Ceroid pigment deposition in circulating blood monocytes and T lymphocytes in Hermansky-Pudlak syndrome: An ultrastructural study

An electron mlcroscoplc study of peripheral leukocytes obtalned from a 39 year old woman with Hermansky-Pudlak ayndrome was performed. Cerold pigment granules were found within tha lysoaomes in 3.5% of monocytes and 5.4% of lymphocytes. Infrequently, pigment granules were also found in the parallel tubular arrays of lymphocytes. The lymphocytes contalning ceroid pigment granules were confirmed to be T cells by immunoelectron microscopy. It was speculated that intralysosomal accumulation of ceroid pigment granules in Hermansky-Pudlak syndrome may be due to lysosomal dysfunction.     

Fifty cases of human immunodeficiency virus (HIV) infection: immunoultrastructural study of circulating lymphocytes.

The peripheral lymphocytes of 50 cases of human immunodeficiency virus (HIV) infection (13 of acquired immune deficiency syndrome (AIDS), 17 of AIDS related complex (ARC), and 20 healthy carriers) were studied immunoultrastructurally. The prevalence of "tubuloreticular structures" and "tubular confronting cisternae" increased with the progression of the disease. Numerous tubular confronting cisternae were noted in patients presenting with a high serum acid labile alpha-interferon values. The patients with depressed natural killer cell activity were characterised by circulating immature natural killer cells with abundant multivesicular bodies that were devoid of "parallel tubular arrays". With an immunogold staining technique the location of HIV antigen was detected ultrastructurally, both at the surface of "hand-mirror" natural killer cell lymphocytes and inside vacuolised cells, probably corresponding to infected T4 lymphocytes. These findings indicate the usefulness of electron microscopic techniques in evaluating the pathology and the pathogenetic outcome of AIDS.     

Mechanisms of corticosteroid action on lymphocyte subpopulations. I. Redistribution of circulating T and b lymphocytes to the bone marrow.

The effect of corticosteroid administration on the redistribution of sirculating lymphocytes was studied in the guinea-pig, since this species closely resembles man in its relative resistance to the lymphopenic effect of corticosteroids. A single intravenous injection of hydrocortisone (either 10 mg or 100 mg/kg) caused a profound but transient lymphocytopenia which was maximal at 4 hours following injection, with a returnto normal counts by 24 hours. There was a proportionately greater decrease in circulating T lymphocytes compared to B lymphocytes, although both populations were diminished. Chronic cortisone acetate treatment (100 mg/kg subcutaneously for 7 days) caused a similiar pattern of lymphocytopenia except that it was sustained during the period of chronically elevated plasma cortisol levels. The lymphocytes remaining inthe circulation during the period of lymphocytopenia responded normally in vitro to the mitogens phytohemagglutinin, concanavalin A, and pokeweek mitogen. There was very littleeffect of corticosteroid administration on the numbers, proportions, or mitogenic response of splenic lymphocytes. There was a dramatic increase in the bone marrow of proportions and absolute numbers of lymphocytes bearing surface T-and B-cell markers, as well as a marked increase in response of bone marrow lymphocytes to mitogenic stimulation during the period of maximal circulating lymphocytopenia caused by the administration of corticosteroids, especially chronic cortisone acetate. There was a preferential homing of reinfused -51Cr-labelled syngeneic peripheral blood lymphocytes to the bone marrow of corticosteroid-treated recipients. These studies demonstrate aredistribution of circulating lymphocytes to the bone marrow during corticosteroid treatment, resulting in an increase in immunocompetence of this compartment, while the peripheral blood lymphocyte compartment is quantitatively immunosuppressed due to a lymphocytopenia.     

The effects of prednisolone in leucocyte function in man. A double blind controlled study.

