Typology of Ohio, USA, tree farmers based upon forestry outreach needs.

This study differentiated groups of Ohio tree farmers through multivariate clustering of their perceived needs for forest management outreach. Tree farmers were surveyed via a mailed questionnaire. Respondents were asked to rate, on a 1-7 scale, their informational needs for 26 outreach topics, which were reduced to six factors. Based on these factors, three clusters were identified-holistic managers, environmental stewards, and pragmatic tree farmers. Cluster assignment of individuals was dependent upon a tree farmer's age, acreage owned, and number of years enrolled in the American Tree Farm System. Holistic managers showed a greater interest in the outreach topics while pragmatic tree farmers displayed an overall lesser interest. Across clusters, print media and in-person workshops were preferred over emails and webinars for receiving forest management information. In-person workshops should be no more than 1 day events, held on a weekday, during the daytime, at a cost not exceeding $35. Programming related to environmental influences, which included managing for forest insects and diseases, was concluded to have the greater potential to impact clientele among all outreach factors due to the information being applicable across demographics and/or management objectives.

HIV-1 infection of placental cord blood monocyte-derived dendritic cells.

Dendritic cells (DC), the most potent antigen-presenting cells (APC), have been implicated as the initial targets of HIV infection in skin and mucosal surfaces. DC can be generated in vitro from blood-isolated CD14(+) monocytes or CD34(+) hematopoietic progenitor cells in the presence of various cytokines. In this study, we investigated whether monocytes obtained from placental cord blood are capable of differentiation into dendritic cells when cultured with a combination of cytokines - granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-4 (IL-4), and tumor necrosis factor-alpha (TNF-alpha). We then examined HIV infection, HIV receptor (CD4, CCR5) expression, and beta-chemokine [macrophage inflammatory protein-1alpha and -1beta (MIP-1alpha, MIP-1beta)] production by placental cord monocyte-derived dendritic cells (MDDC) as compared to that of autologous cord monocyte-derived macrophages (MDM). Monocytes isolated from placental cord blood differentiate into DC after 7 days in culture with the mixture of cytokines, as demonstrated by development of characteristic DC morphology, loss of CD14 expression, and gain of CD83, a marker for mature DC. Mature cord MDDC had significantly lower susceptibility to M-tropic ADA (CCR5-dependent) envelope-pseudotyped HIV infection in comparison to autologous placental cord MDM, whereas there was no significant difference in virus replication in cord MDDC and MDM infected with murine leukemia virus envelope-pseudotyped HIV (HIV receptor-independent). This limited susceptibility of cord MDDC to M-tropic HIV infection may be due to lower expression of CD4 and CCR5 on the cell membrane and higher production of MIP-1alpha and MIP-1beta. These data provide important information toward our understanding of the biological properties of cord MDDC in relation to HIV infection.

Natural killer cell enumeration and function in HIV-infected and high-risk uninfected adolescents.

This is the first report of natural killer cell enumeration and function in HIV-infected and high-risk uninfected adolescents. We examined the association of demographic characteristics of this cohort with three outcomes: CD16+ cell absolute count, lytic units per peripheral blood mononuclear cell (PBMC), and lytic units per natural killer (NK) cell. We also examined the association of CD4, CD38, and antiretroviral therapy (ART) use with these outcomes in the subset of HIV-infected adolescents. Adolescents participating in an on-going longitudinal study (the REACH study) were sampled for CD16+ cell count and NK function. This cross-sectional analysis was performed on 412 subjects with NK cell data available. HIV-positive males had higher numbers of CD3-/CD16+/CD56+ NK cells than HIV-positive females. However, for the HIV-negative subjects, we did not observe a gender-related effect for absolute NK cell numbers. Gender, however, was a significant covariate for the analysis, using lytic units per PBMC as the unit of measurement, with males showing higher values than females. Age was not a predictive covariate for any of the three assessments of NK cell number and function examined. Our observations concerning the HIV-positive individuals indicate that reduced CD4+ T cell counts were associated with decreased circulating CD3-/CD16+/CD56+ NK cells. We also observed an association between elevation of CD8+/CD38+/DR+ lymphocytes and lower NK lytic units per PBMC. The results of our multivariate models indicate that there is a reduced number of NK cells and reduced lytic units per PBMC in patients receiving single or multidrug antiretroviral therapy. There are changes in circulating NK cell number and function in HIV-infected adolescents, in comparison with high-risk HIV-negative adolescents. The data suggest that these changes may occur early in the course of HIV disease but that quantitative changes continue to occur with advancing depletion of the CD4+ T cell pool.

