Human immunodeficiency virus (HIV) and Epstein-Barr virus (EBV) antigens and genome in lymph nodes of HIV-positive patients affected by persistent generalized lymphadenopathy (PGL)

The presence of human immunodeficiency virus (HIV) and Epstein-Barr virus (EBV) antigens and genome has been investigated in 50 lymph nodes involved by persistent generalized lymphadenopathy (PGL). All the patients were HIV infected and most of them (42 of 50) also had anti-EBV serum antibodies. At lymph node level, HIV and EBV antigens were studied by immunohistochemistry using monoclonal antibodies directed against viral core proteins. The HIV p24 protein was detected in 43 of 50 lymph nodes within the B-cell germinal centers with a reticular pattern. Few cells with positive results for EBV antigens were found in only 2 of 50 lymph nodes. These rare EBV-positive centrocyte-like cells were mainly located in the germinal centers. The presence of HIV and EBV genome was also studied in lymph nodes involved by PGL, with the use of in situ and Southern blot hybridization. A positive reaction for HIV genome was detected in only 1 of 14 lymph nodes with the Southern blot hybridization, and the presence of EBV genome was never demonstrated in these lymph nodes with the use of both in situ and Southern blot hybridization. The expression of EBV antigens and genome was also investigated in the peripheral blood of 15 patients with PGL in which cells with positive results for EBV antigens were detected in a single case with a frequency of 1 X 10(-4). No evidence of EBV genome was found with the use of the in situ hybridization. These results suggest that EBV is not present in lymph nodes during the PGL phase and that its possible implication in the pathogenesis of acquired immune deficiency syndrome (AIDS)-associated lymphoma might be a late event.     

HIV is trapped and masked in the cytoplasm of lymph node follicular dendritic cells.

To gain further insight into the pathogenesis of human immunodeficiency virus (HIV) infection, lymph nodes from seven asymptomatic HIV+ subjects were analyzed during the latent phase of disease. Both ultrastructural and immunohistochemical analyses revealed that, in all of the cases, plasma cells producing IgM/gamma were present in germinal centers. Secreted immunoglobulins formed extracellular deposits mimicking the follicular dendritic cell network. Immunoglobulin produced by germinal center plasma cells are specific for HIV because they bind the HIV env protein gp 120. Plasma cells producing antibodies with the same specificity were also abundant in the extrafollicular regions of lymph nodes. During the latent phase of infection, the virus largely accumulates within the germinal centers. Therefore, extracellular immunoglobulin may form immune complexes, as shown by the presence of HIV-specific antibodies, HIV particles, and complement components C3c, C3d, and C1q in the interdendritic spaces. When the ultrastructural localization of HIV in germinal centers was analyzed, abundant virus particles were found in the interdendritic spaces. In addition to this extracellular localization of HIV, receptor-mediated endocytosis of viral particles by follicular dendritic cells was observed. Complete HIV particles were found within the endosomal compartment of the follicular dendritic cells and, as complete viral particles, free in the cytoplasm, indicating that the virus may escape from the endocytic compartment. As the virus is abundant in the cytoplasm, this event leads to formation of a hidden reservoir within follicular dendritic cells. In this location, HIV escapes recognition by cytotoxic T lymphocytes. In contrast, virus budding indicating a productive infection of follicular dendritic cells that would render them susceptible to T-cell-mediated lysis has been seldom observed.     

Persistent and generalized lymphadenopathy: a lesion of follicular dendritic cells? An immunohistologic and ultrastructural study

An immunohistochemical and ultrastructural study of 14 cases of persistent and generalized lymphadenopathy (PGL), acquired immune deficiency syndrome (AIDS) related, revealed florid follicular hyperplasia, follicular dendritic cell (FDC) lysis, lymphoid follicle invaginations, increased presence of T8 cells in germinal center, immature sinus histiocytosis (monocytoid B-cells), and inversion of T4/T8 ratio in the paracortical area. Electron microscopic examination showed viral particles of morphologic characteristics consistent with human immunodeficiency virus (HIV) virions attached to the processes of FDC in three of the nine cases studied. Lesions of the germinal center dendritic cell network are the cardinal feature of PGL. This finding lends support to the idea of a viral aggression directed against FDC as the cause of disregulation of the B-cells.     

