paxillin-GFP-expressing breast cancer cells (1×106) were injected in the epigastric cranials vein of the nude mouse. Paxillin-GFP-expressing breast cancer cells became attached to the inner vessel wall within 3 hours after injection. Rapidly-moving cytosolic paxillin-GFP molecules were imaged with RICS. With the ability to measure the molecular dynamics of paxillin in cancer cells in vitro and in vivo by RICS, we are now capable of studying the role of both slow-moving paxillin in the cell membrane and rapidly-moving cytosolic paxillin in cancer-cell behavior. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5183. doi:1538-7445.AM2012-518

Pregnant and Postpartum Women’s Experiences and Perspectives on the Acceptability and Feasibility of Copackaged Medicine for Antenatal Care and PMTCT in Lesotho

Objective. To improve PMTCT and antenatal care-related service delivery, a pack with centrally prepackaged medicine was rolled out to all pregnant women in Lesotho in 2011. This study assessed acceptability and feasibility of this copackaging mechanism for drug delivery among pregnant and postpartum women. Methods. Acceptability and feasibility were assessed in a mixed method, cross-sectional study through structured interviews (SI) and semistructured interviews (SSI) conducted in 2012 and 2013. Results. 290 HIV-negative women and 437 HIV-positive women (n=727) participated. Nearly all SI participants found prepackaged medicines acceptable, though modifications such as size reduction of the pack were suggested. Positive experiences included that the pack helped women take pills as instructed and contents promoted healthy pregnancies. Negative experiences included inadvertent pregnancy disclosure and discomfort carrying the pack in communities. Implementation was also feasible; 85.2% of SI participants reported adequate counseling time, though 37.8% felt pack use caused clinic delays. SSI participants reported improvement in service quality following pack introduction, due to more comprehensive counseling. Conclusions. A prepackaged drug delivery mechanism for ANC/PMTCT medicines was acceptable and feasible. Findings support continued use of this approach in Lesotho with improved design modifications to reflect the current PMTCT program of lifelong treatment for all HIV-positive pregnant women

Detectable Viral Load in Late Pregnancy among Women in the Rwanda Option B+ PMTCT Program: Enrollment Results from the Kabeho Study.

There are limited viral load (VL) data available from programs implementing Option B+, lifelong antiretroviral treatment (ART) to all HIV-positive pregnant and postpartum women, in resource-limited settings. Extent of viral suppression from a prevention of mother-to-child transmission of HIV program in Rwanda was assessed among women enrolled in the Kigali Antiretroviral and Breastfeeding Assessment for the Elimination of HIV (Kabeho) Study. ARV drug resistance testing was conducted on women with VL\u3e2000 copies/ml. In April 2013-January 2014, 608 pregnant or early postpartum HIV-positive women were enrolled in 14 facilities. Factors associated with detectable enrollment VL (\u3e20 copies/ml) were examined using generalized estimating equations. The most common antiretroviral regimen (56.7%, 344/607) was tenofovir/lamivudine/efavirenz. Median ART duration was 13.5 months (IQR 3.0-48.8); 76.1% of women were on ART at first antenatal visit. Half of women (315/603) had undetectable RNA-PCR VL and 84.6% (510) had36 months compared to those on ART 4-36 months (72/191, 37.7% versus 56/187, 29.9%), though the difference was not significant. The odds of having detectable enrollment VL decreased significantly as duration on ART at enrollment increased (AOR = 0.99, 95% CI: 0.9857, 0.9998, p = 0.043). There was a higher likelihood of detectable VL for women with lower gravidity (AOR = 0.90, 95% CI: 0.84, 0.97, p = 0.0039), no education (AOR = 2.25, (95% CI: 1.37, 3.70, p = 0.0004), nondisclosure to partner (AOR = 1.97, 95% CI: 1.21, 3.21, p = 0.0063) and side effects (AOR = 2.63, 95% CI: 1.72, 4.03, p36 months with genotyping available. Most women were receiving ART at first antenatal visit, with relatively high viral suppression rates. Shorter ART duration was associated with higher VL, with a concerning increasing trend for higher viremia and drug resistance among women on ART for \u3e3 years

Assessing the feasibility, acceptability, and costs of diagnosing HIV at birth in Lesotho and Rwanda