Patterns of plasma human immunodeficiency virus type 1 RNA response to highly active antiretroviral therapy in infected children.

This study examined the rate of decline in plasma human immunodeficiency virus type 1 (HIV-1) RNA levels to <400 and <50 copies/mL in children receiving highly active antiretroviral therapy (HAART) consisting of efavirenz, nelfinavir, and 1 or 2 nucleoside reverse-transcriptase inhibitors. Children receiving HAART achieved a plasma HIV-1 RNA level <400 copies/mL by a median of 4 weeks after initiation of therapy and a decline to <50 copies/mL by 20 weeks. Baseline plasma HIV-1 RNA levels affected the likelihood of achieving potent and sustained virus suppression, and children whose CD4 lymphocyte counts increased >70 cells/microL by 20 weeks on therapy were more likely to achieve durable virological and immunological benefit. These data provide time frames for virus suppression after the initiation of HAART that should be useful in evaluating the potential efficacy and durability of response of newly instituted combination antiretroviral therapy in HIV-1-infected children.

Measurement of cytokine secretion, intracellular protein expression, and mRNA in resting and stimulated peripheral blood mononuclear cells.

Quantitation of cytokine production is a valuable adjunct to standard immunologic assays in defining several pathologic processes. Nevertheless, there is little agreement about which tissues should be assayed, which type of assay should be performed, and which stimulation protocol should be used. As these types of assays enter the clinical arena, there is need for standardization. There is also a need to maximize the amount of information which may be derived from a single sample. We compared secreted interleukin 4 (IL-4), IL-2, IL-6, tumor necrosis factor alpha (TNF-alpha), and gamma interferon proteins as measured by enzyme-linked immunosorbent assay with intracellular cytokine production (IL-2 and gamma interferon) as detected by flow cytometry and quantitative competitive PCR for IL-2, IL-4, TNF-alpha, and gamma interferon mRNA and cDNA. Results from unstimulated cells and cells stimulated with phorbol myristate acetate, phytohemagglutinin, and phorbol myristate acetate plus phytohemagglutin were compared. All three methodologies detected significant stimulation of cytokine production. The combination of phytohemagglutinin and phorbol myristate acetate was overall the most-potent stimulus.

T-lymphocyte subsets in HIV-infected and high-risk HIV-uninfected adolescents: retention of naive T lymphocytes in HIV-infected adolescents. The Adolescent Medicine HIV/AIDS Research Network.