Clinico-immunopathological alterations of lymph nodes from human immunodeficiency virus-infected patients in northern Thailand.

To determine if the immunopathologic alterations of HIV-infected lymph nodes have any correlation with clinical stages in the northern Thai patients, we conducted a comparative analysis of immunopathologic features of lymph nodes between 25 HIV-infected patients from various clinical categories and 25 non-HIV individuals of reactive hyperplasia morphology of lymph node biopsies. The risk factors for HIV infection were all heterosexual. The majority of patients in clinical category A (PGL) showed a histopathologic pattern of explosive follicular hyperplasia, while category C (AIDS) patients demonstrated follicular involution and lymphocyte depletion on lymph node sections. Interestingly, weak reactivity for HIV p24 gag protein was detected within the germinal centers and scattering interfollicular lymphocytes in only 20% of the HIV-infected cases. Morphologically, the presence of MGCs was specific for HIV-infected lymph nodes. MGCs (hematoxylin & eosin stain) were found in 64% of the HIV-infected cases, which was significantly different from 4% found in control cases (p = 0.00002). By S-100 immunostaining, MGCs were demonstrated in all HIV-infected lymph node sections, while they were found in 32% of the control lymph nodes. Immunostaining with S-100 protein also revealed the appearance of syncytial ballooning and countable numbers of MGCs. High numbers of MGCs seemed to correlate with histologic and clinical changes. In conclusion, the HIV-infected patients had high numbers of MGCs or syncytia on lymph node sections in early stage and pre-AIDS conditions, which has never been reported before.     

LAV-like viral particles in lymph node germinal centers in patients with the persistent lymphadenopathy syndrome and the acquired immunodeficiency syndrome-related complex: an ultrastructural study of 30 cases

The detection of LAV- or HTLV III-type viral particles in lymph node germinal centers from patients with the persistent lymphadenopathy syndrome (LAS) or the acquired immunodeficiency syndrome (AIDS)-related complex (ARC) is an important diagnostic factor in the prodromal stages of AIDS. These particles, the morphology of which is defined, are situated solely in the extracellular spaces delimited by cytoplasmic extensions of the dendritic reticular cells. Often few in number, they were found in 26 of the 30 lymph nodes studied, selected uniquely on the basis of light microscopic criteria (predominantly follicular lymphoid hyperplasia). The four negative nodes contained no, or fewer than two, germinal centers in the samples taken for ultrastructural study. The diagnosis of the LAS or the ARC was always confirmed clinically and biologically. Thus, lymph node biopsy and the corresponding ultrastructural study are important steps in the diagnosis of AIDS.     

The immunohistology of the persistent generalized lymphadenopathy syndrome (PGL)

The authors employed a large panel of monoclonal antibodies to characterize and quantitate lymphoid subpopulations within the paracortex, mantle, and germinal centers of frozen sections of lymph nodes from 18 patients with the persistent generalized lymphadenopathy (PGL) syndrome and five heterosexual controls. The authors' data indicate that Leu-3+ phenotypic T-helper cells (TH) are reduced within all three compartments, while T-cytotoxic-suppressor cells (Tcs) are increased. Using the antibody 9.3, which allows dissection of the Leu-2+ Tcs subset into 9.3+ cytotoxic cells (Tc) and 9.3- suppressor cells (Ts), the authors found that the Ts subset is increased in the lymph nodes of these patients. In contrast to acquired immune deficiency syndrome (AIDS) patients, paracortical total T-cells and Leu-8+ cells appear to be preserved in patients with PGL. Study of TH and Tcs subpopulations in peripheral blood in 12 of these patients revealed inverted ratios (mean, 0.59), which did not correlate with those seen in the lymph nodes. Although the paracortical TH/Tcs ratios were significantly reduced (mean, 1.44) they were not inverted, in contrast to some other reported series. In aggregate, these data suggest that, relative to AIDS, there is preservation of the paracortical T-cell microenvironment in PGL. Clinically, this correlates with more intact cell-mediated immunity and the absence of opportunistic infections and Kaposi's sarcoma in this patient group. Follicle lysis was present in 11 patients. Increased HLA-DR+ paracortical cells, aggregates of Leu-6+ dendritic cells, decreased TAC+ cells, increased OKT-10+ plasma cells, and increased interstitial immunoglobulin were among the other features observed in these patients.     

