Human immunodeficiency virus infection and indeterminate western blot patterns. Prospective studies in a low prevalence population.

Interpretation of human immunodeficiency virus (HIV) antibody results that are "indeterminate" rather than clearly positive or negative is problematic for the person delivering the result as well as for the individual being tested. To improve counseling messages for these individuals, we evaluated data collected from a well-characterized cohort of 387 blood donors who had been monitored for up to 2 years. We sought to determine if persons with indeterminate Western blot patterns were infected with HIV, and whether information derived from follow-up monitoring would assist in the development of counseling messages for persons on whom no follow-up information was available. Donors were studied by laboratory assays, clinical evaluation, and assessment of risk for HIV. The absence of HIV infection in 97 of 98 donors with indeterminate Western blot patterns was confirmed by clinical follow-up, Western blot assays of sequential samples, and negative gene amplification results. We propose supplemental guidelines to be used as an adjunct to existing interpretive criteria for counseling individuals when they first present with an indeterminate Western blot finding.

Purification of human peripheral blood colony forming cells (CFUC).

A 10-fold enrichment of colony forming cells (CFUC) from single donor platelet-apheresis residues and from 70-120 ml of peripheral blood of normal donors was achieved by sequential sedimentation on Ficoll-diatrizoate, depletion of cells adherent to plastic, and depletion of cells rosetting with sheep red blood cells (T lymphocytes). Culture of 5 x 10(5) unfractionated mononuclear cells yielded 9 +/- 3 colonies and mononuclear cells depleted of adherent cells and T lymphocytes yielded 53 +/- 6 colonies. The mononuclear cell fraction depleted of adherent cells and T lymphocytes was further enriched for CFUC by isopycnic sedimentation of Percoll gradients. Cells recovered in the 1.0063-1.065 g/cm3 density layer of the gradient formed 146 +/- 9 colonies in culture. The mononuclear cells depleted of adherent cells and T lymphocytes were also enriched for CFUC by depletion of Fc-receptor positive cells using an immune sheep red blood cell rosette sedimentation technique. Cultures of the Fc-receptor depleted fractions yielded 107 +/- 12 colonies, while the Fc-receptor enriched fraction yielded only 2 +/- 1 colonies. CFUC appear to lack surface membrane receptors for sheep erythrocytes and the Fc portion of immunoglobulin as well as the ability to adhere to plastic.

Evaluation of HIV type 1 western blot-indeterminate blood donors for the presence of human or bovine retroviruses.

From 1985 through 1990, 1100 of 500,000 human blood donations in Syracuse, New York were repeatedly reactive by ELISA for antibodies to the human immunodeficiency virus type 1 (HIV-1). Nine hundred of the ELISA-reactive samples were confirmed as negative by Western blot (WB), 40 were confirmed as positive, and the remaining 160 sera were indeterminate, reacting mainly with HIV-1 gag gene products. Twenty donors with the most reactive indeterminate WB were selected for follow-up studies. Four of these 20 donors admitted to retroviral risk factors and, interestingly, 12 (60%) had exposure to dairy cattle and drank unpasteurized milk. These 20 donors were analyzed over a 3-year period for the presence of the pathogenic human retroviruses HIV-1, HIV-2, human T cell lymphoma/leukemia virus types I and II (HTLV-I and HTLV-II), as well as bovine immunodeficiency virus (BIV) and leukemia virus (BLV). Retroviral analyses included serology, plasma antigen capture, virus culture, and the polymerase chain reaction. Only one donor seroconverted and was clearly infected with HIV-1. None of the other 19 donor serological reactivities to HIV-1 changed, nor were they positive for any of the above-mentioned retroviruses. Although we cannot ascertain whether these latter 19 HIV-1 WB-indeterminate donors were exposed to human or bovine retroviral proteins, it is unlikely that their HIV-1 seroreactivity was caused by infection with HIV-1, HIV-2, HTLV-I, HTLV-II, BLV, or BIV.

Cytochemical reactions in resting and activated T-lymphocytes.