BACKGROUND: The capacity of the immune system of adolescents to generate and repopulate naive and memory cell populations under conditions of normal homeostasis and human immunodeficiency virus (HIV) infection is largely unknown. OBJECTIVE: To assess lymphocyte subsets in HIV-infected and high-risk HIV-negative adolescents. DESIGN: The Reaching for Excellence in Adolescent Care and Health Project of the Adolescent Medicine HIV/AIDS Research Network recruits a cohort of HIV-infected and high-risk HIV-uninfected adolescents, aged 13 to 18 years 364 days, into a study of biomedical and behavioral features of HIV infection as seen in the context of full availability of primary care and HIV-related consultative services. Lymphocyte phenotypes were determined using standard 3-color flow cytometry. SETTING: The Reaching for Excellence in Adolescent Care and Health Project is carried out at 16 clinical sites in 14 urban areas. PARTICIPANTS: T-lymphocyte subsets are reported in 192 HIV-positive and 78 HIV-negative youths. RESULTS: For HIV-positive subjects, the total CD4+ cell count and the percentage of CD4+ cells are decreased when compared with those of the HIV-negative controls (P<.001). The reduction in total CD4+ cells reflects a loss of naive, and memory, CD4+ cells compared with HIV-negative youths. Human immunodeficiency virus-infected adolescents, many of whom have been infected recently (ie, those with CD4+ cell counts > or =0.500 x 10(9)/L [500/microL]), have a significant increase in naive CD8+ cells compared with HIV-negative youths (P<.01). There also is a significant increase in memory CD8+ cells at all strata of total CD4+ cells compared with HIV-negative youths (P<.01). The increase in naive CD8+ cells in those subjects with CD4+ cell counts of 0.500 x 10(9)/L or greater is a unique finding in this cohort. CONCLUSIONS: This study demonstrates high levels of naive CD8+ cells in response to HIV infection in adolescents with CD4+ cell counts of 0.500 X 10(9)/L or greater. The presence of high levels of naive CD8+ cells suggests functioning thymic tissue in some adolescents infected with HIV. Furthermore, the normal level of naive CD4+ cells in adolescents with CD4+ levels of 0.500 x 10(9)/L or greater provides additional support for the concept of a more robust immune system in HIV-infected adolescents compared with HIV-infected adults. These observations suggest that the immune system of HIV-infected adolescents may be capable of better responses to neoantigens and cytotoxic T-lymphocyte responses to HIV than the immune system of infected children or adults. Human immunodeficiency virus-infected adolescents may have an immune system that is capable of reconstitution following highly active antiretroviral therapy.

Inhibition of HIV-1 replication in chronically infected cell lines and peripheral blood mononuclear cells by retrovirus-mediated antitat gene transfer.

Among potential genetic targets for intervention in the HIV-1 life cycle, the tat gene product is a key target. We investigated the ability of an antitat gene to inhibit HIV-1 activation and replication in chronically infected promonocyte (U1) and T cell (ACH-2) lines in vitro. U1 and ACH-2 cells were transduced with an antitat gene expressing RNA with dual (polymeric Tat activation response element and antisense-tat) function that interferes with HIV-1 replication. Tumor necrosis factor-alpha (TNF-alpha) plus phorbol 12- myristate 13-acetate (PMA)-induced HIV-1 expression, as determined by reverse transcribed PCR and reverse transcriptase (RT) assays, was significantly inhibited in U1 and ACH-2 cells transduced with the antitat gene, compared with the cells transduced with control vector and untransduced cells. This resistance to TNF-alpha plus PMA-induced HIV-1 expression was demonstrated in antitat gene-transduced U1 and ACH-2 cells maintained in G418-free media for 5 months, suggesting that functional antitat gene may persist for many months in transduced cells and their progeny. Most importantly, we demonstrate that the antitat gene, when introduced into peripheral blood mononuclear cells (PBMC) isolated from patients with HIV-1 infection, inhibited TNF-alpha plus PMA-induced viral replication as determined by RT-PCR and RT activity. In addition, the antitat gene enhanced the survival of CD4+ T lymphocytes from such patients. These data suggest the feasibility of utilizing antitat gene therapy to block activation and replication of HIV-1 in latently infected monocytes and T- lymphocytes in vivo. Gene Therapy (2000) 7, 321-328.

Use of human immunodeficiency virus (HIV) human hyperimmune immunoglobulin in HIV type 1-infected children (Pediatric AIDS clinical trials group protocol 273).

The clinical, immunologic, and virologic effects and the pharmacokinetics of human immunodeficiency virus (HIV) human hyperimmune immunoglobulin (HIVIG) were assessed in 30 HIV-infected children aged 2-11 years. All had moderately advanced disease with an immune complex-dissociated (ICD) p24 antigen >70 pg/mL and were on stable antiviral therapy. Three groups of 10 children received 6 monthly infusions of 200, 400, or 800 mg/kg of HIVIG, and serial immunologic and virologic assays were performed. HIVIG doses as high as 800 mg/kg were safe and well tolerated. The half-life of HIVIG, determined by serial p24 antibody titers, was 13-16 days, the volume of distribution was 102-113 mL/kg, and clearance was 5.6-6.0 mL/kg/day. Plasma ICD p24 decreased during the infusions, but CD4 cell levels, plasma RNA copy number, cellular virus, immunoglobulin levels, and neutralizing antibody titers were minimally affected by the infusions. Clinical status did not change during the 6-month infusion and 3-month follow-up periods.

