Different localization of dendritic cell reservoirs in human immunodeficiency virus-1 subtype B versus subtype E-infected lymph nodes.

The presence of p24 protein was studied in lymph nodes from human immunodeficiency virus (HIV)-positive patients affected by persistent generalized lymphadenopathy. Paraffin-embedded lymph node sections from 50 HIV-1 subtype E-infected lymph nodes from patients in Thailand and 25 HIV-1 presumably subtype B-infected lymph nodes from patients in the United States were immunostained with p24 HIV major core and capsid monoclonal antibodies using the streptavidin-biotin immunoperoxidase technique. Positivity for HIV p24 protein was detected in 20 of 22 HIV-1 subtype B infected nodes in which lymphoid follicles were present, with p24 staining demonstrating a reticular pattern within the germinal centers. Interestingly, no case from 50 clade E-infected lymph nodes containing lymphoid follicles had such a reticular pattern in the germinal centers. This difference could be explained by differential infection of subsets of dendritic cells by the two HIV-1 clades, or perhaps by different routes of initial HIV-1 transmission.

In vivo identification of Langerhans and related dendritic cells infected with HIV-1 subtype E in vaginal mucosa of asymptomatic patients.

In Thailand, the predominant HIV subtype is E, rather than Subtype B as in North America and Europe, and the predominant mode of transmission is heterosexual contact. Subtype E has the ability to replicate in vitro in Langerhans cells. We hypothesized that this cell type might constitute a reservoir for the HIV virus in vaginal mucosa of asymptomatic carriers. To examine this hypothesis, we compared vaginal tissue histology in HIV-1-seropositive cases with seronegative cases and determined the immunophenotype of HIV-1-infected cells, their numbers, and their distribution in vaginal mucosa. Vaginal biopsies were performed at four different sites from six asymptomatic HIV-1 Subtype E-infected persons and from six seronegative cases at necropsy and examined histologically. Immunophenotyping was performed using immunoperoxidase for Gag p24 HIV, CD3, CD20, CD68, CD1a, S-100 and p55 antigens and by double labeling, combining immunoperoxidase with alkaline phosphatase using pairs of the above antigens. Twenty of twenty-four vaginal biopsies (83.3%) from HIV-seropositive cases showed definite inflammation compared to five of twenty-four vaginal necropsies (20.8%) from HIV-seronegative cases. One third of HIV-seropositive biopsies (8/24) demonstrated p24-positive cells in the epithelium, whereas three-fourths (18/24) of the biopsies revealed p24-positive cells in the lamina propria. All seropositive patients showed positive cells in at least one biopsy, but not all biopsies contained positive cells. Infected cells were more frequently observed at sites of greater inflammation. The dendritic cell count in HIV-seropositive vaginal epithelium was significantly higher than that observed in the seronegative cases (P =.004). The majority of p24-positive cells in the vaginal epithelium were Langerhans cells (CD1a+/S-100+), whereas in the lamina propria, about half of p24-positive cells were Langerhans-related dendritic cells (p55+ and S-100+) and half were T lymphocytes. In conclusion, the increased propensity for heterosexual transmission of Subtype E may be related to vaginal inflammation, leading to the accumulation of Langerhans cells and related dendritic cells which, once infected with HIV, can act as a reservoir for further virus transmission.

Chronic inflammation with increased human immunodeficiency virus (HIV) RNA expression in the vaginal epithelium of HIV-infected Thai women.

Thai residents have a greater risk of heterosexual transmission of human immunodeficiency virus (HIV) than do US residents. To analyze host factors associated with heterosexual transmission, vaginal epithelial biopsies from HIV-seropositive Thai and US women were evaluated for tissue virus load and histologic makeup. In all, 84% of Thai and 14% of US women exhibited a chronic inflammatory T cell infiltrate in the vaginal epithelium. In Thai tissue, the infiltrate was associated with elevated levels of HIV RNA in the epidermis. Uninfected Thai women also had vaginal epithelial inflammation. Inflammation did not correlate with sexually transmitted diseases or HIV disease stage. The higher rates and increased risk of heterosexual transmission in Thailand may be due to chronic inflammation at the site where the virus is transmitted, which leads to the accumulation of activated T cells. Such cells might act as targets for initial viral infection and subsequently as reservoirs that support efficient transmission.