Thymic pseudotumorous enlargement due to follicular hyperplasia in a human immunodeficiency virus sero-positive patient. Immunohistochemical and molecular biological study of viral infected cells.

An enlargement of the thymus suggesting a tumor was discovered in a 28-year-old man who had early-stage acquired immune deficiency syndrome. A biopsy was performed. The adipose involuted thymus, with persistence of many Hassall's corpuscles, was judged to be a large lymphoid follicular hyperplasia. This follicular hyperplasia was similar to that previously described for lymph nodes, spleen, and other lymphoid tissues at earlier stages of human immunodeficiency virus infection, before the development of acquired immune deficiency syndrome. Human immunodeficiency virus RNA and p24 human immunodeficiency virus protein were detected in the hyperplastic germinal centers (lymphocytes and follicular dendritic infected cells), and also in many cells that may have been either lymphocytes and/or epithelial cells in the interfollicular areas. The tissue was negative for Epstein-Barr virus DNA sequences, as determined by the polymerase chain reaction. These observations identify the first state of infection of the thymus in a human immune deficiency virus-infected adult, preceding the severe involution with lymphoid depletion observed in all fatal cases of acquired immunodeficiency syndrome in which the thymus has been analyzed.     

Distribution of T-cell phenotypic subsets and surface immunoglobulin-bearing lymphocytes in lymph nodes from male homosexuals with persistent generalized adenopathy: an immunohistochemical and ultrastructural study

Lymph nodes from homosexual men with persistent generalized adenopathy were evaluated for distribution of T-cell phenotypic subsets and surface immunoglobulin(SIg)-bearing lymphocytes. Electron microscopy revealed tubulovesicular structures within lymphocytes but no multivesicular rosettes. Eight to 33 per cent of the lymphocytes within germinal centers were suppressor T cells, compared with germinal centers from control lymph nodes, in which these cells were rare (P = 0.002). Significantly greater percentage of suppressor/cytotoxic T lymphocytes were also present in the paracortex and follicular mantles of lymph nodes from the homosexual group (P = 0.002 and 0.007, respectively). Percentages of helper T lymphocytes were significantly decreased in germinal centers (P = 0.008) and paracortical regions (P = 0.002). Florid follicular hyperplasia with aberrations in follicular architecture was the most common histologic pattern, but one node with diffuse hyperplasia and subtotal effacement of architecture revealed depletion of SIg-bearing lymphocytes and increased numbers of suppressor T cells. Reversed helper-to-suppressor T-cell ratios in lymph nodes from homosexuals with generalized adenopathy may be related to viral infection and contribute to immune deficiency in this group.     

DETERIORATION OF B CELL PROLIFERATION CORRELATES WITH DENDRITIC RETICULUM CELL DESTRUCTION IN GERMINAL CENTERS OF AN AIDS PATIENT. Case Study

A lymph node from a bisexual Caucasian male infected with human immunodeficiency virus (HIV) and in the persistent generalized lymphadenopathy (PGL) stage was studied. Dendritic reticulum cells (DRCs) were well preserved in over half of the germinal centers (GCs), while in the rest, they showed marked destruction, producing patchy or rather wide DRC-depleted areas. Proliferation-associated antigens, i.e., PC antigen and DNA-polymerase a, were demonstrated in nuclei of germinal center B cells in areas where the DRC network was intact, while they were prominently depleted in areas where the DRC network was lost. The p-24 viral core antigen was shown to be localized in DRCs, especially those in the process of degeneration. These results suggest that the DRC in this patient, when infected with HIV, were destroyed, and that the resulting DRC depletion led to the suppression of B cell proliferation in GCs. ACTA PATHOL JPN 38: 1205∼1214, 1988.     

