Plausibility of HIV-1 Infection of Oral Mucosal Epithelial Cells.

The AIDS pandemic continues. Little is understood about how HIV gains access to permissive cells across mucosal surfaces, yet such knowledge is crucial to the development of successful topical anti-HIV-1 agents and mucosal vaccines. HIV-1 rapidly internalizes and integrates into the mucosal keratinocyte genome, and integrated copies of HIV-1 persist upon cell passage. The virus does not appear to replicate, and the infection may become latent. Interactions between HIV-1 and oral keratinocytes have been modeled in the context of key environmental factors, including putative copathogens and saliva. In keratinocytes, HIV-1 internalizes within minutes; in saliva, an infectious fraction escapes inactivation and is harbored and transferable to permissive target cells for up to 48 hours. When incubated with the common oral pathogen Porphyromonas gingivalis, CCR5- oral keratinocytes signal through protease-activated receptors and Toll-like receptors to induce expression of CCR5, which increases selective uptake of infectious R5-tropic HIV-1 into oral keratinocytes and transfer to permissive cells. Hence, oral keratinocytes-like squamous keratinocytes of other tissues-may be targets for low-level HIV-1 internalization and subsequent dissemination by transfer to permissive cells.

Quantitative image analysis of HIV-1 infection in lymphoid tissue.

Tracking human immunodeficiency virus-type 1 (HIV-1) infection at the cellular level in tissue reservoirs provides opportunities to better understand the pathogenesis of infection and to rationally design and monitor therapy. A quantitative technique was developed to determine viral burden in two important cellular compartments in lymphoid tissues. Image analysis and in situ hybridization were combined to show that in the presymptomatic stages of infection there is a large, relatively stable pool of virions on the surfaces of follicular dendritic cells and a smaller pool of productively infected cells. Despite evidence of constraints on HIV-1 replication in the infected cell population in lymphoid tissues, estimates of the numbers of these cells and the virus they could produce are consistent with the quantities of virus that have been detected in the bloodstream. The cellular sources of virus production and storage in lymphoid tissues can now be studied with this approach over the course of infection and treatment.

Kinetics of response in lymphoid tissues to antiretroviral therapy of HIV-1 infection.

In lymphoid tissue, where human immunodeficiency virus-type 1 (HIV-1) is produced and stored, three-drug treatment with viral protease and reverse transcriptase inhibitors markedly reduced viral burden. This was shown by in situ hybridization and computerized quantitative analysis of serial tonsil biopsies from previously untreated adults. The frequency of productive mononuclear cells (MNCs) initially diminished with a half-life of about 1 day. Surprisingly, the amount of HIV-1 RNA in virus trapped on follicular dendritic cells (FDCs) decreased almost as quickly. After 24 weeks, MNCs with very few copies of HIV-1 RNA per cell were still detectable, as was proviral DNA; however, the amount of FDC-associated virus decreased by >/=3.4 log units. Thus, 6 months of potent therapy controlled active replication and cleared >99.9 percent of virus from the secondary lymphoid tissue reservoir.

Relationship between systemic corticosteroids and osteonecrosis.

Numerous reports describe osteonecrosis after oral corticosteroid therapy. It is still uncertain if corticosteroid treatment alone or in combination with other factors leads to the development of this condition. The literature presents controversial clinical and experimental data. The most affected site for osteonecrosis is the femoral head and therefore our considerations are concentrated at this site. Oral corticosteroids are commonly used in dermatology, especially in the treatment of connective tissue diseases and hypersensitive diseases. This clinical review evaluates the relationship between and the onset of femoral head necrosis. Although osteonecrosis of the femoral head can be caused by various conditions such as trauma, excess alcohol and hemoglobinopathies, studies indicate that treatment with corticosteroids is the most common cause of the condition. There is some controversy on the role of underlying disease and total corticosteroid dose administered, in the development of osteonecrosis of the femoral head. MRI scans are used to establish an early diagnosis. There are several surgical and nonsurgical options for disease management, dependent on the stage of disease, the age of the patient and other risk factors. In general, the risk for osteonecrosis is considered to be low under oral corticosteroid therapy. So far, no data can establish a direct relationship, but data still strongly suggest an existing cause and effect relationship. Further investigations are necessary for example, a large controlled prospective long-term study, to further refine an association between the corticosteroid dose, the duration of treatment and other risk factors. Dermatologists who prescribe oral corticosteroids, should always be aware of the potential risk of avascular femoral head necrosis and the patients should be informed about this severe complication of oral coricosteroid therapy.

