Lymphocyte Proliferation in AIDS-Related Complex/Walter-Reed 5 Patients: Response to Herpes Simplex Virus and Tuberculin Antigen and Mitogen during Intravenous Immunoglobulin Treatment

Abstract. In a randomized, controlled double-blind study, 15 patients with AIDS-related complex/Walter-Reed 5 (ARC/WR5) were compared during 6 months intravenous immunoglobulin (IVIG) treatment (0.4 g/kg body weight every 2 weeks) with 15 placebo-treated patients. This study was aimed at the lymphocyte response to T and B cell mitogens and antigens. ³H-thymidine uptake was determined after stimulation with the unspecific mitogens phytohemagglutinin (PHA), pokeweed mitogen (PWM), formalinized Staphylococcus aureus -Cowan I (SAC), and with the antigens tuberculin and herpes simplex virus (HSV) at the onset, on days 85, 183, 267 and 351; IgG and IgM antibodies against HSV were measured by ELISA. In addition, 30 untreated HIV-negative controls were tested. For the T cell mitogen PHA, T-cell-dependent B cell mitogen PWM and B cell mitogen SAC, no differences between the two patient groups were observed before therapy nor in the course of therapy or the 6-month observation period thereafter. The entire patient group showed significantly impaired mitogenic response on day 1 as compared to the controls. There was no significant difference in response to tuberculin between the patients and HIV-negative controls, nor for both patients groups before and in the course of treatment. All patients had IgG antibodies against HSV. Three of them showed blastogenic lymphocyte response to HSV on day 1. Among 19 seropositive controls, 7 individuals showed positive HSV lymphocyte response; but for both patient groups, there was no significant difference before and in the course of the treatment and observation period. We concluded that, in spite of some clinical improvement regarding fever and fatigue during IVIG treatment of ARC/WR5 patients, there is no influence on lymphocyte function, as measured by response to mitogens and antigens.     

Interferon Production in Leukaemia

Leucocyte interferon production in vitro and circulating interferon levels were studied in healthy subjects and in 80 patients with acute or chronic leukaemia. Circulating interferon was not found in either group. Interferon synthesis in response to a virus was normal in patients with acute leukaemia and appeared to be enhanced in some. In chronic leukaemia reduced levels were common particularly in CLL, in which condition normal results were rarely found; lymphocyte transformation to PHA was also depressed in this group. No clinical or haematological correlation with the interferon levels was found and no consistent effect of treatment was shown. The possible factors which could account for these findings and their significance in relation to pathogenesis and treatment of leukaemia are discussed.     

Inhibition of mitogen- and antigen-induced lymphocyte blastogenesis by herpes simplex virus.

Lymphocytes were separated from peripheral blood of adult human donors by Ficoll-Hypaque gradient centrifugation and cultured in the presence of nonspecific mitogens (phytohemagglutinin[PHA] and concanavalin A) or specific microbial anatigens (herpes simplex virus [HSV], mumps virus, streptococcal enzymes, and Candida albicans). Exposure of lymphocyte cultures to infectious HSV resulted in almost complete inhibition of blastogenesis ([3/H]thymidine uptake) induced by each of the mitogens and antigens, a finding which suggests that a common mechanism may underlie the inhibitory effect. Several characteristics of the effect of virus on blastogenesis were noted: (1) virus inactivated by heat or ultraviolet irradiation was ineffective; (2) inhibition (is greater than 90%) was greatest in cultures exposed to HSV on or before the addition of PHA; (3) lymphocyte preparations washed free of HSV continued to be refractory to stimulation, an observation indicating that the presence of unabsorbed virions or viral products was not essential; and (4) inhibition was independent of the cell donor's state of humoral immunity to HSV.     

Selective Increase in Lymphocyte Interferon Response to Vaccinia Antigen after Revaccination

Viral antigen prepared by heat inactivation of vaccinia virus stimulated production of interferon in association with transformation of sensitized human lymphocytes in vitro. Involvement of a macrophage-lymphocyte interaction in production of interferon stimulated by viral antigen was found in which macrophage greatly augmented the amount of interferon produced by lymphocytes. Reimmunization with live vaccinia virus resulted in a selective increase in the ability of lymphocytes to produce interferon in the presence of viral antigen 4-7 weeks later without a corresponding increase in the degree of already significant lymphocyte transformation. There was no correlation between the extent of lymphocyte transformation and the amount of interferon produced. The augmented interferon response after reimmunization described in this study may be a significant component of the protective effect of immunization with vaccinia against disease occurring after exposure to small-pox.     