Peripheral blood T-lymphocytes from normal persons were studied before and after incubation with phytohemagglutinin (PHA) for the presence of beta-glucuronidase, alpha-naphthyl acetate esterase, alpha-naphthyl butyrate esterase, acid phosphatase, and periodic acid-Schiff (PAS)-positive material. The number of T-lymphocytes containing beta-glucuronidase, alpha-naphthyl acetate esterase, and alpha-naphthyl butyrate esterase was reduced (P < 0.005) after incubation with PHA. No significant change in the number of T-lymphocytes positive for acid phosphatase was observed. PAS-positive material was markedly increased (P < 0.005) in activated T-lymphocytes. The data suggest that the cytochemical profile of resting T cells differs markedly from that of activated T-lymphocytes.

Laboratory diagnosis of parasitic and fungal diseases of the central nervous system.

This review is presented to bring attention to those fungal and parasitic organisms that have been associated with central nervous system (CNS) infection and to offer an approach for handling their laboratory diagnosis. Treatment of the cerebrospinal fluid (CSF) to yeild best results on direct smear examination and culture are discussed. Culture procedures and staining methods to be done are given in chart form. Those immunologic tests useful in supporting the diagnosis of fungal or parasitic CNS infections are also included.

Immunological studies in hairy cell leukemia.

Cytochemical and immunological studies were performed on tissues and mononuclear cell suspensions from ten patients with hairy cell leukemia. In all cases studied, tartrate-resistant acid phosphatase was noted within the cytoplasm of hairy cells (HCs). In two thirds of the cases, alpha naphthyl acetate esterase was observed in HCs. In addition, HCs did not form spontaneous rosettes with sheep erythrocytes. A variable number of HCs displayed complement receptors. The nonspecific binding of conjugated immunoglobulin to HCs probably reflected the presence of a high concentration of Fc receptors on the HCs surface. In three cases, the conjugated immunoglobulin reacted predominantly to one light chain, thus suggesting the presence of a monoclonal immunoglobulin. In four of six cases, mononuclear cell suspensions of HCs demonstrated latex phagocytosis. In one case, HCs displayed resynthesis of surface membrane immunoglobulin, the presence of B cell antigen, and phagocytosis of latex. These findings suggest that HCs are distinctive and contain properties of both B lymphocytes and monocytes.

Effects of vitamin E on mixed lymphocyte cultures.

When lymphocytes from individuals ingesting vitamin E were used as responding cells in the mixed lymphocyte culture (MLC) the proliferative response was normal indicating that T lymphocyte reactivity to allogeneic antigen was undisturbed. However, the use of lymphocytes from individuals receiving vitamin E as stimulating cells in the MLC resulted in a diminished proliferative response suggesting that large doses of vitamin E may effect B cells and/or macrophages as they interacted with T lymphocytes.

Studies of lymphocyte proliferation in hairy cell leukaemia: activity in mixed lymphocyte reaction and responses to mitogens.

Subpopulations of splenic lymphocytes from patients with hair cell leukaemia (HCL) were compared with similar subpopulations of lymphocytes from reference individuals for their ability to respond to mitogens and to participate in allogenic and autologous mixed lymphocyte reactions. T cell enriched subpopulations were obtained by double passage of mononuclear cells through mylon wool columns. Non-T cell subpopulations were collected by eluting adherent cells from nylon wool columns and by incubating them with sheep erythrocytes followed by density gradient centrifugation. Unfractionated mononuclear cells, T enriched and non-T subpopulations were compared. Enriched T cell subpopulations from HCL and reference patients responded similarly to allogeneic antigens and phytohaemagglutinin. Splenic non-T cells from reference patients produced a stronger stimulus in the allogeneic mixed lymphocyte reaction than did the unfractionated or the T enriched cells. In contrast, the non-T subpopulations from patients with HCL produced a reduced response compared to that of reference splenic cells when mixed with allogeneic lymphocytes. In addition, non-T cells from HCL patients failed to respond to pokeweed mitogen. Neither reference nor HCL splenic cells produced a significant response in the autologous mixed lymphocyte reactions. The data suggest that splenic non-T cells from patients with HCL either suppress the stimulatory capacity of normal B lymphocytes or fail to stimulate allogeneic lymphocytes in the mixed lymphocyte reactions.