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.

Immunohistochemical characterization of a new monoclonal antibody reactive with dengue virus-infected cells in frozen tissue using immunoperoxidase technique.

This paper presents a novel monoclonal antibody shown to react with cytoplasmic antigens in various dengue infected human frozen organs from autopsy and necropsy specimens. Strong reactivity was found in hematopoietic cells, including immunoblasts, lymphocytes, plasma cells and macrophages of spleen, lymph node, lung, kidney and stomach. Strikingly, strong positivity was demonstrated in cerebral cortex neurones, Purkinje cells, choroid plexus and blood vessels in addition to astrocytes and microglia. Neurotropism of the virus could explain the meningitis, encephalitis, mononeuropathy and polyneuropathy observed by direct toxicity, but noted especially after an activation of mononuclear phagocytes and amplification of the immune response with subsequent vascular inflammation and formation of immune complexes.

Non-Hodgkin's lymphomas in Thailand. A retrospective pathologic and clinical analysis of 1391 cases.

BACKGROUND: Geographic variations in the histopathologic pattern of non-Hodgkin's lymphoma (NHL) are well documented. Insight into this epidemiologic data might shed light on the underlying etiology. Currently, there is a paucity of information regarding the pattern of NHL occurring in Thailand and Southeast Asia. The current nationwide study was undertaken to obtain a clearer definition of the disease among Thais and to compare this information with data from other Asian and Western countries. METHODS: A retrospective analysis of histopathologic subtypes and clinical features was conducted in 1391 patients (age > or = 15 years) with NHL, who were treated at 6 major medical centers in 4 representative areas of Thailand. One hundred and thirty unselected cases were immunohistochemically studied. The reports from other countries used for comparison were identified through a computerized search on MEDLINE. RESULTS: Of the total cases studied, follicular lymphomas constituted 3.8% and diffuse lymphomas 91.4%. The frequency of low and intermediate grade (including large cell immunoblastic, using the Working Formulation) were 12.8% and 72.9%, respectively. The most common histologic subtype was the diffuse large cell and large cell immunoblastic entity, which constituted 39.9% of the cases studied. The rate of small lymphocytic and diffuse small cleaved subtypes were higher than in the United States. Of the 130 cases studied, T-cell lymphoma comprised 16.1%, which was much less than in Japan, China, or Taiwan. CONCLUSIONS: The histopathologic pattern of NHL in Thailand was characterized by the features noted for Asia, i.e., a low rate of the follicular entity and a preponderance of the diffuse aggressive subtypes. In addition, the frequency of the small lymphocytic and diffuse small cleaved subtypes were increased compared with the Western population and the rate of T-cell lymphoma appeared to be less than in Far East Asia.

Kaposi sarcoma in a Thai boy with acquired immunodeficiency syndrome.

Kaposi sarcoma is rare in children with acquired immunodeficiency syndrome (AIDS). We report a 3-year-old boy with AIDS and Kaposi sarcoma of the skin and lymph node. This patient is the only one with this disease among 278 children with AIDS who have been seen at Chiang Mai University Hospital. He responded well to intravenous vincristine.

Histopathologic spectrum of AIDS-associated lesions in Maharaj Nakorn Chiang Mai Hospital.