The effect of prednisolone on various immunological parameters was studied in patients with ulcerative colitis in complete remission. The study was designed as a double blind trial in which patients received either prednisolone or a dummy preparation and the following observations were made:(1) The mean lymphocyte count fell from 1738 cells per mm3 to 501 cells/mm3 4 hr after prednisolone was given but by 24 hr was significantly elevated to 2399 cells/mm3; thereafter it returned to normal levels. (2) Surface marker assays of lymphocytes forming spontaneous sheep cell (E), Fc (EA), and C3 (EAC) rosettes; and cells bearing surface immunoglobulin fluctuated in approximately the same pattern as the total lymphocyte count. (3) The mitotic response to a sub-maximal stimulating dose of phytohaemagglutinin (PHA) was significantly depressed 4 hr after steroid administration but returned to normal by 24 hr. (4) Spontaneous and PHA-induced lymphocyte mediated cytotoxicity fell significantly by four hours and remained depressed to the end of steroid administration. The PHA-induced cytotoxicity was still significantly depressed 7 days after steroid administration was stopped. (5) K-cell cytotoxicity did not follow the general pattern and was only slightly reduced at four hours being lowest after 24 hr and still depressed 7 days after cessation of steroid administration. (6) The number of plasma cells in the rectal lamina propria showed no significant change after one week of steroid administration. (7) No significant changes occurred in any of the above assays, in the control group. (8) Polymorphonuclear leucocyte counts rose sharply by 4 hr in the patients receiving prednisolone. There was also a smaller but significant rise in the control group. They remained elevated for 7 days in the group receiving prednisolone, and subsequently fell to normal levels. The control group had returned to initial levels by 24 hr.     

Complement receptors on normal human lymphocytes containing parallel tubular arrays

Membrane complement receptors have been identified on a subpopulation of normal lymphocytes containing cytoplasmic inclusions called parallel tubular arrays (PTA) using two different rosetting techniques. The first technique utilizes as indicator cells erythrocytes that were coated with complement by the classic pathway of complement activation (EAC rosettes). The second technique utilizes as indicator cells Salmonella typhi, which were coated with complement by the alternate pathway of complement activation (FBC rosettes). In the latter technique, lipopolysaccharide material in the bacterial cell wall directly activates complement without the use of a sensitizing antibody. This eliminates binding of marker particles by lymphocytes having Fc receptors. The presence of PTA lymphocytes at the center of EAC rosettes and FBC rosettes was demonstrated by electron microscopy, indicating that the PTA lymphocyte has a complement receptor. Examination of FBC rosettes revealed that the adherent complement-coated bacteria were usually partially surrounded by pseudopodal extensions of the PTA lymphocyte. In addition, some PTA lymphocytes phagocytized the complement-coated bacteria but not the complement-inactivated bacteria. These phagocytic cells were placed in the lymphocytic series instead of the monocytic series by virtue of complete lack of endogenous peroxidase activity.     

Immunosuppressive activity of BCG: effects of adjuvant disease, lymphocyte subpopulations, and homing of thoracic duct cells in rats.

Administration of BCG by various dosage schedules suppressed adjuvant disease in rats. BCG administration produced an initial increase, followed by a depression, of the phytohemagglutinin response of purified blood lymphocytes. An increase in absolute and relative numbers of bursa-equivalent (B)-cells followed BCG administration, concurrent with a decrease in the phytohemagglutinin responsiveness. With adjuvant alone, there was a diminution in phytohemagglutinin response and an increase in number of B-cells; the latter occurred immediately after adjuvant injection and also when the generalized disease appeared. When both BCG and adjvant were present, parallel increases of phytohemagglutinin responsiveness and B-cell numbers resulted. The pattern of tissue localization of radioactively labeled thoracic duct cells from normal or BCG-treated donors given to normal, BCG-treated, adjuvant-injected, and BCG-treated + adjuvant-injected syngeneic recipients indicated significantly greater homing to the thymus and decreased localization to the bone marrow when BCG had been given to either donors or recipients. When labeled thymus cells were used, only the decreased bone marrow localization was noted. These observations suggest that the suppressive effect of BCG may be mediated through modification of the lymphocyte recirculation pattern, possibly resulting from alterations in lymphocyte recognition sites.     

Effect of in vitro x-irradiation on human peripheral blood T and B lymphocytes.