The effects of a promoter of cell differentiation and selected hormones on human cytomegalovirus infection using an in vitro cell system.

The influence of factors that can regulate cellular developmental or metabolic processes in host tissue on cytomegalovirus (CMV) replication in vitro was determined. Hydrocortisone treatment of cells before viral infection resulted in a 12- to 13-fold increase in the expression of immediate early proteins at 4 h after virus inoculation. The addition of a phorbol diester 1.5 h after CMV infection resulted in an 8- to 13-fold increase in production of viral progeny. In contrast, beta-human chorionic gonadotropin treatment generally resulted in a 25%-72% suppression of both CMV-specific proteins and progeny. Effects on CMV infection with either progesterone or estradiol were minor and generally suppressive. The stimulating or suppressive effects of these factors on CMV replication in vitro may be important to CMV reactivation in humans. Further study of regulatory factors may lead to the development of therapeutic approaches to the prevention of CMV reactivation in patients at risk for severe disease.

Factors in the liquid portion of stored blood inhibit the proliferative response in mixed lymphocyte cultures.

The transfusion of blood may suppress the immune responses of patients with renal transplants and with malignant disorders. To study the in vitro suppressive effects of banked blood, 4 units of blood were stored in CPDA-1 and ADSOL at 4 degrees C for 14 days. Lymphocytes and plasma or ADSOL supernatants were harvested on Days 0, 4, 7, 10, and 14. Subpopulations of lymphocytes were enumerated by flow cytometry. Recalcified and heat-treated plasma and supernatants from the units of blood were added to mixed lymphocyte cultures (MLC) composed of cells from normal individuals. No significant changes were noted in the proportions of T or B cells from blood stored under these conditions. A 60 +/- 3 percent inhibition in the proliferative response was observed when plasma from CPDA-1 units was added to MLCs (p less than 0.02). Supernatants from ADSOL units demonstrated a 29 +/- 4 percent inhibition (p less than 0.10) of the proliferative response, and this inhibition of response was observed on all 14 days of the study. When appropriate concentrations of dextrose or adenine were added to other MLCs, adenine (at the concentration found in ADSOL) caused a significant inhibition of the proliferative response. This inhibition was not, however, as marked as that observed with recalcified, heat-treated plasma from CPDA-1 units. We conclude that adenine plus some additional factor(s) found in the liquid portion of stored blood inhibits the proliferative response of normal lymphocytes. It is possible that these factors contribute to the immune suppression observed in vivo in some patients who receive blood transfusions.

The development of acute myelomonocytic leukemia in a patient with acute lymphocytic leukemia.

A diagnosis of acute lymphocytic leukemia (ALL) was made from a peripheral blood and bone marrow specimen from a 59-year-old woman. Typical-appearing lymphoblasts were positive for periodic acid-Schiff (PAS) reaction, but negative for peroxidase, Sudan black B (SBB) and non-specific esterase (NSE) stains. Lymphoblasts failed to form non-immune rosettes and had no surface membrane immunoglobulins. However, lymphoblasts exhibited an "Ia-like" membrane antigen and markedly stimulated allogeneic lymphocytes in a mixed lymphocyte reaction (MLR). These cytochemical and immunologic studies were considered characteristic of null-cell subtype of ALL. Thirteen months later, the peripheral blood and bone marrow specimens contained numerous myelomonoblasts characterized by a weak or negative PAS stain and strongly positive peroxidase, SBB, and NSE reactions. Electron micrographs of the bone marrow suggested that the majority of leukemic cells were myelomonocytic and a minority of cells were lymphoblasts. In addition, myelomonoblasts in liquid cultures appeared to differentiate into mature macrophages. These data suggest the development of acute myelomonocyte leukemia in a previous case of ALL.

Diminished autologous mixed lymphocyte reaction in patients with Hodgkin disease: evidence for non-T cell dysfunction.