Deterioration of B cell proliferation correlates with dendritic reticulum cell destruction in germinal centers of an AIDS patient. Case study

A lymph node from a bisexual Caucasian male infected with human immunodeficiency virus (HIV) and in the persistent generalized lymphadenopathy (PGL) stage was studied. Dendritic reticulum cells (DRCs) were well preserved in over half of the germinal centers (GCs), while in the rest, they showed marked destruction, producing patchy or rather wide DRC-depleted areas. Proliferation-associated antigens, i.e., PC antigen and DNA-polymerase alpha, were demonstrated in nuclei of germinal center B cells in areas where the DRC network was intact, while they were prominently depleted in areas where the DRC network was lost. The p-24 viral core antigen was shown to be localized in DRCs, especially those in the process of degeneration. These results suggest that the DRC in this patient, when infected with HIV, were destroyed, and that the resulting DRC depletion led to the suppression of B cell proliferation in GCs.     

Expression of RNA and antigens of human immunodeficiency virus type-1 (HIV-1) in lymph nodes from HIV-1 infected individuals

The presence of proteins (p17 and p24 core proteins, gp41 envelope protein) and mRNA (gag/pol and env gene segments) of human immunodeficiency virus type-1 (HIV-1) was analyzed on frozen tissue sections of lymph nodes from HIV-1 infected individuals. Thirty-one lymph nodes were categorized in the stages of follicle hyperplasia (n = 18), follicle degeneration (n = 5), and total depletion (n = 8). The follicle dendritic cells in germinal centers showed the presence of core proteins and, to a lesser extent, gp41. The staining patterns, being similar to those of immunoglobulins, suggested that they occur in the form of immune complexes. In addition there were solitary cells expressing viral protein, in particular gp41, and mRNA. The number of mRNA-positive cells was very low: about five positive cells were observed in a tissue section with about ten (hyperplastic) follicles. HIV-1-mRNA-positive cells were observed both in follicles and interfollicular areas and showed no differences between various stages. The extent and intensity of distinct HIV-1 proteins and HIV-1-mRNA gene segments in follicles were significantly correlated, as was their presence in interfollicular areas. No significant correlation was found between the presence of HIV-1 components in follicles and in interfollicular areas. This indicates that processes involving HIV-1 components occur in a segregated manner in both lymph node compartments. The presence of HIV-1 components did not correspond to any clinical classification (CDC criteria), nor to other histochemical characteristics. An exception was the correlation between gp41-positive cells and CD1-positive interdigitating cells in the interfollicular areas.     

Reactive lymphadenopathy in Ugandan patients and its relationship to EBV and HIV infection

In Uganda, a large number of biopsied enlarged lymph nodes is diagnosed as reactive lymphoid hyperplasia (RLH) not indicative of a specific etiologic agent. The aim of this study was to examine the spectrum of RLH in lymph node biopsies in Ugandan patients and their possible association with HIV and EBV infection. Ninety biopsies were retrieved and included in the study. The predominant RLH type was follicular, found in 45 (50.0%) of the cases. Positive staining for LMP‐1 was found in six cases (6.7%), EBNA‐1 in 36 cases (40.0%) and HIV1‐p24 in 15 cases (16.7%), respectively. A combination of EBV and HIV positivity was found in 46 (52.2%) of the cases. EBV infection was associated with hyperplastic germinal centers (p<0.01). HIV1‐p24 positive staining was associated with follicle fragmentation (p<0.01) but not hyperplastic GC (p=0.08). In conclusion, RLH in Ugandan patients is frequently associated with EBV and HIV infection. The histologic features of the lymph nodes are not specific for any individual infection, but a high number of EBV‐positive cases are associated with hyperplastic GC, and follicular fragmentation is characteristic of HIV infection.     

