Identification of human B and T lymphocytes by scanning electron microscopy.

In this study a variety of human lymphocytes of known B or T cell type, obtained from multiple sources, were prepared for scanning electron microscopy (SEM) by the critical point drying method. Distinction between normal B and T lymphocytes was relatively easy in most instances, on the basis of their surface architecture. Using immunological methods, between 20 and 30% of normal peripheral blood lymphocytes (PBL) were identified as B cells and from 69 to 82% as T cells. SEM results showed that 20% of the PBL had a complex villous surface and approximately 80% of cells were smaller and had a relatively smooth surface. Comparison of the above data and enrichment of B cells from PBL, by centrifugation after T cell rosettes had formed, indicated that the "villous" cells were B lymphocytes and the "relatively smooth" cells were T lymphocytes. T cells obtained from two human thymuses were also of the generally smooth cell type. Further evidence for the distinction of B and T lymphocytes, on the basis of surface morphology, was obtained from the examination of cultured lymphoid cell lines of known B or T cell derivation. Cells from cases of chronic lymphocytic leukemia also provided support for the above interpretations. Five of six untreated cases were clearly of B cell type by immunologic and SEM criteria. One unusual case showed the presence of T and B lymphocytes in almost equal numbers by SEM and a mixture of B and T cells by immunologic markers. An additional case that had received chemotherapy showed numerous atypical cells that were difficult to classify by SEM. Detailed examination of the smoother T cells showed that at least half of them had a moderate number of surface digitations and a small proportion had an intermediate surface morphology with a relatively large number of surface digitations. The latter presented difficulties in classification and may correspond to different stages of differentiation and represent subpopulations of lymphocytes. The distinction between human B and T lymphocytes on the basis of their surface architecture can be made by SEM of critical point dried samples, with relative ease in most but not all instances. The effects of stimulation, cell cycle, differentiation, intercellular contact, and density of cell population, on the surface architecture of lymphoid cells, remain to be determined.

The nature of the principal type 1 interferon-producing cells in human blood.

Interferons (IFNs) are the most important cytokines in antiviral immune responses. "Natural IFN-producing cells" (IPCs) in human blood express CD4 and major histocompatibility complex class II proteins, but have not been isolated and further characterized because of their rarity, rapid apoptosis, and lack of lineage markers. Purified IPCs are here shown to be the CD4(+)CD11c- type 2 dendritic cell precursors (pDC2s), which produce 200 to 1000 times more IFN than other blood cells after microbial challenge. pDC2s are thus an effector cell type of the immune system, critical for antiviral and antitumor immune responses.

Suppression of B-cell differentiation by leukocytes from hypogammaglobulinemic patients.

Leukocytes from patients with several forms of immunodeficiency characterized by apparently differing defects in B-lymphocyte maturation produced few or no plasmacytoid cells in vitro, and were capable of suppressing the generation of plasma cells in co-culture with cells of normal persons, in the presence of pokeweed mitogen. Such inhibition was commonly observed in cultures which included cells from patients with primary immunoglobulin deficiency, but was not seen to a significant degree in identical co-cultures of cells from normals. The suppression observed was not dependent upon mixed leukocyte culture reactivity. Both sheep erythrocyte-rosetting lymphocytes and adherent cells appeared to participate in these effects in some patients. In one patient with common variable immunodeficiency, but not in several others, removal of suppressing cells permitted the patient's remaining cells to differentiate into plasma cells in vitro. Because of the diverse syndromes in which suppression was observed, it is likely that, in at least some hypogammaglobulinemic patients, the suppression is secondary to the disease process rather than being the primary pathogenic mechanism.

Apoptotic depletion of CD4+ T cells in idiopathic CD4+ T lymphocytopenia.

