Gregory J. Naus

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

Memory CD4+ T Cells Are the Earliest Detectable Human Immunodeficiency Virus Type 1 (HIV-1)-Infected Cells in the Female Genital Mucosal Tissue during HIV-1 Transmission in an Organ Culture System

ABSTRACT The virologic and cellular factors that are involved in transmission of human immunodeficiency virus type 1 (HIV-1) across the female genital tissue are poorly understood. We have recently developed a human cervical tissue-derived organ culture model to study heterosexual transmission of HIV-1 that mimics the in vivo situation. Using this model we investigated the role of phenotypic characteristics of HIV-1 and identified the cell types that are first infected during transmission. Our data indicate that the cell-free R5 HIV-1 was more efficiently transmitted than cell-free X4 HIV-1. Cell-free and cell-associated HIV-1 had comparable transmission efficiency regardless of whether the virus was of R5 or X4 type. We have demonstrated that memory CD4+ T cells and not Langerhans cells were the first HIV-1 RNA-positive cells detected at the epithelial-submucosal junction 6 h after virus exposure. Multicolor laser confocal microscopy demonstrated a globular distribution of HIV-1 gag-pol mRNA in the cytoplasm, and the distribution of CD4 and the CD45RO isoform was irregular on the cellular membrane. At 96 h postinoculation, in addition to memory CD4+ T cells, HIV-1 RNA-positive Langerhans cells and macrophages were also detected. The identification of CD4+ T cells in the tissue at 6 h was confirmed by flow cytometric simultaneous immunophenotyping and ultrasensitive fluorescence in situ hybridization assay on immune cells isolated from disaggregated tissue. Furthermore, PMPA {9-[2-(phosphonomethoxy)propyl] adenine}, an antiretroviral compound, and UC781, a microbicide, inhibited HIV-1 transmission across the mucosa, indicating the utility of the organ culture to screen topical microbicides for their ability to block sexual transmission of HIV-1.

A case‐matched molecular comparison of extraovarian versus primary ovarian adenocarcinoma

Extraovarian müllerian adenocarcinoma (EOM) resembles primary ovarian carcinoma (POC) both histologically and clinically, yet little is known regarding the molecular genetic characteristics of this entity. The objective of this study was to compare the expression of three molecular markers of tumor behavior in EOMs and POCs.

Prognostic value of flow cytophotometric DNA content analysis in single treatment stage IB-IIA squamous cell carcinoma of the cervix.

DNA content was measured flow cytometrically in archival tissue from 65 single-treatment stage IB and IIA squamous cell carcinomas of the cervix with at least 5 years of clinical follow-up. Thirty-five cases were treated exclusively by hysterectomy and thirty exclusively by radiation therapy. Tumors were categorized into four groups on the basis of DNA content and cell cycle distribution. DNA content was measured relative to the position of the first resolvable cell peak. G2/M and S-phase fractions were estimated as percentage of cells with DNA contents greater than or equal to relative position 1.70 and percentage of cells with relative positions between 1.20 and 1.70, respectively. The 40 tumors characterized as either aneuploid or nondemonstrably aneuploid with high S-phase fraction estimate had a 5-year recurrence rate significantly higher than that of the 25 tumors categorized as tetraploid or nondemonstrably aneuploid with low S-phase fraction estimate (52 and 4%, respectively; chi 2 = 15.8, P less than 0.001). Similar results were found when radiation and surgically treated tumors were considered independently (chi 2 = 7.95, P less than 0.005 and chi 2 = 5.7, P less than 0.025, respectively). These data suggest that an increased 5-year recurrence rate is associated with both abnormal DNA content and elevated S-phase fraction in stage IB-IIA squamous cell carcinoma of the cervix, and that this relationship is largely independent of treatment method.

Reply to ‘ In vitro models of mucosal HIV transmission’