Transfer of immune complexes from lymphocytes to follicular dendritic cells

Antigens in the form of immune complexes are retained on the membranes of follicular dendritic cells (FDC) for long periods of time. To examine how immune complexes reach germinal centers, where FDC are located, we injected mice with anti-2,4-dinitrophenyl (DNP) antibodies complexed to DNP-myoglobin-coated gold particles. The distribution of the particles in spleens or draining lymph nodes was then determined with the electron microscope. The vast majority of the particles were cell bound. Shortly after injection they were phagocytized by macrophages or fixed on lymphocytes. The latter were found even in the corona of lymph follicles but not in germinal centers. Already 30 min after injection, FDC in contact with the corona were faintly positive but were negative in the center. FDC precursor cells were occasionally observed but in too small a number to account for the transport of immune complexes to the germinal centers. Twenty-four hours after injection colloidal gold particles were found in phagolysosomes of macrophages or on cytoplasmic extensions of FDC in all parts of the germinal centers. Experiments performed on isolated FDC showed that they are not only able to take up free immune complexes but are also able to adsorb immune complexes from pulsed lymphocytes. These results strengthen the idea that lymphoid cells binding immune complexes by their Fc receptors may transport these complexes inside germinal centers.     

The immunohistology of non-T cells in the acquired immunodeficiency syndrome.

The authors employed a panel of monoclonal antibodies to characterize B cells, histiocytes, and natural killer cells in lymph node biopsies obtained from 7 homosexual men with the acquired immune deficiency syndrome (AIDS) and 5 heterosexual controls. Six of the AIDS patients, each with cutaneous and/or nodal Kaposi's sarcoma, exhibited reactive follicular hyperplasia, as did the 5 controls. The seventh AIDS patient had opportunistic infections and exhibited a lymphocyte depletion pattern in lymph nodes. In AIDS patients, reactive B-cell follicles often exhibited attenuation of their mantle zones. Many were regressively transformed and composed predominantly of dendritic reticulum cells. Occasional germinal center dendritic reticulum cell networks exhibited fragmentation reminiscent of the "follicle lysis" described previously in the persistent generalized lymphadenopathy syndrome. In the 1 AIDS patient with the lymphocyte depletion pattern of lymph node histology, germinal center elements, including dendritic reticulum cells, were totally absent. In all cases, the mononuclear-cell subsets exhibited normal patterns of antigen expression. Quantitative studies, however, revealed a significant increase (P less than or equal to 0.01) in natural killer cells and histiocytes within the paracortical T-cell domain of lymph nodes obtained from patients with AIDS. There was no significant difference in the number of natural killer cells within the mantle or germinal center B-cell domains. While there was no significant difference in the absolute number of paracortical B cells, they were relatively increased due to an absolute decrease in T cells. It is concluded that quantitative alterations in mononuclear-cell subsets in patients with AIDS are not restricted to T cells and that these alterations are microenvironmentally specific.     

Lymph node pathology of acquired immunodeficiency syndrome (AIDS)

There has been a recent notable increase in the number of patients in the United States seropositive for the human immunodeficiency virus (HIV) and also an increase in the number of otherwise healthy homosexuals with persistent generalized lymphadenopathy (PGL). Lymphoid tissue appears to be a favorite target for the initial viral infection, subsequent opportunistic infections, and associated neoplasms. Therefore, evaluation of PGL is important in understanding the nature of the disease. Biopsies of the acquired immunodeficiency syndrome (AIDS) lymph nodes show a spectrum of abnormal lymphoid proliferations, eventual lymphoid depletion, Kaposi's sarcoma, and malignant lymphoma. Although the individual features of AIDS-related lymphadenopathy may not be specific, the constellation of histologic, immunologic, ultrastructural, and fine needle aspiration findings is characteristic.     

HTLV-III/LAV Viral Antigens in Lymph Nodes of Homosexual Men With Persistent Generalized Lymphadenopathy and AIDS

The presence of core antigens of retrovirus HTLV-III/LAV, referred to as "AIDS-related virus" (AV), has been sought in lymph node samples of patients with persistent generalized lymphadenopathy (PGL, 28 patients), prodromal AIDS (1 patient) and AIDS with Kaposi sarcoma (3 patients). In 30 patients the deposition of viral antigens, detected by monoclonal antibodies to HTLV-III and LAV, could be observed within the germinal centers (GCs) primarily within the extracellular network of immune complexes, and the two patients who were negative were atypical. No AV could be found in normal tonsil or in samples with follicular hyperplasia of unknown etiology (20 cases). These findings, taken together with the ultrastructural identification of typical retrovirus particles in all 9 PGL and 2 AIDS cases studied, indicates that the network of follicular dendritic (FD) cells is an important reservoir of AV virus antigen at this site. The persistence of this retrovirus inside the GCs helps explain how the follicular hyperplasia affecting FD cells and B blasts in PGL may in progressive cases be accompanied by destruction of FD cells and gradual development of T4+ lymphopenia. T4+ T cells may circulate through the GCs and become infected with AV there. In addition, the identification of retrovirus antigen in situ may be of diagnostic value.     