[Physical strain during tocolysis of premature labour (author's transl)]. / Körperliche Belastung während Tokolyse vorzeitiger Wehentätigkeit.

A group of 29 patients who had premature labour contractions were chosen to take part in this study. All were cardiovascularly healthy. Their performance during a step climbing exercise was examined in relationship to a Fenoterol-tocolysis (30-60 mg/day) and to the amount of exercise. The fetal heart rate showed no pathological changes following the exercise, as compared to the preexercise values. An external tocometry showed the uterine activity also to be uninfluenced.

Artificial disruption of skin barrier prior to irritant patch testing does not improve test design.

BACKGROUND: Irritant patch testing is often performed as a 24- or 48-h occlusive patch test with low concentrations of sodium lauryl sulphate (SLS). OBJECTIVES: The aim of this study was to investigate potential ways to shorten this test procedure and obtain precise test results. PATIENTS AND METHODS: Thirty-six healthy volunteers underwent irritant patch testing with different pretreatments (PT) of the test fields. Occlusive test chambers were applied on the upper back with SLS 0.5%, 1%, 2% and 5% in large Finn Chambers(R). The patches were removed after 4 and 24 h, respectively, depending on the concentration used. Test fields were pretreated as follows: PT 0, field without any PT (control); PT 1, prick with lancet; PT 2, prick with test stamp; PT 3, scratch with lancet; PT 4, incision with standardized incision instrument (0.1-0.2 mm depth). Skin reactions were evaluated by transepidermal water loss (TEWL), skin erythema and skin hydration and as well by a visual score (VS) at 4, 24 and 72 h. RESULTS: Our data show an obvious distinction between PT 0-2 and PT 3-4 at all measurement methods. The average TEWL values with PT 3-4 were higher than those with PT 0-2, especially on the 4-h course. This distinction may derive from the shape and size of the skin impairment achieved by PT 3-4, leading to a mechanical barrier disruption. However, SLS may infiltrate directly into deeper skin layers supported by capillarity. Consequently, no or little penetration through the epidermis and interaction with its structures occurs, which is responsible for irritant skin reactions. The SLS dose in the upper skin layers is therefore lower at these PTs. The lower remaining dose of SLS also explains this distinction, especially for the VS. Additionally, there are presumed reactions in deeper layers of the epidermis and dermis at PT 3-4. CONCLUSIONS: In summary, all data suggest a different reaction pattern from the classical irritant response. Therefore, application without any PT seems to be best suited for irritancy skin testing, especially for visual assessment. PTs prior to irritant patch testing have been shown to be unjustifiable.

Enhancement of immune status by high levels of dietary vitamin B-6 without growth inhibition of human malignant melanoma in athymic nude mice.

The effects of dietary vitamin B-6 supplementation on the development of human malignant melanoma (M21-HPB) xenografts and on in vitro responses of leukocytes were examined. Male athymic nude mice, five weeks old, were divided into two groups of 48 each and fed 20% casein diets containing pyridoxine (PN) at 4.1 (control diet) and 61.6 mg/kg diet for 10 weeks. After four weeks of dietary treatment, 20 animals from each dietary group were injected subcutaneously with 3 x 10(7) melanoma cells. After 4, 8, and 10 weeks of dietary regimen, animals from each group were killed and blood, liver, and spleen samples were obtained. Food consumption and mouse body weights were similar between groups, and no difference was noted in tumor incidence or volume. Noninjected and tumor-bearing mice given the PN 61.6 diet generally exhibited greater oxygen radical production by phagocytic cells from blood and spleen than did animals fed the PN 4.1 diet. Spleen and blood B lymphocyte proliferation in response to lipopolysaccharide (LPS) was enhanced (10 and 30%) in the noninjected animals given the PN 61.6 diet. In addition, tumor-bearing mice fed the PN 61.6 diet had significantly greater LPS-induced spleen cell proliferation at eight weeks when compared with mice consuming the PN 4.1 diet. Despite immune enhancement, tumor incidence and progression was not modified by a high level of dietary vitamin B-6. Therefore, it is tempting to speculate that tumor inhibition by high dietary vitamin B-6 may be mediated by T lymphocyte-dependent mechanisms that are lacking in these genetically immuno-deficient mice.

The relationship between tumor necrosis factor and human immunodeficiency virus gene expression in lymphoid tissue.