The histopathological alterations in various organs and the presence of AIDS-associated lesions were studied in 86 biopsy and 29 necropsy specimens of AIDS patients. The most common cancer seen in this study were malignant lymphomas (4% of cases) with development of extensive extranodal lymphomatous involvement from the outset. Although a preponderance of high grade B-cell pathologic subtypes is found in AIDS-associated lymphoma, we also report the first case of T-lymphoblastic lymphoma with a picture of acute lymphoblastic leukemia (T-ALL). Tuberculosis (34% of cases) was the most common opportunistic infection presented in tissue sections, and the majority of tissue biopsies revealed poorly organized granulomas and extensive necrosis with numerous bacilli. Penicilliosis (20% of cases) appeared to be the most common cutaneous lesion with multiple organ involvement. The involved organs showed a partially anergic tissue reaction characterized by poorly formed granulomas with diffuse infiltrate of fungi-laden macrophages and lymphoid cell depletion. This organism has to be distinguished from Histoplasma capsulatum and other yeast-form fungi. Co-existing cytomegalovirus and P. carinii infections were the predominant findings in lung necropsy specimens from pediatric patients who died from AIDS. A major pathologic feature in this group was diffuse alveolar damage stage II to III with heavy loads of organism and extensive lymphoplasmacytic infiltration.

Epidemiologic characteristics of leprosy reactions.

An 8-year prospective study of a cohort of 176 newly diagnosed leprosy patients was conducted to examine the possible influence of age, sex, multidrug therapy (MDT), and duration of illness on the risk of either type 1 or type 2 reactions. Patients were enrolled over a 5-year period (1984-1989) and followed for a minimum of 3 years. All reactions studied were severe enough to warrant hospital admission. Overall, 45% of this cohort developed a reaction; 32% of patients considered at risk developed type 1 reactions, and 37% of patients considered at risk developed type 2 reactions. Despite the predominance of men among the leprosy patients, type 1 reactions occurred with significantly greater frequency in women, and did not appear to be influenced by age of onset of leprosy. Individuals experiencing one type 1 reaction were not likely to experience a recurrence, suggesting that the immunologic mechanisms of this reaction may be limited or regulated by genetic or immunologic factors. Type 2 reactions, on the other hand, occurred with equal frequency in both males and females, but were highly associated with onset of leprosy in the second decade of life. Individuals who experienced type 2 reactions often had one or more recurrence of the reaction. No increased risk was seen for either reaction with longer duration of leprosy or longer duration of treatment. The mechanisms by which these differences relate to the pathogenesis of leprosy reactions remains unclear, but future studies of clinical and immunological parameters of leprosy reactions may benefit from stratification of data by gender and age of onset of leprosy in addition to the routine grouping of results by leprosy classification.

Sudden death in Stevens-Johnson syndrome: a case report.

We report a case of a 28-year-old male who died suddenly on the way from his house to the hospital. He was said to have a gurgling sound in this throat followed by a seizure while sleeping at night. His wife tried to wake him up but he never returned to consciousness. She brought him to the hospital where a doctor in the emergency room pronounced him dead upon arrival. The autopsy revealed various types of mucocutaneous lesions, involving conjunctiva, lips, face, trunk and extremities, compatible with Stevens-Johnson syndrome. The eruptions covered only 1-2% of the total body surface area. The internal examination disclosed severe glossitis, epiglottitis and inflammation of the upper respiratory tract with prominent laryngeal edema. The microscopic sections of some skin lesions confirmed the previous diagnosis, and the cause of sudden death was believed to be acute upper airway obstruction which complicated the disease. This is probably the first report of Stevens-Johnson Syndrome resulting in death prior to disease recognition by medical personnel.

Immune complexes and antibody levels in blisters over human leprosy skin lesions with or without erythema nodosum leprosum.