Progressive loss of CD4+ T lymphocytes, accompanied by opportunistic infections characteristic of the acquired immune deficiency syndrome, ahs been reported in the absence of any known etiology. The pathogenesis of this syndrome, a subset of idiopathic CD4+ T lymphocytopenia (ICL), is uncertain. We report that CD4+ T cells from seven of eight ICL patients underwent accelerated programmed cell death, a process facilitated by T cell receptor cross-linking. Apoptosis was associated with enhanced expression of Fas and Fas ligand in unstimulated cell populations, and partially inhibited by soluble anti-Fas mAb. In addition, apoptosis was suppressed by aurintricarboxylic acid, an inhibitor of calcium-dependent endonucleases and proteases, in cells from four of seven patients, The in vivo significance of these findings was supported by three factors: the absence of accelerated apoptosis in persons with stable, physiologic CD4 lymphopenia without clinical immune deficiency; detection of serum antihistone H2B autoantibodies, one consequence of DNA fragmentation, in some patients; and its selectivity, with apoptosis limited to the CD4 population in some, and occurring among CD8+ T cells predominantly in those individuals with marked depletion of both CD4+ T lymphocytes linked to clinical immune suppression have evidence for accelerated T cell apoptosis in vitro that may be pathophysiologic and amenable to therapy with apoptosis inhibitors.

Opportunistic infections in acquired immune deficiency syndrome result from synergistic defects of both the natural and adaptive components of cellular immunity.

We evaluated the cellular immunity of 408 clinically stratified subjects at risk for acquired immune deficiency syndrome (AIDS), to define the role of interferon-alpha production deficits in the pathogenesis of opportunistic infections (OI). We followed 115 prospectively for up to 45 mo. Onset of OI was associated with, and predicted by, deficiency both of interferon-alpha generation in vitro, and of circulating Leu-3a+ cells. Interferon-alpha production is an index of the function of certain non-T, non-B, large granular lymphocytes (LGL) that are independent of T cell help. Leu-3a+ cell counts are a marker of T cell function. OI did not usually develop until both of these mutually independent immune functions were simultaneously critically depressed, leading to a synergistic interaction. These data suggest that the AIDS virus affects a subset of LGL, and that cytokine production by these cells is an important component of the host defense against intracellular pathogens that becomes crucial in the presence of severe T cell immunodeficiency.

In vitro studies of axillary lymph node cells in patients with breast cancer.

A total of 170 axillary lymph nodes were obtained from fresh mastectomy specimens from 81 women with breast cancer. Lymph node cells were tested in vitro for T and B cells by the rosette technique and immunofluorescence microscopy and for functional capacity by response to the mitogens phytohemagglutinin (PHA) and concanavalin A. T cells showed a wide range of relative values: 32-80 percent, with a mean of 63.5 percent. B cells defined by the presence of surface immunoglobulins ranged from 14 to 61 percent (mean, 35.8 percent); those defined by the presence of C3 receptors, from 8 to 54 percent (mean, 24.9 percent); and those defined by the presence of IgG-specific (Fc) receptors, from 10 to 45 percent (mean, 27.5 percent). Cells with the C3 and Fc receptors constituted approximately two-thirds of the cells not binding spontaneously to sheep red blood cells (non-SRBC-R), whereas virtually all non-SRBC-R stained for surface immunoglobulins. The proportion of T and B cells and the response to mitogens varied widely among nodes and among patients. Differences were significant between values observed in young and old patients, nodes with and those without metastatic disease, and lymph nodes with different morphology. Lymph nodes from patients over 60 years old showed a higher proportion of B cells and a lower proportion of T cells than did those from patients 45 years of age or younger. Lymph nodes with disease metastic to them also showed a higher percent of B cells and a lower percent of T cells than the nodes that did not have metastatic disease. Lymph nodes with lymphocyte predominance showed a relatively high proportion of T lymphocytes, a high PHA response, and a low content of B lymphocytes. By contrast, lymph nodes with germinal-center predominance showed a relatively low content of T cells, a low PHA response, and a relatively high proportion of B lymphocytes.