In tissue culture models of chronic human immunodeficiency virus type 1 (HIV-1) infection, cytokines such as tumor necrosis factor alpha (TNF-alpha) activate viral gene expression. We sought evidence that TNF-alpha might similarly regulate viral gene expression in vivo in the major lymphoid tissue (LT) reservoir. We used in situ hybridization, quantitative image analysis, and double-label techniques to compare cytokine and HIV-1 RNA levels in sections of tonsil and lymph node tissues obtained from individuals in early and later stages of HIV-1 infection. The levels of TNF-alpha gene expression in LT from HIV-1-infected an uninfected individuals were indistinguishable, and we found no correlation between TNF-alpha gene expression in LT and the level of HIV-1 gene expression in LT. There is thus little evidence that in vivo TNF-alpha significantly influences HIV production in LT.

Vascular endothelial growth factor expression in early stage ovarian carcinoma.

BACKGROUND: Tumor angiogenesis is essential for solid tumor growth. Yet, the importance of any particular factor in neoplastic proliferation is poorly defined. This study examines the clinical significance of increased expression of one of the angiogenic factors, vascular endothelial growth factor (VEGF), in early stage ovarian carcinoma. METHODS: Tumor specimens from 68 patients with International Federation of Gynecology and Obstetrics Stage I and II ovarian carcinoma were evaluated for VEGF expression. Antisense and corresponding sense (control) RNA probes were transcribed from the pCRII construct (Invitrogen, San Diego, CA), which contained human VEGF cDNA. The antisense probe was designed to include a highly conserved region of the VEGF coding sequence and thus detect all known variants. After in situ hybridization, sections were assessed for overexpression of VEGF. RESULTS: Twenty-nine of the tumor samples overexpressed VEGF, whereas 39 specimens did not. In patients whose tumors demonstrated elevated VEGF expression, 25% were without evidence of disease recurrence at last follow-up. In contrast, 75% of the patients whose tumors did not overexpress VEGF were without evidence of disease at last follow-up (P < 0.001). Median disease free survival for the VEGF positive group was 22 months, compared with > 108 months for the VEGF negative group (P < 0.001). When borderline tumors were excluded from the survival analysis, median disease free survival for the VEGF positive group was 18 months, compared with >120 months for the VEGF negative group (P < 0.001). Other possible prognostic variables had minimal impact on survival; these included age, stage, grade, cytology, and tumor size (P > 0.05). Assignment to a high risk group, as defined by the Gynecologic Oncology Group of the National Cancer Institute, was somewhat predictive of a shorter relapse free interval (P = 0.056). In a multivariate analysis, however, only elevated VEGF expression was associated with poorer survival. CONCLUSIONS: In this analysis, patients with early stage ovarian carcinoma with increased VEGF expression had a poorer prognosis. Further study of VEGF may ultimately lead to identification of patients with high risk lesions whose tumor biology portends a worse prognosis and who therefore may benefit from aggressive adjuvant therapy.

Proviral load and expression of avian leukosis viruses of subgroup C in long-term persistently infected heterologous hosts (ducks).

Proviral DNA load and expression of avian leukosis viruses of subgroup C (ALV-C) in ducks infected in mid embryogenesis were studied using quantitative PCR, RT-PCR, in situ hybridization employing ALV-specific riboprobe, and immunohistochemistry. A group of long-term surviving, non-reviremic ducks was selected for the study and compared to control reviremic animals in order to obtain information about persisting retroviruses in different duck tissues. A widespread distribution of proviruses in the tested tissues was found, but the proviral load was significantly lower in non-reviremic in comparison to reviremic animals. The only exception were brain and blood cells, in which no significant difference in the quantity of integrated proviruses was found between both categories of ducks, thus indicating an exceptional position of the brain and blood cells among all tested tissues. Contrary to reviremic, the proviruses were not transcribed in non-reviremic ducks, with the exception of brain and thymus. In the majority of non-reviremic ducks viral RNA was revealed in the brain, but no infectious virus could be recovered from this tissue. The opposite situation was observed in the thymus, where infectious virus was recovered but viral RNA remained below the detection limit of the assay. As revealed by in situ analysis, infected cells were either disseminated or focally distributed in tissues. From the long-term follow up of ALV-C in intraembryonally infected ducks we conclude that this model is suitable for the study of retrovirus persistence accompained both by the presence and absence of reviremias. The possible consequences of transmission and long-term persistence of retroviruses in the heterologous host for retroviral evolution are discussed.