Rapid method for determining the rate of DNA synthesis and cellular proliferation.

A new method has been developed for determination of DNA synthesis during cell proliferation. The method is based on the metabolism of [U-(13)C(6)]glucose to deoxyribose (DR) and then incorporation of [U-(13)C(5)]DR into newly synthesized DNA. Extracted cellular DNA is subjected to HCl hydrolysis (2 h at 100 degrees C), which converts DR into levulinic acid. The (13)C enrichment in DR is determined in the trimethylsilyl derivative of levulinate using gas chromatography-mass spectrometry. The method is rapid and sensitive. It can precisely determine (13)C enrichment below 1 at.% excess in as little as 4 ng DNA. We have used this method to determine the rate of cell proliferation in vitro and the level of DR in a given amount of DNA. The current approach has significant advantages over previously described methods and overcomes several difficulties related to the determination of DNA synthesis both in vivo and in vitro.

Combination therapy with efavirenz, nelfinavir, and nucleoside reverse-transcriptase inhibitors in children infected with human immunodeficiency virus type 1. Pediatric AIDS Clinical Trials Group 382 Team.

BACKGROUND: Consistent long-term viral suppression has been difficult to achieve in children with human immunodeficiency virus type 1 (HIV-1) infection. We tested the safety and antiviral efficacy of a novel combination consisting of efavirenz, nelfinavir, and one or more nucleoside reverse-transcriptase inhibitors in 57 children previously treated with only nucleoside reverse-transcriptase inhibitors. METHODS: The children were monitored for 48 weeks after the initiation of therapy. We assessed plasma concentrations of efavirenz and nelfinavir, plasma HIV-1 RNA levels, and lymphocyte subpopulations. RESULTS: At base line, the 57 HIV-1-infected children (age range, 3.8 to 16.8 years) had a median of 699 CD4 cells per cubic millimeter and 10,000 copies of HIV-1 RNA per milliliter of plasma. The most common treatment-related effects of at least moderate severity were rash (in 30 percent of children), diarrhea (in 18 percent), neutropenia (in 12 percent), and biochemical abnormalities (in 12 percent). Serious side effects were uncommon. The mean values for the area under the curve for efavirenz and nelfinavir corresponded to expected values. In an intention-to-treat analysis, 76 percent of children had plasma HIV-1 RNA levels of less than 400 copies per milliliter after 48 weeks of therapy and 63 percent had levels of less than 50 copies per milliliter. A high plasma HIV-1 RNA level at base line significantly decreased the likelihood that plasma levels of HIV-1 RNA would become undetectable during treatment. CONCLUSIONS: In HIV-1-infected children who were previously treated with nucleoside reverse-transcriptase inhibitors, the combination of efavirenz, nelfinavir, and nucleoside reverse-transcriptase inhibitors was generally well tolerated and had a potent and sustained antiviral effect.

Human immunodeficiency virus infection of human placental cord blood CD34+AC133+ stem cells and their progeny.