Reduction of virus burden-induced splenectomy in patients with liver cirrhosis related to hepatitis C virus infection

AIM: To examine the hepatitis C virus (HCV) levels and immunological markers in cirrhotic patients after splenectomy. METHODS: HCV RNA titers as well as cellular and humoral immune markers were determined in 20 cirrhotic patients after splenectomy and in 32 cirrhotic controls with an intact spleen. RESULTS: Serum HCV RNA titers were lower in the splenectomized patients than in the controls (186±225×103 copies/ml vs 541±417×103 copies/mL, P<0.01). HCV RNA was judged to have been spontaneously eradicated in 4 splenectomized patients, but in none of the controls. Natural killer cell activity was higher in the splenectomized patients than in the controls (41.2±19.3% vs 24.7±15.3%, P<0.01), and natural killer cell activity was negatively correlated to HCV RNA titers in the splenectomized patients except in those with serotype 2-related infection. The CD4/CD8 ratio was significantly lower in the splenectomized patients than in the controls. CONCLUSION: The findings suggest that splenectomy may diminish virus burden in cirrhotic patients with HCV infection at least in part, through augmentation of natural killer cell activity.     

Immune responses to administration of a vaccine against Haemophilus influenzae type B in splenectomized and non-splenectomized patients.

OBJECTIVES: we investigated the cause of hypo-responsiveness to vaccines in splenectomized subjects. METHODS: we evaluated the immune responses to a Haemophilus influenzae type b vaccine and the sizes of lymphocyte subpopulations in 25 splenectomized and 45 non-splenectomized thalassaemic patients, in 12 individuals who had been splenectomized after trauma and in 20 controls. RESULTS: the immune response in the controls was significantly higher (P < 0.001) than in splenectomized patients after trauma and in both, the response was higher (P < 0.001) than in thalassaemic patients. In asplenic subjects after trauma, percentages of CD3 and CD4 cells were lower (P < 0.001) than in patients in the other groups; the controls had higher percentages of CD8 cells (P < 0.001) than patients in the other groups. The natural logarithm of the mean percentage of (CD19 showed a quadratic trend from thalassaemic patients through asplenic subjects to controls (P < 0.001). Levels of CD16+ natural killer (NK) cells were higher (P < 0.001) only in asplenic subjects after trauma. CONCLUSIONS: the significant decrease in the immune response of the splenectomized thalassaemic patients vs. non-splenectomized thalassaemic patients may, in part, be due to their basic immunological condition. Thus, the best strategy for protecting these subjects is to vaccinate them before the splenectomy.     

Humoral and cell-mediated immunity in neonates with herpes simplex virus infection

Fifty-nine neonates with herpes simplex virus (HSV) infection were evaluated with use of assays for neutralizing antibody (NAb), lymphocyte transformation (LT), alpha interferon production, and virus-specific antibody (immunoblots). Infants with disseminated disease or onset in the first week of life were more likely to lack NAb. Patients treated with vidarabine were more likely than those treated with acyclovir to develop a fourfold rise in NAb titer. Infants with encephalitis showed a broader spectrum of IgG and IgM antibody reactivity against HSV proteins by immunoblotting than did those who had earlier onset of mucocutaneous illness. Only 10 of 33 infants had HSV-specific LT, compared with eight of eight adults with primary HSV. Neonates with positive LT were more likely to show a fourfold rise in NAb titer. In vitro alpha interferon production was diminished in infants, compared with values in adults.     

The relationship between responsiveness to splenectomy and clinical course after splenectomy in chronic idiopathic thrombocytopenic purpura (ITP)

Fifty-four ITP patients were treated by the protocol proposed by Japanese Idiopathic Disease of Hematopoietic Organ Research Committee. Forty-eight patients were treated with corticosteroid (CS). Twenty-eight patients out of 30 patients who were refractory to CS were splenectomized. To study natural courses of splenectomized patients, 25 patients were splenectomized without elevation of platelet counts at the operation and were followed up their platelet counts and PAIgG for one month without treatment. Clinical courses after splenectomy were classified into four types by the platelet increment followed by splenectomy and the platelet counts at one month after splenectomy; type I (markedly over-shooted platelet peak and normalized platelet counts), type II (moderate platelet peak and 50 approximately 100 x 10(3)/microliters platelets), type III (minimal platelet peak and less than 50 x 10(3)/microliters platelets), and type IV (elevation of platelet counts at day 7 or later of splenectomy and normalized platelet counts). The results of analyses from 25 patients indicated that the numbers of type I, II, III and IV were 10, 5, 6 and 4, respectively. The clinical course of type IV suggests that platelet production may be impaired by anti-platelet antibodies.     

In Vitro Stimulation of Sensitized Lymphocytes by Herpes Simplex Virus and Vaccinia Virus

Splenic lymphocytes from rabbits immunized with herpes simplex virus (HSV) were incubated in vitro with ultraviolet light-inactivated HSV, and the degree of lymphocyte transformation was determined by measurement of the incorporation of [(3)H]thymidine into acid-insoluble material. Lymphocytes from immunized rabbits were stimulated as much as 30-fold, whereas lymphocytes from control rabbits were unaffected. Lymphocyte sensitization occurred within 3 days after immunization; sensitized lymphocytes could still be detected 120 days after immunization. The antigenicity of the ultraviolet light-inactivated crude virus pool was found to reside primarily in the virion. Infectious virus, in comparison with inactivated virus, produced less lymphocyte stimulation. Studies on the interaction of the humoral and cellular immune responses showed that incubation of anti-HSV antibody with HSV antigens did not reduce the capacity of the viral antigens to stimulate sensitized lymphocytes. Other experiments revealed that lymphocytes from both the spleen and peripheral blood of animals immunized with vaccinia virus could be stimulated by vaccinia, but not by HSV. Conversely, lymphocytes from animals immunized with HSV could not be stimulated by vaccinia. The transformation of sensitized lymphocytes by viral antigens appears to be a simple, highly specific, and quantitative in vitro technique for the study of the cellular immune response to viral infections.     