In the autologous mixed lymphocyte reaction (AMLR), T lymphocytes are stimulated to proliferate by autologous non-T mononuclear cells. In five untreated patients with Hodgkin disease, the AMLR was diminished. In addition, in the same five patients, T cell response PHA was inhibited by a cell in the non-T cell fraction, the response of non-T cells to PWM was diminished, and there was a diminished ability of the non-T cell population to stimulate in allogeneic MLR. However, the response of T cells from patients with Hodgkin disease to allogeneic antigen was normal. The AMLR and allogeneic MLR were then studied in an additional five untreated patients before and after monocyte depletion of the stimulating non-T mononuclear cell population. In this second group of Hodgkin disease patients, the AMLR was again diminished when T cells were incubated either with non-T cells or non-T cells depleted of monocytes. In the Hodgkin patients, monocyte depletion did not alter the T cell response in the AMLR. In the controls, monocyte depletion greatly diminished the proliferative response. The diminished AMLR in untreated Hodgkin disease patients may be the result of a failure of adequate monocyte stimulation of autologous T cells.

Association of HLA-DR antigens with the autologous mixed lymphocyte reaction.

A study was performed to evaluate the association of HLA-DR antigens with the proliferative response of T cells in autologous mixed lymphocyte cultures. Peripheral blood mononuclear cells from 100 normal healthy individuals were typed for HLA-DR antigens and autologous mixed lymphocyte cultures were established. A low proliferative response from autologous cultures was found with individuals bearing HLA-DR3 antigens and in individuals with only one identifiable HLA-DR antigen. In contrast, a strong proliferative response was associated with HLA-DR6 and two identifiable HLA-DR antigens. These data are consistent with the hypothesis that HLA-DR3 antigens are associated with a weak immune response gene and HLA-DR6 antigens are associated with a strong immune response gene.

HLA antigens and childhood acute lymphocytic leukaemia.

HLA-A and -B antigens were determined for 94 children with acute lymphocytic leukaemia (ALL) and for 376 normal controls. Sixty-four of these 94 patients were typed for lymphocyte surface markers and 59 were defined as 'null cell' ALL. There was no difference in the distribution of the HLA-A or -B locus antigens between the control group and the entire group of patients with ALL or the 'null cell' subgroup. Patients with HLA-A9 determinants had a significant increase in early, first remission duration compared to patients without HLA-A9. This was particularly evident in the 'null cell' ALL subgroup. In addition, HLA-A9 appeared to be an independent factor affecting the length of first remission since there was no correlation between known prognostic factors such as patient age, sex or WBC and the presence or absence of the HLA-A9 antigen. Survival for the first 12-18 months was also greater in the HLA-A9 group than in the non-HLA-A9 population. Thus, the presence of HLA-A9 appears to be associated with some protective effect among patients with ALL.

Studies of mixed lymphocyte reactions, surface B cell antigens, and intracytoplasmic immunoglobulins in "null cell" acute lymphocytic leukemia.

Lymphoblasts from "null cell" acute lymphocytic leukemia (ALL) were analyzed for the pattern of proliferation displayed in a mixed lymphocyte reaction (MLR), the presence of a B cell surface antigen, and for the presence of intracytoplasmic immunoglobulin (ICIg). "Null cell" ALL was defined by cytologic and cytochemical criteria and by the absence of spontaneous rosette formation and surface membrane immunoglobulin in cell suspensions of the malignant lymphocytes. In eleven of fourteen patients the proliferative characteristics of lymphoblasts in the MLR were similar to those observed with normal B enriched lymphocytes. In eleven cases studied, anti-B cell serum reacted with a majority of the lymphoblasts. None of the ten cases examined displayed ICIg in the lymphoblasts. We conclude that the "null" lymphoblast from most cases of ALL is a B cell in an early stage of development.

Detection of antibody to cytomegalovirus-induced early antigens and comparison with four serologic assays and presence of viruria in blood donors.

Five hundred blood donors were evaluated for cytomegalovirus (CMV) viruria, antibody to CMV early antigens (EA-ab), CMV seropositivity by two screening assays, and CMV-specific immunoglobulin M by two methods. Three donors were viruric, EA-ab positive, and seropositive; two viruric donors were immunoglobulin M positive.

Detection of cytomegalovirus-specific IGM in renal transplant recipients.