The AC133 is a novel antigen selectively expressed on primitive CD34bright stem cells and is a valuable marker for the selection of long-term culture-initiating cells (LTC-ICs) and severe combined immunodeficiency (SCID)-repopulating cells. Human placental cord blood (HPCB) is a rich source of CD34+AC133+ cells. Since AC133 antibody is likely to be used as an alternative to CD34 for the selection of stem cells in transplant and gene therapy situations, we examined the susceptibility of HPCB-isolated CD34+AC133+ stem cells to infection with free and cell-associated HIV-1 in vitro. Freshly isolated HPCB CD34+AC133+ stem cells were not susceptible to HIV-1 infection as determined by PCR and reverse transcriptase assays. Inoculation with HIV-1 did not affect the viability and clonogenic ability of HPCB CD34+AC133+ cells. Although the highly purified HPCB CD34+AC133+ stem cells contained mRNA for CD4 and CXCR4 receptors, CD4 and CXCR4 proteins were not expressed on these cells. Similarly, CCR5 protein, the major macrophage-tropic HIV-1 coreceptor, was not expressed in freshly isolated HPCB CD34+AC133+ stem cells, although the transcript for CCR5 was identified in these cells. Expression of CD4, CXCR4, and CCR5 receptor proteins on the progeny derived from HPCB CD34+AC133+ stem cells was detected and correlated with susceptibility to HIV-1 infection in vitro. These findings suggest that freshly isolated HPCB CD34+AC133+ stem cells are not susceptible to HIV-1 infection and may not be a viral reservoir. These data have important implications for the use of AC133 antibody as a means of enriching for primitive hematopoietic stem cells from placental cord blood and in the design of stem cell or progenitor cell-based gene therapeutic strategies for perinatal HIV-1 infection.

Deficient antibody-dependent cellular cytotoxicity against human immunodeficiency virus (HIV)-expressing target cells in perinatal HIV infection.

Peripheral blood mononuclear cells (PBMC) of human immunodeficiency virus (HIV)-infected children, age-matched HIV-seronegative controls, and HIV-infected asymptomatic and symptomatic adults were compared for their ability to mediate antibody-dependent cellular cytotoxicity (ADCC) and natural killer (NK) cell-mediated cytotoxicity against target cells expressing HIV or herpes simplex virus (HSV) antigens. Target cells consisted of CD4 lymphocytes purified from PBMC of HIV-seronegative adults and incubated with the IIIB strain of HIV, HUT78 cells chronically infected with IIIB, and HSV-infected human fibroblasts. PBMC of asymptomatic HIV-infected adults were generally able to lyse CD4 cells expressing HIV antigens. Direct correlation was found between the magnitude of lysis and absolute CD4 cell counts in these individuals. In contrast to these results, PBMC from HIV-infected children were generally unable to lyse IIIB-expressing CD4 cells, regardless of the children's clinical status, age, or absolute CD4 cell counts. Cells from HIV-seronegative adults and children did not directly lyse these target cells either but, in contrast to cells of HIV-seropositive children, were able to mediate cell lysis when serum from an HIV-seropositive adult was added. However, effector cells from these HIV-infected children were able to mediate both ADCC against HSV-infected fibroblasts and NK cell-mediated cytotoxicity against IIIB-infected HUT78 cells. Reduced ability of PBMC from vertically HIV-infected children to mediate ADCC against HIV antigen-expressing CD4 cells may contribute to rapid progression to AIDS.

Recombinant human gamma interferon in human immunodeficiency virus-infected children: safety, CD4(+)-lymphocyte count, viral load, and neutrophil function (AIDS Clinical Trials Group Protocol 211).

Nineteen children with human immunodeficiency virus (HIV) infection were treated with recombinant human gamma interferon (rIFN-gamma) (50 microg/m2 subcutaneously three times each week during weeks 1 through 12 and 100 microg/m2 subcutaneously three times each week during weeks 13 through 24) in a phase I/II clinical trial. All children continued to receive previously prescribed therapy with oral zidovudine or didanosine. Children were assessed clinically and with laboratory studies during 24 weeks of study treatment and for 12 weeks after completion of rIFN-gamma therapy. In general, rIFN-gamma therapy was well tolerated. There were two clinical or laboratory adverse events thought to be possibly or probably study drug associated. One child developed acute pancreatitis; another child developed granulocytopenia. Median CD4(+)-lymphocyte counts and plasma HIV RNA concentrations did not change significantly during therapy. In vitro neutrophil bactericidal activity against Staphylococcus aureus and superoxide production were not significantly affected by rIFN-gamma therapy. We conclude that rIFN-gamma therapy in HIV-infected children receiving single-agent antiretroviral therapy is safe and does not produce consistent changes in CD4(+)-lymphocyte count, plasma HIV RNA concentration, or in vitro neutrophil function.