Impaired interferon alpha response in hairy cell leukemia is corrected by therapy with 2-chloro-2'-deoxyadenosine: implications for susceptibility to opportunistic infections.

Patients with hairy cell leukemia (HCL) are susceptible to opportunistic intracellular infections, suggesting defects in cellular immunity. Prior studies have indicated an association between failure of IFN-alpha generation by peripheral blood mononuclear cells (MNC) and susceptibility to such infections. We here present results on IFN-alpha generation in HCL patients pre- and post-therapy. Prior to treatment with 2-chloro-2'-deoxyadenosine (CdA), MNC from 24 HCL patients with active disease produced little or not IFN-alpha (geometric mean < 40 IU/ml) compared with controls (n = 140, geometric mean 1730 IU/ml, p < 0.0005). After treatment with CdA, IFN-alpha generation was studied in 16 patients, with a geometric mean value of 650 IU/ml (p < 0.0005 compared with pre-CdA levels). The severe depression of IFN-alpha generation improved progressively following CdA therapy-induced clinical remission. We propose that deficiency of IFN-alpha production may play a role in the susceptibility to intracellular infections of patients with active HCL.

Isoferritins in HIV infection: relation to clinical stage, CD8 lymphocyte binding and the pathogenesis of AIDS.

Placental isoferritins (PLF), known to be immunosuppressive in Hodgkin's disease and other states, were found to be increased in sera of subjects infected with HIV. We assayed for PLF using a 'sandwich' antigen capture enzyme-linked immunosorbent assay (ELISA) employing two monoclonal antibodies. Individuals with lymphadenopathy, with or without symptoms suggestive of AIDS-related complex, had the highest serum levels, which declined with progressive immunodeficiency. Total (normal) ferritins, in contrast, increased progressively with stage of disease. PLF was found on a subset of CD8 lymphocytes and appeared to block detection of the CD8 antigen by specific monoclonal antibodies. Elution of PLF by incubation with levamisole, but not by culture medium alone, led to the unblocking of the CD8 determinant on these cells. Profiles of isoferritins in HIV infection may provide clues to prognosis. PLF, a physiologic down-regulator of hematopoiesis and cellular immunity, could play a role in the progressive immune deficiency, marrow suppression and HIV expression that lead to AIDS.

Interferon-alpha generation and immune reconstitution during antiretroviral therapy for human immunodeficiency virus infection.

OBJECTIVES: To quantify the effect of HIV infection and HIV-suppressive therapy on interferon-alpha (IFN-alpha) production by human blood mononuclear cells; to compare, in parallel, effects on CD4+ T-cell numbers; and to ascertain the relationship of these interferon and CD4 parameters to resistance to opportunistic infections. DESIGN: Serial studies of 294 unselected patients with HIV infection during therapy, with outcomes analysis. METHODS: Determination of IFN generation by blood mononuclear cells via bioassay, and T-lymphocyte subset analysis via flow cytometry; serial studies of individual patients; linear regression and chi2 contingency table analysis. RESULTS: HIV burden is inversely related to interferon-alpha generation, much as it is to CD4+ T-cell counts. Both of these recover during HIV-suppressive therapy. Reconstitution of IFN-alpha generation to levels commensurate with protection against opportunistic infection occurs prior to similar restoration of CD4 counts. In the outcomes analyses, such immune reconstitution was associated with protection from recurrent or new opportunistic infection. Conversely, viral suppression without such immunologic recovery was not protective against opportunistic infection. CONCLUSIONS: Rapidly responding IFN-alpha generating cells appear to participate in resistance to opportunistic intracellular infection. Recovery of IFN-alpha generation may be an early marker of immune reconstitution in AIDS.

Dose-limiting toxicity of rifabutin in AIDS-related complex: syndrome of arthralgia/arthritis.