The effect of in vitro irradiation with increasing in logarythmic progress X-ray doses on lymphocyte viability and on T and B lymphocyte populations was studied in normal adults, patients with myasthenia gravis and in patients undergoing long-term steroid therapy. Decrease in numbers of lymphocytes carrying T or B lymphocyte surface markers was higher than viable cell loss. The decrease showed no linear correlation with X-ray doses applied, which might reflect the existence of radioresistant T and B lymphocytes. A higher so called early radiosensitivity of B lymphocytes was demonstrated. In patients with myasthenia gravis early radioresistance of T lymphocytes was detected. In patients undergoing long-term steroid therapy, an increase in numbers of cells lacking markers of any of lymphocyte populations was found in parallel with a decrease in T lymphocyte number which, in these patients, showed a higher radiosensitivity.     

Correlation between in vivo and in vitro studies of modulation of resistance to experimental Candida albicans infection by cyclophosphamide in mice.

Mice receiving a single injection of cyclophosphamide (150 mg/kg) 1 to 6 days before inoculation with viable Candida albicans showed an increased susceptibility to the challenge accompanied by a reduction in peripheral blood polymorphonuclear leukocytes and lymphocytes as well as in spleen cellularity. Several immunological in vitro functions also appeared to be dramatically depressed. Most of these hematological and functional parameters returned to control values by day 9 after cyclophosphamide administration, at a time when resistance to C. albicans infection appeared to be unchanged. However, when exposure to cyclophosphamide occurred 12 to 21 days before inoculation with the live yeast, enhanced resistance was observed with the majority of the animals surviving challenge. To gain some insight into the mechanisms underlying this late increase in resistance to C. albicans infection after cyclophosphamide administration, we analyzed a series of immunological functions, including the in vitro candidacidal activity of polymorphonuclear neutrophils and plastic-adherent and nonadherent spleen cells as well as the activity of natural killer cells and alloreactive T lymphocytes. The results show that a numerical rebound of blood polymorphonuclear neutrophils and the appearance of a highly candidacidal cell population in the spleen may be among the factors underlying the late increase in resistance to C. albicans after administration of cyclophosphamide.     

A quantitative ultrastructural study of peripheral blood lymphocytes containing parallel tubular arrays in Epstein-Barr virus and cytomegalovirus mononucleosis.

In the normal peripheral circulation there exists a subpopulation of lymphocytes that is ultrastructurally distinct. This lymphocyte is identified with the electron microscope by the presence of cytoplasmic microtubulelike inclusions called parallel tubular arrays (PTAs) and contains Fc-receptors for cytophilic antibody. In this study, lymphocytes containing PTAs (PTA-lymphocytes) were quantitated from serial peripheral blood specimens obtained from two patients with Epstein-Barr virus (EBV) mononucleosis and two patients with cytomegalovirus (CMV) mononucleosis. These data were then correlated with the clinical state of the patient. It was determined that both the percentage and absolute number of PTA-lymphocytes were highest during the acute phase of the illness. In follow-up specimens, three of the four patients' absolute lymphocyte count fell to within normal limits before the absolute PTA-lymphocyte count. In one patient, the absolute PTA-lymphocyte count was significantly elevated 13 months after the initial clinic visit. Although the PTA-lymphocyte count was highest during the acute phase of the illness, there was no consistent correlation with the clinical state of the patient during follow-up. The estimation of absolute PTA-lymphocyte counts was determined to be valid after a morphometric analysis of the cellular areas occupied by PTAs during the acute and convalescent phases of the disease revealed no statistical differences. Electron microscopy was also performed on the peripheral blood of a patient with syphilis. Although a hematologic workup of this patient during the acute phase of his illness revealed a large number of atypical lymphocytes, electron-microscopic examination of the same specimen revealed both a normal number and a normal percentage of PTA-lymphocytes. The immunologic role of this ultrastructurally distinct third population (non-T, non-B) of lymphocytes, or "killer cells," in the course of infectious mononucleosis is discussed.     