Pseudolymphoma of the thyroid

An unusual case of pseudolymphoma of the thyroid is reported. The tumor was histologically characterized by good demarcation, mature lymphocytes with prominent follicle formation and germinal centers, and mature plasma cells. Immunohistochemical staining revealed a similar distribution of T- and B-cells to reactive lymph nodes and a polyclonal nature of the plasma cell infiltrate. These findings are closely related to the entity, pseudolymphoma in other organs. Differential diagnosis from follicular lymphoma or ectopic thymic tissue is discussed.     

The immunoarchitecture of cutaneous pseudolymphoma

The immunoarchitecture of five cutaneous pseudolymphomas was studied by staining serial sections for T- and B-cell and dendritic reticulum cell (DRC) antigens with monoclonal antibodies, and compared with that of reactive lymph nodes and cutaneous lymphoma. In four cases compartmentalization of B and T cells was observed, analogous to findings in reactive lymph nodes. In two of these cases the immunoarchitectural features were strikingly similar to those of reactive lymph nodes. Both had distinct follicles with germinal centers, and in one distinct mantle zone formation was seen. B cells in the follicles were polyclonal, with kappa chain predominance. The germinal centers showed the expected intercellular and/or dendritic pattern of immunoglobulin heavy chain, B2, and DRC-antigen expression. T cells admixed in the germinal centers were overwhelmingly of the T-helper type. The B-cell compartments in the other two cases showed some subtle immunologic evidence of aberrance, but the weight of evidence suggested reactive/aberrant rather than malignant processes. The T-cell compartments in all four cases showed a predominance of T-helper and a minority of T-suppressor/cytotoxic cells. All contrasted with the lymphomas, which showed B-cell monoclonality, markedly deranged T-subset proportions, or novel T-cell phenotypes. Although the main focus of this study was cases involving substantial populations of both B and T cells, preliminary observations were made in one case in which a predominance of T cells and prominent epidermotropism simulated mycosis fungoides. Quantitative ultrastructural analysis in this case suggested a reactive T-cell process. Leu-6-positive Langerhans cells were increased in the epidermis and dermis in all five cases, and in the dermis they were found almost exclusively in T-cell compartments. It is proposed that this distribution is the anatomic correlate to the known functional role of Langerhans cells in antigen processing/presentation and T-cell activation. In the cutaneous "lymph node equivalent," Langerhans cells are analogous to interdigitating reticulum cells of reactive lymph nodes in distribution and, probably, in function. The DRC found in the germinal centers in two cases were probably antigenically identical and functionally analogous to those in germinal centers of reactive lymph nodes. Immunologic phenotyping of serial cutaneous sections may aid in distinguishing reactive from neoplastic lymphoid lesions. Immunoarchitectural analysis promises to be a powerful tool for the study of lymphoproliferative disease.     

Dissemination of SIV after Rectal Infection Preferentially Involves Paracolic Germinal Centers

Homosexual transmission remains a major mode of contamination in developed countries. Early virological and immunological events in lymphoid tissues are known to be important for the outcome of HIV infections. Little data are available, however, on viral dissemination during primary rectal infection. We therefore studied this aspect of rectal infection in rhesus macaques inoculated with the biological isolate SIVmac251. We show that infection is established initially in lymph nodes draining the rectum. Infected cells and virions are localized mainly in germinal centers at that stage. With increasing viral burden, infected cells are found throughout the lymph node parenchyma. In addition the difference in viral load between lymph nodes draining the rectum and other lymph nodes is attenuated or abolished. We discuss this pattern of viral dissemination with respect to the physiology of the mucosal immune system. The pattern and kinetics of viral dissemination after rectal infection have important implications for the development of efficient mucosal vaccines.