Erythema nodosum leprosum (ENL) is a serious complication of lepromatous leprosy, affecting skin and peripheral nerves in a large percentage of these patients, and is presumed to result from spontaneous immunologic changes. Its pathogenesis is poorly understood, although histopathologic features have suggested immune complex (IC)-mediated injury. Abundant circulating antibody is present but no convincing correlation has been established between circulating IC and ENL. We have examined cutaneous leprosy lesions in vivo using blisters induced by prolonged gentle suction to determine whether or not IC are demonstrable in lesions with or without ENL, using an IC assay based on monoclonal rheumatoid factor binding. We also examined whether antibodies involved in such IC are produced locally or reach the skin via the circulation. Surprisingly large quantities of IC were found in ENL lesions, and in some cases the quantities were significantly higher than in matching serum. Total IgG, IgA, and IgM in the skin were not higher than expected, however. Attempts to demonstrate increases in intracutaneous levels of specific anti-Mycobacterium leprae antibodies were unsuccessful. This is the first report of the demonstration of IC in suction blister fluid. The results indicate that large quantities of IC may be present in cutaneous leprosy lesions and are consistent with the hypothesis that they are formed in situ when circulating antibody combines with antigen in the skin. The nature of the antigen in these IC remains undefined.

Studies of human leprosy lesions in situ using suction-induced blisters: cell changes with IgM antibody to PGL-1 and interleukin-2 receptor in clinical subgroups of erythema nodosum leprosum.

To examine the immunopathogenesis of type 2 erythema nodosum leprosum (ENL) reactions in leprosy, we studied cellular and soluble immunologic components of skin lesions in 57 patients with reactions (19 acute ENL and 38 chronic ENL), 61 active patients without reactions, and 33 control patients whose leprosy had been treated and cured. Cells, IgM antibody to PGL-1 and Tac peptide levels were obtained from fluid aspirated from blisters induced by suction directly over representative skin lesions. During ENL reactions: a) the lesions in chronic ENL showed a decreased number of CD8+ (T-suppressor) cells and increased helper/suppressor ratio as compared to those in acute ENL and non-reactional leprosy; b) Tac peptide and IgM antibody to PGL-1 levels were elevated in the chronic ENL lesions; c) and systemic administration of corticosteroids appeared to cause a reduction in the intralesional CD4+ cell population and IgM antibody to PGL-1 but did not change CD8+ cell population and the levels of Tac peptide in the lesions. The elevated levels of Tac peptide were localized in the skin lesions while increased levels of IgM anti-PGL-1 seemed to be filtered from the peripheral blood. We conclude that spontaneous lymphocyte activation in situ, primarily of decreased CD8+ and relatively increased CD4+ cells, are important features of chronic, recurrent ENL reactions and may be an intermittent or cyclic phenomenon during the reaction. Understanding the mechanisms of these spontaneous changes in immunity in leprosy will enlarge our knowledge of reactions and of the underlying determinants of delayed type hypersensitivity and cell-mediated immunity in leprosy, which in turn will allow us to realize the potential for artificially manipulating these responses as proposed with vaccines or immunotherapy.

Detection of estrogen and progesterone receptor bindings in mammary carcinoma cells: a comparative study using immunohistochemical and biochemical methods.

A comparative study of estrogen and progesterone receptor bindings of breast carcinoma tissue was done by immunoperoxidase and dextran-coated charcoal (DCC) methods. Fifteen cases of paraffin embedded formalin fixed tissue of mammary carcinomas which had previously been evaluated by the DCC method were selected. Twelve cases were ductal carcinoma and 3 were of lobular origin. Lymph node tissue showing metastasis was available in 3 cases. By immunoperoxidase technique, 12 and 10 (80 and 66.7%) cases were positive for estrogen and progesterone receptor respectively compared with 8 and 3 (53.3 and 20%) cases by the DCC technique. Corresponding results of both methods to detect estrogen and progesterone bindings were 9 and 8 (60 and 53.3%) of all cases, respectively. Five cases for estrogen and 6 cases for progesterone positive by immunoperoxidase could not be detected by the DCC technique. Only one case of estrogen negative by the immunoperoxidase gave a positive result with the DCC technique. Variability of staining occurred between primary and metastatic lesions, 2 out of 3 cases displayed positive staining in both sites; one remaining case was positive only in the lymph node metastasis. Immunoperoxidase is a relatively simple, swift and inexpensive technique in comparison to the DCC technique. Using fixed embedded tissue makes it possible for retrospective studies and providing a permanent record for reevaluation. Moreover, morphology of the tumor can be determined at the same time as detection of hormonal receptor bindings.