We studied the tolerance of humans to rifabutin, a rifamycin with antimycobacterial and in vitro anti-HIV activity. Sixteen subjects with AIDS-related complex were treated for 4-66 weeks with stepwise increasing oral doses of rifabutin from 300 to 2400 mg/day. The highest dose attained was twice that previously reported for humans. Serum and cerebrospinal fluid levels of drug were detected by high-pressure liquid chromatography. A reversible syndrome of arthritis/arthralgia, not previously described, was seen in most (nine out of 10) of those given doses exceeding 1050 mg/day. Uveitis and aphthous stomatitis developed at doses of approximately 1800 mg in two of those with joint manifestations. Typical manifestations of Reiter's syndrome were not seen in any patient. An orange-tan skin pigmentation was almost universal. Other toxicities resembled those previously associated with rifampin. Serum levels did not approach those found to inhibit HIV significantly in vitro. No consistent antiviral or immunological effects were observed; even at the highest doses, rifabutin did not appear to inhibit cellular immunity. Rifabutin was well tolerated at daily doses blow 1 g.

Efficacy of rifabutin in the treatment of AIDS-related complex.

We performed a phase 1-2 antiviral dose escalation trial of rifabutin, a rifamycin antibiotic with anti-HIV-1 activity in vitro. We followed 16 men with AIDS-related complex (ARC) for a mean duration of 29 weeks; the maximum toxicity-limited dose of rifabutin was 2400 mg/day, which was achieved in two patients. There was some evidence of anti-HIV-1 activity in two patients, one of whom had an improvement in immune status, but 11 of the 16 patients showed a deterioration in either virologic or immunologic status. The majority of the patients under study remained clinically stable during the trial, but there was clinical deterioration in the three who entered with CD4 cell counts of less than 100 x 10(6)/l. On the basis of this trial, rifabutin as a single antiviral agent does not appear to be beneficial to ARC patients.

Peripheral blood of AIDS patients contains cells capable of providing accessory function for the natural killer cell-mediated, lysis of herpes simplex virus-infected targets despite low interferon-alpha production.

We have previously demonstrated that in vitro production of interferon-alpha (IFN-alpha) in response to herpes simplex virus (HSV) by peripheral blood mononuclear cells PBMCs from patients infected with the human immunodeficiency virus (HIV-1) decreases dramatically with disease progression, with extremely low levels of IFN-alpha preceding and predictive of opportunistic infections. Natural killer (NK) lysis, however, was found to decay later in disease and often was within normal limits even when IFN-alpha production was severely compromised. The NK lysis of HSV-infected fibroblasts (HSV-FS) is dependent on an HLA-DR+ accessory cell (AC) population that shares the phenotype of the predominant IFN-alpha-producing cell (IPC) population. To determine whether there is a correlation between AC activity and IFN-alpha production in these patients, we tested the ability of PBMCs from AIDS patients to provide AC help to NK cells from heterologous donors. While NK cells were highly sensitive to gamma irradiation, AC activity was relatively radioresistant. Therefore, NK cells from healthy donors were depleted of HLA-DR+ ACs and added to irradiated PBMCs from either healthy or AIDS donors to test for the function of ACs in the irradiated populations. Irradiated cells from AIDS patients were found to provide normal AC activity despite decreased IFN-alpha production in the majority of the patients. We failed to observe NK augmenting activity in supernatants of irradiated PBMCs from IFN-deficient patients that had been stimulated with HSV-FS.(ABSTRACT TRUNCATED AT 250 WORDS)

Hyponatremia in patients with acquired immune deficiency syndrome.