Protective role of beta-chemokines associated with HIV-specific Th responses against perinatal HIV transmission.

To examine the protective role of cellular immunity in the vertical transmission of HIV, we analyzed HIV-specific IL-2 and CTL responses, as well as beta-chemokine expression in HIV-infected and uninfected infants of HIV+ mothers. Our results showed that HIV envelope (env) peptide-specific IL-2 responses associated with beta-chemokine production were detectable at birth in the majority of uninfected infants of HIV+ mothers. The responses falling to background before the infants were 1 yr old were rarely associated with HIV-specific CTL activity. Conversely, HIV-specific Th and CTL cellular responses were absent at birth in HIV-infected infants. Infants with AIDS-related symptoms exhibited undetectable or very low levels of HIV-specific cellular immunity during the first year of life, whereas those with a slowly progressive disease showed evidence of such immunity between their second and ninth month. The latter group of infected infants tested negative for plasma HIV RNA levels shortly after birth, suggesting lack of intrauterine exposure to HIV. The presence of HIV-specific Th responses at birth in uninfected newborns of HIV+ mothers, but absence of such activities in HIV-infected infants without evidence of intrauterine HIV infection, suggests that in utero development of HIV-specific Th responses associated with beta-chemokines could mediate nonlytic inhibition of infection during vertical transmission of HIV.

Inhibition of HIV type 1 replication in chronically infected monocytes and lymphocytes by retrovirus-mediated gene transfer of anti-Rev single-chain variable fragments.

We investigated a strategy for gene therapy, intracellular expression of anti-HIV-1 Rev single-chain variable fragments (SFvs), in promonocytic (U1) and T (ACH-2) cell lines latently infected with HIV-1. The cellular and molecular mechanisms leading to activation of latent integrated HIV-1 provirus in U1 and ACH-2 cells have been well delineated. These cells produce HIV-1 in response to stimulation with certain cytokines. U1 and ACH-2 cells were transduced with a murine retroviral shuttle vector that expresses anti-Rev SFv (pLXSN-D8SFv-Rev) or with a control murine leukemia virus (MLV) vector (pLXSN). Tumor necrosis factor alpha (TFNalpha)-, interleukin 6 (IL-6)-, and phorbol myristate acid (PMA)-induced HIV-1 expression, as determined by reverse transcriptase (RT) assay, was significantly inhibited in cells transduced with pLXSN-D8SFv-Rev, compared with cells transduced with pLXSN. In addition, pLXSN-D8SFv-Rev-transduced cells, when incubated with monokine-enriched supernatants of human peripheral blood monocyte cultures, produced significantly less HIV-1 than did cells transduced with pLXSN. This resistance to cytokine-induced HIV-1 expression was demonstrated in SFv-transduced U1 and ACH-2 cells maintained in G418-free medium for 2 months. These data suggest that feasibility of utilizing various anti-HIV-1 SFvs to block activation of HIV-1 infection in vivo.

Safety and immunogenicity of the Towne strain cytomegalovirus vaccine.

BACKGROUND: Women with naturally acquired serum antibodies to cytomegalovirus (CMV) are usually protected against both frequent secondary infection and giving birth to infants severely affected by intrauterine CMV infection. OBJECTIVE: To determine the feasibility of using a live attenuated strain of CMV (Towne) to achieve immunity similar to that provided by wild-type infection, we evaluated a new lot of the Towne strain of CMV in 3 open label trials involving 68 men, 63 women of childbearing age and 13 children, respectively. RESULTS: Mild local reactions occurred among approximately one-third of subjects. There were no systemic reactions. All 45 subjects tested developed lymphoproliferative responses to CMV. CD8+ class I-restricted cytotoxic T cell responses specific for CMV antigens were detected in three of four subjects and persisted for 6 months. Neutralizing titers were maximal at 2 to 4 months postimmunization, were dose-dependent and were comparable to those induced by natural infection. CONCLUSION: These results support further evaluation of the Towne strain of CMV in women at risk for acquiring CMV infection during pregnancy or among children transmitting CMV to pregnant women.