Infant HIV diagnosis as early as possible in a child’s life followed by immediate antiretroviral treatment (ART) could stem the progressive rise in infant mortality among HIV-positive infants, particularly as several studies have suggested that an increased proportion of perinatal infections may occur in utero when maternal ART is received during pregnancy. This Project SOAR study leveraged two existing cohort studies to address some critical questions related to very early infant diagnosis. The objective was to determine the feasibility, acceptability, and costs associated with the addition of birth HIV testing to the routine testing algorithm for infants born to HIV-positive women. Findings will contribute to the global guidance on whether current early infant diagnosis guidelines should be reconsidered to add birth HIV testing as standard of care

Effectiveness of an integrated multilevel early child development intervention on caregiver knowledge and behavior: a quasi-experimental evaluation of the Malezi program in Tanzania

Background The quality of caregiving and the parent-child relationship is critical for early child development (ECD) and has been shown to be modifiable. This study evaluated an ECD project in Tanzania, assessing the effectiveness of radio messaging (RM) alone and a combined radio messaging/video job aids/ECD (RMV-ECD) intervention. Methods This two-arm pre-post evaluation study enrolled a cohort of caregivers of children 0–24 months in four districts of Tabora region, following them for 9 months. ECD radio messages were broadcast on popular stations at least 10 times/day reaching all study districts. In two districts, community health workers (CHW), trained in UNICEF’s Care for Child Development package, used ECD videos in home- and facility-based sessions with caregivers. We used McNemar’s testing (pre-post pairs) within intervention group to describe how the intervention was associated with change in five outcomes: ECD knowledge, early stimulation, father engagement, responsive care, and environment safety. Logistic regression was used to describe the relative benefits of the combined intervention package (RMV-ECD) compared to radio messaging (RM). Results In the RMV-ECD arm, all outcomes at endline except environment safety significantly improved after the intervention with the largest change seen in ECD knowledge (35.8% increase, p \u3c .0001) and the smallest in father engagement (6.7%, p = .015). In the RM arm, ECD knowledge (5.7%, p = .031) and environment safety (18.1%, p = \u3c.0001) improved. High measures of parenting stress were associated with lower likelihood of having good ECD knowledge (AOR 0.50, 95%CI: 0.35, 0.71), father engagement (AOR 0.72, 95%CI: 0.52, 0.99) and responsive care (AOR 0.31, 95%CI: 0.18, 0.54). Conclusions An intervention that includes mass media, educational video content and CHWs who counsel caregiv- ers in their homes and health facilities was associated with significant improvements in ECD parenting knowledge and behaviors but a relationship with responsive care could not be established. The less costly mass media-only intervention was associated with improved parenting knowledge and household environment safety. Parenting interventions targeting young children could be improved by incorporating more messaging and caregiver coaching in managing parental stress

Antibodies to C1q in systemic lupus erythematosus: Characteristics and relation to FcγRIIA alleles

Antibodies to C1q in systemic lupus erythematosus: Characteristics and relation to FcγRIIA alleles. Autoantibodies to the collagen-like region of the first complement component (C1qAB) are found in patients with systemic lupus erythematosus (SLE), particularly those with renal disease. In a cohort of 46 SLE patients with diffuse proliferative glomerulonephritis, we found declining C1qAB titers in 77% of treatment responders and in only 38% of treatment non-responders (P < 0.03). To further characterize this autoantibody, we tested 240 SLE patients for the presence of C1qAB. Positive titers were found in 44% of patients with renal disease and 18% of patients without renal disease (χ2 P < 0.0003). Analysis of IgG subclass revealed IgG2 C1qAB alone in 34%, IgG1 C1qAB alone in 20%, and both IgG1 and IgG2 in 46% of patients. Fewer than 10% of patients had measurable titers of IgG3 or IgG4 C1qAB. The pathogenic role of these IgG2-skewed C1qAB may relate to impaired immune complex clearance by the mononuclear phagocyte system: IgG2 antibodies are efficiently recognized by only one IgG receptor, the H131 allele of FcγRIIa (FcγRIIa-H131). In contrast, FcγRIIa-R131, which is characterized by minimal IgG2 binding, has recently been associated with lupus nephritis. In our C1qAB positive patients, the presence of FcγRIIA-R131 was associated with an increased risk for renal disease. Autoantibodies to C1q may have pathogenic significance in SLE patients with genetic defects in the ability to clear IgG2 containing immune complexes