Immunogenetic and immunologic aspects of gliosarcoma growth in rats

Growth of the chemically induced, transplantable rat brain tumor gliosarcoma 9L (GS-9L) is under immunogenetic control. Both susceptible and resistant rats produce an immune response to the tumor, but the response is qualitatively different in the two groups. The intraperitoneal administration of gliosarcoma-9L cells in resistant KGH rats causes the production of cytotoxic lymphocytes and macrophages, and in susceptible F344 rats suppressor lymphocytes are produced. After gliosarcoma-9L cells were administered to (KGH x F344)F1 and backcross rats, tumor susceptibility or resistance and the nature of the immune response correlated well with the histocompatibility type, indicating the parallel genetic control of both traits. However, a second gene or gene complex, not linked to the major histocompatibility complex, may participate in the regulation of tumor growth.     

Interleukin 2 synthesis in the presence of steroids: a model of steroid resistance.

In this study interleukin 2 (IL-2) synthesis by human lymphocytes in the presence and absence of prednisolone in a group of normal subjects has been assessed. An association between suppression in vitro of induced phytohaemagglutinin-blastogenesis by prednisolone and synthesis of IL-2 was found. Those subjects whose lymphocytes are identified as steroid-resistant have significantly higher IL-2 activity in the supernatants of both steroid and non-steroid treated lymphocyte cultures than steroid sensitive subjects. The addition of exogenous IL-2 was found to ablate the suppressive effects of steroids on lymphocyte blastogenesis. These results suggest that significantly greater activity of IL-2 in the culture supernatants of steroid resistant subjects may represent a mechanism for glucocorticoid resistance in vitro and help explain the relationship between increased loss of grafts and steroid resistance in renal allograft recipients.     

Lymphocyte tubular structures in rheumatoid arthritis.

Two types of lymphocyte tubular structures were studied by electron microscopy in 80 patients with classic or definite rheumatoid arthritis (RA). Fifteen patients with unequivocal systemic lupus erythematosus (SLE) and 10 healthy persons were studied as controls. Lymphocyte tubulo-reticular structures were found in 13 of the 80 patients with RA and in 10 of the 15 patients with SLE. No tubuloreticular structures were found in any of the healthy subjects. In the RA patients antinuclear antibodies, LE cell phenomenon, and chlorambucil treatment were associated significantly with these inclusions but disease activity was not related to their presence. In cases in which tubuloreticular structures were present the number of cells containing inclusions was on average much lower in patients with RA than in those with SLE. Lymphocyte tubular parallel arrays were found in all the patients with RA and SLE and in all the healthy persons. In all three groups the average number of cells containing parallel tubular arrays was similar.     

Distinct leucocyte redistribution after glucocorticoid treatment among difficult-to-treat asthmatic patients

Difficult-to-treat asthma (DTA) represents a heterogeneous subgroup of asthma. Up to now, the lack of specific diagnosis not only complicates appropriate specification and control of asthma, but also makes targeted research difficult. The aim of this study is to categorize this heterogeneous group of DTA patients (n=27; referring to the GINA guidelines) based on the distinct leucocyte redistribution (LR) after glucocorticoid (GC) treatment. Furthermore, the effect of adjuvant therapies was investigated for its impact on LR. The frequency of CD3+, CD4+, CD8+, CD14+, CD19+ and NK cells was analysed in peripheral blood before and 3 h after systemic GC treatment, along with the markers of activation HLA-DR and CD25. Within 3 h of GC administration, a significant average decrease of 16% in CD3+CD4+ (P < or = 0.001) and a 12% increase in NK-cell frequency (P < or = 0.001) clearly distinguished two groups of patients: LR-responsive and LR-unresponsive patients. The CD3+CD8+ T-cell number and activation marker remained unchanged. Patients who received adjuvant therapy, such as methotrexate or interferon-alpha, because of poor clinical response to GC showed an LR similar to that showed by responsive patients. DTA patients comprise at least two immunologically distinct groups: patients showing an immediate decrease in CD3+CD4+ T cells and an increase in NK cells following GC administration and patients lacking an immediate change. Analysis of LR not only may allow the identification of immunologic steroid resistance, but also may be of value for immunologic determination of effective steroid doses.     