We performed prospective and retrospective studies of 96 consecutive patients with acquired immune deficiency syndrome (AIDS) or AIDS-related complex (ARC) to determine the incidence, pathogenesis, and clinical significance of hyponatremia, defined as serum sodium levels less than or equal to 130 mmol/L on more than one occasion. Thirty (31.3%), six with ARC and 24 with AIDS, had hyponatremia, and it developed in 20 as outpatients. Age, gender, duration of illness, and weight loss did not differ between groups. The hyponatremic patient had more opportunistic illnesses, including Pneumocystis carinii pneumonia and cytomegalovirus infections, and had a mortality of 70% as compared to 36.4% of the patients without hyponatremia. The probability of 50% survival after diagnosis of human immunodeficiency virus (HIV) infection in the hyponatremic group was 11.5 months, as compared to 39 months for those without hyponatremia, p less than 0.001. The probability of 50% survival after development of hyponatremia was 4.5 months and the median length of time to development of hyponatremia was 12.5 months after diagnosis of HIV infection. Eighty-eight percent had hypovolemia and 12% normovolemia. Seventeen of 21 with hypovolemia had no evident source of fluid loss. Two had Addison's disease, and 15 had unexpectedly high urine sodium concentration without evidence of renal or adrenal insufficiency. Hyponatremia occurs commonly in ambulatory patients with ARC or AIDS, appears in patients with higher mortality and morbidity, and does not represent a terminal event. Most patients had hypovolemia and unexpectedly high urine sodium concentration, suggesting defective renal sodium conservation.

Fatal disseminated adenovirus 11 pneumonia in an agammaglobulinemic patient.

Adenovirus type 11, an organism not previously associated with pneumonia, caused the death of a patient with infantile x-linked agammaglobulinemia who had normal cell-mediated immunity. Despite long-standing, regular therapy with immune globulin, his serum lacked neutralizing antibody to the virus. This case confronts the conventional view that viral infections are primarily resisted by cellular immune reactions and reemphasizes the importance of antibody in the host defense against adenoviruses. It further demonstrates the continued vulnerability of such patients to certain pathogens in the presence of presumably adequate standard-dose passive immunization.

Association of a chromosomal abnormality with lymphocytes having both T and B markers in a patient with lymphoproliferative disease.

The lymphocytes of a patient with leukemic lymphosarcoma were found to have an unusual surface phenotype in that they bound both sheep erythrocytes (a T cell marker) and complement-coated erythrocytes (a B cell marker) but lacked other B cell surface characteristics. Marker chromosomes were present in these cells, but not in other, phenotypically normal cells from the same patient. This case may provide a clue to the chromosomal origin of some lymphocyte surface markers in man.

Cell replication in an immunologically(?) Stimulated cell population in human bone marrow.

In AIDS, although there is a lack of humoral responsiveness in vitro and in vivo, many patients persistently have an increased number of B cells which continue to produce increased amounts of immunoglobulin. An objective, reproducible morphologic classification scheme for B cells was devised. Comparison of cell kinetic parameters in various disease states will require such a classification. Although not immunologically responsive to new stimuli, the marrow B cells in the AIDS patients were shown to be replicating and turning over. The latter may be due to either death in situ or to migration. Plasmacytic lymphocytes and lymphocytic plasma cells, morphologic transitions between lymphocytes and mature plasma cells, had the largest fractions in DNA synthesis. Because of their relative cell numbers, the lymphocytic plasma cells contained most of the cells in DNA synthesis. The position of plasmablasts in the sequential compartments is unclear. Only small numbers are dividing. Within a given morphologic category, large cells were more likely to be in DNA synthesis than smaller cells. These studies can serve as a basis for comparison with marrow B-cell proliferation in other disease states.

Deficiency of interferon-alpha generating capacity is associated with susceptibility to opportunistic infections in patients with AIDS.

Acquired immune deficiency syndrome (AIDS) is a newly described syndrome in which patients are susceptible to certain malignancies and opportunistic infections (OI) usually found only in immunosuppressed individuals. Patients with AIDS have been found to have deficiencies of virtually all of their host defense systems. In this report, the natural resistance systems have been discussed. Although a deficiency of NK-cell function has been found in many patients with AIDS, this deficiency failed to distinguish patients susceptible to OI or malignancy from male homosexual controls. A deficiency of interferon-alpha generation by mononuclear cells upon exposure to HSV-1 infected fibroblasts was the best correlate with susceptibility to OI in AIDS patients. This deficiency failed to correlate with serum levels of acid-labile interferon-alpha in these patients. Although the interferon generating deficiency may be caused by the infections in these patients, it is more likely that the deficiency lays the groundwork for the establishment of the opportunistic infections.