To the editor—Collins et al. published in your April issue a report of an in vitro model showing transmission of human immunodeficiency virus (HIV) to cells of the female genital tract. Using a model in which cervical tissue was polarized in 3% agarose, Collins et al. reported that within 24 hours, up to 30% of cell-free virus added to the apical surface of the epithelium passed through the tissue. Given what is known about the ultrastructure and permeability of the cervical epithelium, such data are unexpected. The validity of epitheliumcontaining tissue culture model for transport studies hinges on the presence of tissue confluent in such a way that inoculating virus can gain access to tissue only through the epithelium and not through undetected pores or unsealed edges of the tissue. No convincing data were provided to show what controls were used to eliminate the possibility of such access. Pores, or gaps in the sealing process, however small, can provide access to the underlying stromal tissue and would allow virus to circumvent potential barrier effects of the epithelium. Collins et al. used blue dextran (molecular mass, 2 × 10 daltons) as a control to detect defects in sealing around edges of the polarised tissue. However, this does not rule out the possibility of the presence of pores or gaps that would allow passive transport of smaller molecules. Previous studies on tissue from nonhuman primates have demonstrated that cervical epithelium is impermeable to the small tracer molecules lanthanum nitrate (433 kDa in molecular mass) and horseradish peroxidase; this is also true of human cervical tissue (Fig. 1). Furthermore, intact cervical epithelium, mechanically sealed in an Ussing chamber, is impervious to the passive diffusion of cell-free or cell-associated HIV (ref. 3). Collins et al. provided no explanation for the rapid transport of free virus across cervical epithelium in their model. In agreement with extended studies, the possible involvement of epithelial infection or transcytosis in viral transmission was excluded. The effect reported by Collins required infectious virus, and inactivated HIV was not readily transmitted. The rapid kinetics of transmission of cell-free virus (maximal in the first 24 hours) indicate it is unlikely to depend on de novo infection. Furthermore, possible cross-linking of proteins after inactivation of HIV by ultraviolet irradiation and psoralen could reduce the amount of p24 released from treated virus. Two possible mechanisms for such rapid transmission of the virus remain. For the first mechanism, the virus is picked up by resident Langerhans and T cells within the epithelium, which subsequently migrate through the tissue and release virus from the basolateral surface. Although epithelial Langerhans and T cells within the epithelium may bind virus and rapidly migrate through tissue, such a mechanism is unlikely to account for transport of up to 30% of the input virus. This is confirmed by the authors’ observation that dendritic cell makers remained unchanged throughout the culture period. In the alternative mechanism, virus can cross the epithelium by passive diffusion. Passive diffusion of virus (80–100 nm in size) through the paracellular pathway would seem unlikely given the results of previous studies, unless integrity of the epithelium had been damaged before experimentation, or paracellular permeability had been artifactually increased. This could occur during surgical removal of or obtaining punch biopsies, known to change the size of intercellular spaces within the epithelium; washing of tissue, which may remove both protective mucous and superficial protective cells; loss of integrity of suprabasal layers, known to contribute to cervical paracellular resistance; incorporation of cholera toxin, known to modify epithelial permeability, into the culture medium; or inefficient sealing of the epithelium with agarose. Collins et al. suggested their observation that transmission of the virus occurred in 70% of examined tissue was in keeping with an estimated in vivo transmission rate of 0.01%. An alternative interpretation of this figure would be an expectation that in vitro transmission would only occur in 1% of the tissue examined. Thus the possibility remains that only the 30% of the tissue reported to exclude virus was in fact appropriately sealed. Finally, although the authors attempted to determine the primary target of infection in their model, the use of labeled oligonucleotides to detect HIV gag–pol RNA makes it impossible to distinguish whether positive cells are productively infected or merely binding virus on their surface. Although the reported model is a promising development, more work is needed before it can be accepted as a credible model to study transmission of HIV in the female genital tract. In the meantime, it would be premature to extrapolate the findings from this model to the in vivo situation.

Estimation of hepatic hematopoiesis in second and third trimester singleton gestations using flow cytometric light scatter analysis of archival autopsy tissue

The purpose of this investigation was to develop a simple, quantitative, reproducible and objective method for estimating fetal hepatic hematopoiesis using flow cytometric light scatter measurements and to use this methodology to determine standard values for singleton gestations. Percent hepatic hematopoiesis was estimated from autopsy tissue both flow cytometrically using forward angle and side light scatter characteristics and histologically (single observer) in 67 s and third trimester singleton gestations without evidence of infection, congenital malformation, chronic maternal or placental disorders, or growth retardation. Correlation of flow cytometric and histologic estimates was 0.70 with flow cytometric estimates showing less variability than histologic estimates, especially during the second trimester. Flow cytometric estimates of hepatic hematopoiesis were relatively constant at 50-70% between 16 and 27 weeks gestational age and decreased during the third trimester to a level of approximately 25-30% at term. These results confirm and quantitate the predicted decrease in hepatic hematopoiesis between the second and third trimesters of gestation as well as its persistence at term. In addition, they demonstrate that flow cytometric light scatter analysis is an objective, valid and simple method for estimating hepatic hematopoiesis in archival autopsy tissue and provides objective standard values for comparison with estimates in pathologic gestations.

Endodermal sinus tumor of the ovary: A case series with flow cytometric DNA content analysis

Endodermal sinus tumors (EST) are rare germ cell ovarian malignancies occurring primarily in young women. A retrospective review of the Magee-Womens Hospital tumor registry revealed eight cases of pure EST and two mixed tumors in which the EST component was predominant. Mean patient age was 18.2 years. Abdominal pain was the most common presenting symptom and a pelvic mass was palpable in all patients. Four patients are currently alive and well with no evidence of disease. All were treated with surgery and combination chemotherapy. Flow cytometric DNA content analysis of paraffin-embedded tumor tissue identified similar aneuploid cell populations in three of five tumors analyzed with relative peak positions of 1.72, 1.62, and 1.70. The management of women with endodermal sinus tumor remains controversial with regard to type of chemotherapy employed and the use of second-look laparotomy. The prognosis role of flow cytometric DNA content analysis is yet to be determined.