Studies of human leprosy lesions in situ using suction-induced blisters. 2. Cell changes and soluble interleukin 2 receptor (Tac peptide) in reversal reactions.

To examine the pathogenesis of type 1 (reversal) reactions in leprosy, we studied cellular and soluble immunologic components of skin lesions in 10 patients with reactions, 24 active patients without reactions, and 33 control patients whose leprosy had been treated and cured. Cells and Tac-peptide levels were obtained from fluid aspirated from blisters induced by suction directly over representative skin lesions. During reversal reactions: a) the lesions contained an increased number and percentage of CD4+ (T-helper) cells; b) Tac-peptide levels were elevated in half of the lesions; c) the increases in Tac peptide and CD4+ cells were directly correlated; and d) systemic administration of corticosteroids appeared to cause a reduction in the intralesional CD4+ cell population. These findings were localized to the skin, and do not represent simple filtration of these components from the peripheral blood. We conclude that spontaneous lymphocyte activation in situ, primarily of CD4+ cells, is an important feature of reversal reactions, and may be an intermittent or cyclic phenomenon during the reaction. Findings in active patients without reactions are consistent with the hypothesis that differing states of immunologic equilibrium have been established in different portions of the leprosy spectrum. In reversal reactions we may, therefore, be examining immunologic processes set in motion when a pre-existing equilibrium has been upset by spontaneous, natural events. The mechanism of such spontaneous changes in immunity in leprosy is of considerable interest, not only to understand the reaction, but also to examine the underlying determinants of delayed-type hypersensitivity and cell-mediated immunity in leprosy and the potential for artificially manipulating these responses, as proposed with vaccines or immunotherapy.

Cell changes and soluble interleukin-2 receptors (Tac peptide) in leprosy reversal reactions using suction-induced blisters.

The cellular contents and soluble interleukin-2 receptor (IL-2R) [Tac peptide] of skin blisters induced by suction over 7 reversal reaction (RR) patients were examined using immunoperoxidase and ELISA techniques respectively. The helper T activity (CD4+ cells) and helper:suppressor ratio were significantly greater in borderline lepromatous (BL) lesions with RR than in quiescent BL lesions. Interestingly, the intracutaneous levels of Tac peptide were elevated and directly correlated with the increases in CD4+ cells. The systemic administration of corticosteroids revealed a reduction in the numbers of CD4+ cells in the lesions. These results indicate that RRs are manifestations of a spontaneous increase in delayed type hypersensitivity (DTH) and possibly cell mediated immunity (CMI) in leprosy patients. The mechanism of such changes in immunity is of considerable value in understanding reversal reactions and the underlying determinants of DTH and CMI in leprosy and this in turn will have a bearing on the potential for proposed vaccines or immunotherapy.

Immunohistochemical analysis of non-Hodgkin's lymphomas with the monoclonal antibody to follicular center lymphocytes reactive in paraffin sections.

The monoclonal antibody designated LN-1 was used in an attempt to identify the antigen in follicular center cell lymphomas using tissue sections fixed in formalin. The LN-1 antibody has been shown in previous studies to identify follicular center cells and give reproducible results in tissue fixed in B5. We used the ABC peroxidase technique to examined formalin-fixed, paraffin embedded sections representing 52 cases of various histologic subgroups of non-Hodgkin's lymphomas based upon the Lukes-Collins classification. Following immunostaining with LN-1 using overnight incubation of the antibody and papain treated sections, 37 cases, and all of the 38 cases previously diagnosed as follicular center cell lymphomas, gave a positive reaction to the LN-1 monoclonal antibody.