Progressive nemaline rod myopathy in a woman coinfected with HIV-1 and HTLV-2.

Nemaline-rod myopathy was recently reported in eight young males infected with human immune deficiency virus type 1 (HIV-1). A 41-year-old woman had a 2-year history of progressive proximal-muscle weakness. Muscle biopsy demonstrated the presence of nemaline rods, predominantly in type 1 fibers. She was coinfected with HIV-1 and HTLV-2, as evidenced by positive polymerase chain reaction and serology. There was no lymphopenia or CD4 lymphopenia, despite an abnormal T-cell subset ratio, high CD8 count, skin anergy, and depressed in vitro response to mitogens. This case raises the possibility that dual infection may play a role in the pathogenesis of the rare nemaline-rod myopathies of HIV-infected patients.

Absence of age-related increase in systolic blood pressure in ambulatory patients with HIV infection.

BACKGROUND: Systolic blood pressure is well known to increase significantly with age and is strongly correlated with stroke and coronary artery disease. We and other investigators have reported a low prevalence of hypertension in subgroups of patients with HIV infection. In the present study, we examined an ambulatory population of patients with HIV infection to determine whether in the outpatient setting they may lack an age-related increase in systolic blood pressure. METHODS: In an ambulatory outpatient practice, medical records of 178 consecutive patients with HIV infection and those of 200 control subjects were examined. Systolic and diastolic blood pressure and other clinical and laboratory variables were recorded. Scatter plots were generated to compare age with systolic blood pressure. Spearman rank correlation analysis was carried out to determine the relationship between systolic blood pressure and age and other variables. RESULTS: Patients ranged in age from 13 to 69 years. There was only a very slight increase (which did not achieve statistical significance) in systolic blood pressure with aging in the patients with HIV infection, in contrast to the control population, in which an age-related increase in systolic blood pressure was seen that was comparable to published Framingham data. Mean systolic blood pressure for the group as a whole was 118.2 +/- 1.1 mm Hg. Mean serum albumin was 4.2 +/- 0.04 g/dL and was only slightly diminished in older patients. Mean serum cholesterol was 176.8 +/- 3.4 mg/dL and this bore no relationship to aging. More advanced stages of HIV infection also did not correlate with the lack of age-associated systolic hypertension. CONCLUSION: The present population of ambulatory patients infected with HIV seem to lack an age-related increase in systolic blood pressure; this may be caused by such variables as autonomic dysfunction or factors that may attenuate the development of atherosclerosis.

Acquired immunodeficiency syndrome. Distinctive features of bone marrow biopsies.

Bone marrow biopsies from 30 patients with acquired immunodeficiency syndrome (AIDS), diagnosed according to accepted clinical criteria, were studied in order to determine if characteristic histopathologic features were present. The biopsies were compared with 20 biopsy specimens submitted from patients with fever of unknown origin or with known neoplastic disease, including four biopsies from patients with a history of narcotics addiction being treated in the methadone clinic, and with biopsies from three homosexual men who did not have AIDS. Characteristic biopsy features were recognized in 31 (86%) of 36 biopsy specimens from AIDS patients. Nineteen of 36 patients (53%) showed a distinctive pattern of hypercellularity in which hematic cells (immature granulocytic cells, megakaryocytes, eosinophils, and large lymphocytes) separated, but did not efface, fat cells; these cells were designated as "AIDS-pattern." Twelve specimens showed a less complete (probable-AIDS) bone marrow change. Reticulin fibers were increased in 28 of 36 biopsies.