We have compared two IgM-specific cytomegalovirus (CMV) antibody assays, an immunofluorescence assay (IFA-M) and an enzyme-linked antigen immunoassay (ELA-M), with an assay for CMV total antibody (ELISA) and viral culture for the detection of active CMV infection in renal transplant recipients. Of 75 patients (49 ELISA negative pretransplant, 26 ELISA positive), CMV-specific IgM was detected in 35 (27 ELISA negative pretransplant, 8 ELISA positive) using the IFA-M assay and in 25 (16 ELISA negative pretransplant, 9 ELISA positive) using the ELA-M test. Of the 25 patients identified as positive by ELA-M, 21 had positive viral cultures post-transplant, two seronegative patients had evidence of infection indicated by post-transplant seroconversion, and two patients were seropositive pretransplant but remained viral culture negative throughout the follow-up period. ELA-M and CMV total antibody ELISA detected primary infection in renal transplant recipients equally well, but ELA-M was found to be superior to ELISA and IFA-M for detecting reinfection and reactivation infections.

Examination of whether persistently indeterminate human immunodeficiency virus type 1 Western immunoblot reactions are due to serological reactivity with bovine immunodeficiency-like virus.

The bovine lentivirus, known as bovine immunodeficiency-like virus (BIV), is genetically, structurally, and antigenically related to human immunodeficiency virus type 1 (HIV-1). It is not known whether sera from persons exposed to BIV proteins would show either positive or indeterminate reactivity on HIV-1 antibody tests. We used a BIV Western blot (immunoblot) analysis to examine human sera characterized as HIV-1 antibody positive, HIV-1 antibody negative, HIV-1 persistently indeterminate, HIV-1 p17 antibody positive only, HIV-1 p24 antibody positive only, human T-cell leukemia virus type 1 (HTLV-1) p19 antibody positive only, or HTLV-1 p24 antibody positive only. None of these sera were positive by Western blot to BIV-specific proteins. Many of these sera, however, displayed strong reactivities to bovine cell culture antigens on blots prepared from both mock-infected and BIV-infected cell cultures. The HIV-1 p17 and p24 antibody-positive and the HTLV-1 p19 and p24 antibody-positive sera were further examined by Western blot to bovine leukemia virus (BLV) and were found to be negative. We examined sera from laboratory personnel at risk for BIV exposure, including two laboratory workers who were exposed to BIV by accidental injection with BIV-infected cell culture material, and found no evidence of seroconversion to BIV-specific proteins. We tested 371 samples of fetal bovine sera, each sample representing serum pooled from one to three fetuses. All samples were negative by BIV Western blot. To date, we have not detected any human sera with antibody to BIV-specific proteins. Our data indicate that persistently indeterminate results on HIV-1 Western blot are not caused by a human antibody response to BIV proteins.

Amplification and analysis of specific DNA and RNA sequences of bovine leukemia virus from infected cows by polymerase chain reaction.

Bovine leukemia virus (BLV) is the etiologic agent of leukemia in cattle and is believed to cause decreases in milk productivity, fertility, and life span in infected cows. BLV is a type C retrovirus in the Oncovirinae subfamily. It is most closely related to human T-cell lymphoma/leukemia virus type I (HTLV-I) and type II (HTLV-II). Since the polymerase chain reaction (PCR) provides rapid and efficient amplification of DNA sequences, primers were designed to amplify regions of the polymerase (pol) and pX genes specific for BLV targets. These sets of primers consistently amplified as few as 10 copies of BLV DNA contained in a plasmid in the background of 1 microgram of either human or bovine chromosomal DNA. In addition, no amplification products were detected from cell lines infected with HTLV-I, HTLV-II, or human immunodeficiency virus type 1 or 2 by the BLV PCR systems. Samples of peripheral blood mononuclear cells from 18 cows, previously determined to be serologically positive or negative, were correctly identified in a blind study as containing proviral DNA by use of the BLV primers and probes. Cloning and sequencing of amplified products revealed finite sequence variations among a previously cloned BLV isolate, the wild-type virus, and the published genome. Reverse transcriptase-directed PCR with the primers for both BLV pol and BLV pX was performed on plasma from a BLV-infected cow and detected in vivo BLV RNA expression. In summary, we have developed a specific and sensitive assay using PCR for the detection and identification of BLV infections; this assay can now be applied to clinical and basic research questions in veterinary medicine.