Influence of estrogen on host resistance: increased susceptibility of mice to Listeria monocytogenes correlates with depressed production of interleukin 2.

Mice given pharmacological levels of the synthetic estrogen diethylstilbestrol demonstrated a marked increase in susceptibility to infection with Listeria monocytogenes. Experiments were performed in an effort to determine the mechanism(s) by which estrogen treatment increases the susceptibility of mice to L. monocytogenes infection. Estrogen exposure depressed the in vivo proliferative response of splenic lymphocytes to L. monocytogenes, which correlated with the decreased in vitro response of these cells to phytohemagglutinin. Interleukin 2 (IL 2) production by splenic lymphocytes from estrogen-treated mice was decreased, although these cells were capable of proliferating normally in response to exogenous IL 2. Interleukin 1 production by peritoneal macrophages was not depressed by estrogen exposure. The number of bacteria observed in the spleens of estrogen-exposed mice challenged with L. monocytogenes was reduced by IL 2 administration. Thus, estrogens may decrease host resistance to L. monocytogenes by inhibiting IL 2 production and the subsequent proliferation of antigen-sensitized T lymphocytes required for recovery.     

The effect of acute and prolonged administration of prednisolone and ACTH on lymphocyte subpopulations.

The effect of acute and prolonged three week administration of prednisolone and ACTH on the numbers and function of T- and B-lymphocyte subpopulations in patients requiring corticosteroid therapy was studied. Prednisolone caused severe reduction in E-rosette-forming lymphocytes, phytohaemagglutinin response, EAC-rosette-forming lymphocytes and surface-membrane mu-positive B-lymphocytes maximal at 4--6 hr after administration with reversal sometimes to supernormal levels by 24 hr. Prolonged administration resulted in a similar pattern of response. Acute but not prolonged prednisolone administration caused a reduction in the percentage of E-rosette-forming lymphocytes maximal at 4 hr. ACTH caused moderate reduction in these parameters at 4 and 6 hr which remained low at 24 hr after prolonged administration.     

Cisplatin nephrotoxicity: experimental and clinical studies

Cisplatin is currently one of the most used agents in the treatment of cancer and it is essential in the treatment of germ cell cancer. The use of the drug is hampered by side effects - especially renal toxicity which is dose limiting. The present work was undertaken to elucidate the pathophysiological mechanisms involved in cisplatin induced nephrotoxicity. Immediately after administration of cisplatin to dogs, renal blood flow (RBF) and glomerular filtration rate (GFR) remained unchanged, while proximal reabsorption rates decreased significantly. The cisplatin induced nephrotoxicity is thus initiated by an acute, mainly proximal tubular impairment, preceding alterations in renal hemodynamics. These data were confirmed in a micropuncture study in rats. At 48 to 72 hours after administration of cisplatin depressed renal function could be attributed to impairment of proximal as well as distal tubular reabsorptive capacities, now associated with increased renal vascular resistance. After administration of 4 cycles of 20 mg cisplatin/m2 d. for 5 days in humans, a small but significant decrease in 51Cr-EDTA clearance was observed. In the high-dose cisplatin group (40 mg/m2 d. for 5 days) a severe progressive decrease in GFR was observed during treatment and GFR remained decreased for up to 2 years after termination of treatment. The observation of an acute increase in N-acetyl-beta-D-glucosaminidase and beta-2-microglobulin indicates a primary tubular effect of cisplatin also in humans. A marked reduction of proximal tubular reabsorptive capacities of sodium and water was also observed in this group, together with a decrease in distal tubular function. These changes persist for at least 6 months after treatment. In the high-dose group proteinuria developed. This was mainly of tubular origin during cisplatin infusion and of glomerular origin between treatment cycles. Cisplatin remains one of the most potent antineoplastic agents ever developed. Further work should be performed to reduce its potential for renal toxicity.     

Morphological studies on the synthesis of secretory granules in convoluted tubules of mouse submandibular gland.