L-2-oxothiazolidine-4-carboxylic acid inhibits human immunodeficiency virus type 1 replication in mononuclear phagocytes and lymphocytes.

We investigated the effects of L-2-oxothiazolidine-4-carboxylic acid (OTC; Procysteine), a cysteine prodrug, on human immunodeficiency virus type 1 (HIV-1) expression in both adult peripheral and cord blood mononuclear phagocytes and lymphocytes. OTC suppressed HIV-1 expression in monocyte-derived macrophages (MDM) and lymphocytes in a dose-dependent fashion as determined by HIV-1 reverse transcriptase (RT) activity. This inhibitory effect of OTC occurred with three HIV-1 strains (two laboratory-adapted strains and one primary isolate). Addition of OTC to chronically HIV-1-infected MDM cultures also suppressed RT activity by 40 to 50% in comparison to untreated controls. The inhibitory effects of OTC on HIV-1 were not caused by toxicity to MDM or lymphocytes because there was no change in cell viability or cellular DNA synthesis, as evaluated by trypan blue dye exclusion and [3H]thymidine incorporation, at doses of OTC that inhibit virus replication. These observations indicate that OTC has the potential to limit HIV-1 replication in mononuclear phagocytes and lymphocytes and may be useful in the treatment of HIV-1 infection and AIDS.

Cytokines released by human peripheral blood mononuclear cells inhibit the production of early and late cytomegalovirus proteins.

Cytomegalovirus-infected human fibroblasts are susceptible to lysis by natural killer cells and cytotoxic T cells. The purpose of this study was to determine whether non-lytic mechanisms might also contribute to the control of cytomegalovirus infection. The appearance of cytomegalovirus proteins in infected fibroblasts was determined by flow cytometry. Infected fibroblasts incubated with peripheral blood mononuclear cells for 3 days expressed less early and late proteins than fibroblasts incubated without peripheral blood mononuclear cells. Supernatants generated by the cocultivation of peripheral blood mononuclear cells with cytomegalovirus-infected fibroblasts inhibited the production of cytomegalovirus early and late proteins. The soluble factors in supernatants which contributed to the inhibitory effect were identified as interferons alpha, beta and gamma, and tumor necrosis factors alpha and beta. The ability of supernatants to inhibit the production of cytomegalovirus early protein was mimicked by combinations of corresponding recombinant cytokines. The inhibition of cytomegalovirus protein production by cytokines produced by peripheral blood mononuclear cells may contribute to early containment of cytomegalovirus infection.

Application of the polymerase chain reaction to the diagnosis and management of neonatal herpes simplex virus disease. National Institute of Allergy and Infectious Diseases Collaborative Antiviral Study Group.

Cerebrospinal fluid (CSF) specimens from 77 neonates with herpes simplex virus (HSV) disease were evaluated retrospectively by polymerase chain reaction (PCR). Samples were collected from 202 infants enrolled in a National Institute of Allergy and Infectious Diseases Collaborative Antiviral Study Group trial that compared vidarabine with acyclovir for the treatment of neonatal HSV infection. HSV DNA was detected in the CSF of 26 (76%) of 34 infants with CNS disease, in 13 (93%) of 14 infants with disseminated infection, and in 7 (24%) of 29 with skin, eye, or mouth (SEM) involvement. One of the 7 PCR-positive SEM patients subsequently developed severe neurologic impairment. Eighteen (95%) of 19 infants with positive CSF PCR results after the completion of 10 days of antiviral therapy experienced significant morbidity or mortality. Application of PCR to neonatal HSV disease may provide additional information on which clinical decisions may be based, although its diagnostic utility outside the research setting is unproven.