Kaposi's sarcoma-associated herpesvirus gene expression in endothelial (spindle) tumor cells.

The recent discovery of DNA sequences of a new human herpesvirus in Kaposi's sarcoma (KS) has fueled speculation that this virus might cause KS. The mere presence, however, of a virus in a complex multicellular tumor like KS could just as well be construed as evidence of a passenger agent. We sought stronger evidence linking the KS-associated herpesvirus (KSHV) to tumor formation by using in situ hybridization to investigate the specificity, constancy, and timing of KSHV gene expression in KS tumor cells. Here we document expression of a 700-nucleotide viral RNA in every KS tumor examined, from the earliest histologically recognizable stage to advanced tumors in which the vast majority of identifiable spindle tumor cells contain this transcript. Two other KSHV RNAs were also detected in a smaller fraction of the tumor cells in all but the earliest lesion. These viral RNAs were expressed to relatively low levels in this subset; because one of these RNAs encodes a major viral capsid protein, these cells may be producing KSHV. We did not find these KSHV genes expressed in a variety of other tumors and proliferative processes, but we did detect viral gene expression in prostatic tissue, supporting a possible mechanism for sexual transmission of KSHV. The close relationship between KS and KSHV gene expression is consistent with the hypothesis that KSHV is directly involved in the etiology and pathogenesis of KS.

Productive infection of T cells in lymphoid tissues during primary and early human immunodeficiency virus infection.

Current models suggest that during human immunodeficiency virus type 1 (HIV-1) transmission virions are selected that use the CCR5 chemokine receptor on macrophages and/or dendritic cells. A gradual evolution to CXCR4 chemokine receptor use causes a shift in the proportion of productively infected cells to the CD4 cell population. Productively infected cells during acute and early infection in lymphoid tissue were assessed, as well as the impact of productive infection on the T cell population in 21 persons who had biopsies performed on days 2-280 after symptoms of acute HIV-1 seroconversion. Even in the earliest stages of infection, most productively infected cells were T lymphocytes. There were sufficient infected cells in lymphoid tissue (LT) to account for virus production and virus load in plasma. Despite the relatively high frequency of productively infected cells in LT, the impact on the size of the T cell population in LT at this stage was minor.

Kinetics of CD4+ T cell repopulation of lymphoid tissues after treatment of HIV-1 infection.

Potent combinations of antiretroviral drugs diminish the turnover of CD4+ T lymphocytes productively infected with HIV-1 and reduce the large pool of virions deposited in lymphoid tissue (LT). To determine to what extent suppression of viral replication and reduction in viral antigens in LT might lead correspondingly to repopulation of the immune system, we characterized CD4+ T lymphocyte populations in LT in which we previously had quantitated viral load and turnover of infected cells before and after treatment. We directly measured by quantitative image analysis changes in total CD4+ T cell counts, the CD45RA+ subset, and fractions of proliferating or apoptotic CD4+ T cells. Compared with normal controls, we documented decreased numbers of CD4+ T cells and increased proliferation and apoptosis. After treatment, proliferation returned to normal levels, and total CD4+ T and CD45RA+ cells increased. We discuss the effects of HIV-1 on this subset based on the concept that renewal mechanisms in the adult are operating at full capacity before infection and cannot meet the additional demand imposed by the loss of productively infected cells. The slow increases in the CD45RA+ CD4+ T cells are consistent with the optimistic conclusions that (i) renewal mechanisms have not been damaged irreparably even at relatively advanced stages of infection and (ii) CD4+ T cell populations can be partially restored by control of active replication without eradication of HIV-1.

Reversibility of the pathological changes in the follicular dendritic cell network with treatment of HIV-1 infection.

Over the course of HIV-1 infection, the lymphoid follicles where the humoral immune response is generated initially increase in size and number and then progressively involute. In advanced disease, the network of the processes of follicular dendritic cells (FDCs) that serve as antigen repositories and anatomical substrate for B and T cells and antigen to interact is destroyed, contributing to the breakdown of the immune system. Because destruction of FDCs is associated with deposition of HIV-1, and much of the virus can be cleared from the network with antiretroviral therapy, we investigated the reversibility of damage. We measured the immunohistochemically stainable FDC compartment by quantitative image analysis, and we documented changes in this compartment at different stages of disease. We show that treatment, initiated even at advanced stages of HIV-1 disease, can slowly reverse pathological changes in the FDC network.