The synthesis of zymogen-like secretory granules in convoluted tubules of mouse submandibular gland (SMG) was investigated by histometry, light microscopy and electron microscopy. In normal males secretory granules in the SMG increased greatly from 25 days after birth and reached a maximum level 50 days after birth. Castration of adult male mice markedly decreased the level, but it was completely restored by testosterone administration. A parallel was found between change in the granule level and the amount of rough endoplasmic reticulum (RER) in the convoluted tubular cells during development or after various treatments. Development of the Golgi apparatus was also observed in the cells when the granules increase. Both the increase in the granules and in the RER induced by testosterone were prevented by actinomycin D or puromycin. These results indicate that the granule contents are synthesized on the RER under the control of testosterone, and then condensed in the Golgi apparatus.     

The immunological competence of subjects with sarcoidosis

Immunological competence in sarcoidosis was measured by in vivo and in vitro immunological testing of eighteen subjects who had never been treated with prednisone, and eleven subjects treated with prednisone. Three of the latter group had been studied before steroid therapy. Cutaneous delayed hypersensitivity was assessed by intradermal skin testing with five antigens. Thymus-derived `T' lymphocytes and bone marrow-derived `B' lymphocytes were enumerated and the ability of lymphocytes to respond to stimulation with phytohaemagglutinin (PHA), Candida albicans and allogeneic irradiated lymphocytes (mixed lymphocyte reaction) was measured and compared to results obtained with lymphocytes from healthy subjects.

The non-steroid-treated and the steroid-treated groups did not respond as well as a control group to intradermal challenge. The responses of lymphocytes from patients with untreated sarcoidosis to PHA and Candida albicans was normal but the response to allogeneic lymphocytes was reduced. In comparison stimulation indices for lymphocytes from the group who were receiving steroids were significantly reduced, with eight of eleven individuals having abnormal responses. Significant reduction in T lymphocytes was observed in only three of eleven of the non-steroid-treated subjects and five of ten of the steroid-treated subjects that were studied.

Studies on patients before and after steroid therapy suggest that steroid therapy and not sarcoidosis was responsible for the poorer responses of the steroid-treated group. The most striking finding of this study was the marked disparity between the cutaneous reactivity to Candida albicans, which was abnormal, and the in vitro lymphocyte response to this antigen which was normal. This suggests that the anergy of sarcoidosis cannot be attributed to an inherent lymphocyte dysfunction or to a depletion of these cells.

     

The cellular basis of the local graft-versus-host reaction in rat kidney

Injection of parental strain rat lymphocytes under the kidney capsule of semi-allogeneic F1 recipients causes a local graft-versus-host reaction (GVHR) characterized by a heavy mononuclear cell infiltrate and renal tubular destruction. Since the cellular events involved may have relevance to allogeneic tissue damage in GVH disease and allograft rejection, a detailed analysis of the rat renal GVH reaction was performed. A purified CD4+ lymphocyte subpopulation was as effective in mediating a local GVHR as unfractionated parental lymphocytes, but neither naive CD8+ nor specifically sensitized CD8+ lymphocytes produced a detectable renal GVHR. Mononuclear cells harvested from renal GVHR lesions induced by CD4+ lymphocytes were able to lyse natural killer (NK)-sensitive targets when tested in vitro, but showed no allospecific cell-mediated cytotoxicity. Experiments using recombinant PVG rats demonstrated that the ability of the injected cells to cause a GVHR was dependent upon a disparity in MHC class II antigens and that an isolated disparity of MHC class I antigens alone was not a sufficient stimulus to provoke a response. The use of chimaeric rats demonstrated that F1 MHC alloantigens present on kidney parenchyma (but absent on bone marrow-derived cells) were not sufficient to stimulate injected parental lymphocytes, even in the presence of markedly increased amounts of MHC antigens on vascular endothelium and renal tubular cells following in vivo administration of interferon-gamma (IFN-gamma). These results suggest that the renal GVHR in the rat is mediated principally by the interaction of parental CD4+ lymphocytes recognizing and responding to class II F1 alloantigens on bone marrow-derived cells. The resulting tissue damage is most likely a result of a delayed-type hypersensitivity (DTH) phenomenon.