Immunohistochemical characterization of two new monoclonal antibodies (LN-4, LN-5) reactive with human macrophage subsets and derived malignancies in B5-fixed, paraffin-embedded tissues.

Two monoclonal antibodies (LN-4, LN-5) reactive to human macrophages in B5 formalin-fixed, paraffin-embedded tissue sections have been produced by using deparaffinized cell extracts of peripheral blood mononuclear cells. Both monoclonal antibodies were initially identified on paraffin-embedded sections of hyperplastic lymph nodes by using the immunoperoxidase staining procedure. Specificity screens on normal human tissues show that LN-4 and LN-5 stain the cytoplasm of macrophages and histiocytes in hematopoietic organs including Kupffer's cells of the liver and Langerhans' cells of the skin. LN-4 also showed strong positivity with acini of the stomach, whereas LN-5 was positive with mantle zone B lymphocytes of the lymph node and spleen, spermatogonia, and chief cells of the stomach. Both antibodies were strongly reactive with cases of true histiocytic lymphoma but, except for infiltrating macrophages, were entirely negative in Hodgkin's disease and non-Hodgkin's lymphomas. In all cases of nodular sclerosis Hodgkin's disease, LN-4 was positive in macrophagelike cells present in the collagen bands surrounding the Hodgkin's lesions. Both monoclonal antibodies were also positive in macrophages and histiocytes present in a variety of benign lymphoid lesions including persistent generalized lymphadenopathy, Gaucher's disease, sinus histiocytosis, and dermatopathic lymphadenopathy. Because of their specificity for human macrophages, and their ability to stain B5-fixed, paraffin-embedded tissues, LN-4 and LN-5 are important new reagents for the diagnosis and classification of malignant and benign histiocytic lesions.

The differentiation antigens of macrophages in human fetal liver.

Macrophage populations from human fetal liver were examined for the sequential appearance of different antigenic determinant during maturation. Frozen sections of liver, from 12 to 21 weeks gestation were analyzed using a series of four monoclonal antibodies with known specificity. The macrophage monoclonal antibodies used were OKM-1, which defines monocytes, macrophages, and granulocytes; Leu M-3 and MO-2, which identify monocytes and macrophages; and 6B8, a new macrophage monoclonal antibody which binds to tissue macrophages. The staining pattern described by each of these monoclonal reagents was compared with the distribution of morphologically distinguishable tissue macrophages in fetal liver, based on the expression of surface and/or cytoplasmic antigens. The data indicate that the antigens defined by OKM-1 and 6B8 are present on large numbers of cells as early as 12 weeks gestation. In contrast, the antigenic determinants identified by Leu M-3 and MO-2 are present only on cells in 15 to 21 weeks of gestation; thus these antigens are mature differentiation antigens. Furthermore, double-staining studies confirmed that with the increase in fetal age unique macrophage populations can be identified based on the matrix of antigenic determinants. Thus, macrophage heterogeneity in the fetal liver may be a function of maturation.

Distribution of cells bearing the Tac antigen during ontogeny of human lymphoid tissue.

The monoclonal antibody anti-Tac, which binds to the interleukin 2 (IL 2) receptor, was used to identify this antigen in human fetal and adult lymphoid tissue. Liver, spleen, thymus, lymph node, and peripheral blood were examined for Tac-positive cells with the use of frozen sections or cytocentrifuge preparations. The results show that cells in the fetal and neonatal thymus express the Tac antigen; these cells are predominantly located in the medulla. The liver and spleen of both fetus and adult exhibit very few Tac-positive cells. Double staining demonstrates that cells bearing the Tac-antigen stain with Leu-4, an anti-T cell antibody. In adult lymph node tissue, the Tac-bearing cells are predominantly distributed in the interfollicular area, with positive cells also present in the germinal center and mantle zone. The Tac antigen is present on both T and B cells. Few Tac-positive cells are present in the circulating peripheral blood.