Effect of DDS on phytohemagglutinin-induced lymphocyte transformation

The influence of DDS on PHA-induced lymphocyte transformation was investigated in leukocyte cultures from two samples of healthy Caucasoid individuals. In one sample the sulfone-treated cultures differed from the controls in that they contained 0.4 mug/ml of other sample, the treated cultures contained DDS in concentrations of 4 mug/ml, 8 mug/ml and 16 mug/ml. The frequency of lymphocyte transformation induced by PHA was significantly reduced by DDS in all concentrations used. The data obtained are a strong indication that the plasma levels of dapsone among leprosy patients may contribute to the depression of the blastogenic capacity of their lymphocytes when stimulated by PHA.     

Lymphoid Cell Sets and Serum Immunoglobulins in Patients with Thalassaemia Intermedia: Relationship to Serum Iron and Splenectomy

S ummary Peripheral blood lymphocyte sets and serum immunoglobulins were examined in 15 patients with thalassaemia intermedia, aged between 5 and 30 years. They were divided in three clinical groups: Group I, non-splenectomized, with serum iron (SI) lower than 200 μg/dl; Group II, splenectomized with serum iron lower than 200 μg/dl; and Group III, splenectomized with serum iron levels greater than 200 μg/dl. High absolute lymphocyte counts were observed in the majority of patients, whether splenectomized or not. Following splenectomy, marked increases were observed in absolute number of lymphocytes and percentages of circulating surface immunoglobulin (SIg) bearing B lymphocytes, mouse rosette forming cells (MRFC) and 'null' cells. The increases in circulating B lymphocytes following splenectomy were due exclusively to cells bearing SIgM and/or SIgD. No changes were observed in numbers and proportions of circulating SIgA or SIgG bearing cells. Serum IgA levels were high in the majority of patients studied whether splenectomized or not. Following splenectomy increases in serum IgA and serum IgG were observed which seemed to relate to increasing SI levels. No significant changes occurred in serum IgM levels.
Absolute numbers of circulating T lymphocytes were unaffected by splenectomy or SI levels. Changes were observed, however, in distribution of T lymphocyte subsets with decreases in number of Tμ cells and concomitant increases in numbers of non-Tμ, non-Tγ cells. Tγ cells were the least affected by splenectomy and increasing SI. The results are compatible with the proposition that iron participates in the regulation of lymphoid cell migration and in the modulation of expression of cell surface markers. The findings also illustrate the role of the spleen in the control of lymphocyte migration and immunoglobulin production.     

Autoantibodies in splenectomized patients as a consequence of abdominal trauma.

The presence of autoantibodies was examined in 100 adult patients splenectomized because of abdominal trauma and without systemic disease, and the results were compared with those observed in a healthy control group. Thirty-seven percent of the patients studied presented some type of autoantibody. Those observed were antinuclear, anti-smooth muscle and, in 1 case, anti-reticulin. Patients with antinuclear antibodies have no antibodies against native DNA, but in 5 cases, anti-Ro (SS-A) and anti-La (SS-B) antibodies were observed. Rheumatoid factors were also found, but their incidence was similar in patients and controls. The incidence of autoantibodies proved not to be related to either the period of time since splenectomy or to age of the patient; they were observed in patients many years after splenectomy and in individuals of both sexes and all ages. Although the presence of autoantibodies seemed to have no effect on our patients, all of whom were asymptomatic, it is important to be aware of this phenomenon when evaluating the laboratory results obtained from splenectomized patients.     

Cellular and humoral immunity in Hodgkin's disease. I Patients in continuous long-term remission

Cell-mediated and humoral immunity to herpes simplex virus (HSV), cytomegalovirus (CMV) and varicella zoster virus (VZV) were examined in 37 patients with Hodgkin's disease in continuous long term remission. This group had lower blast-transformation than a matched control group to all 3 antigens. Patients originally showing B-symptoms had higher transformation to VZV than those with A-symptoms. Patients treated with irradiation only had higher transformation than those treated with either chemotherapy or a combination of chemotherapy and irradiation. There was a clear tendency towards lower transformation in patients having been in remission for 2 years or less. Phytohaemagglutinin (PHA) stimulation gave lower response in the patient group than in the control group. Patients with B-symptoms had lower response than those with A-symptoms. Interferon production was specially impaired in patients with B-symptoms. The patient group had higher CF titers against HSV and CMV while the control group had higher titers against VZV. B-symptom patients had higher titers against VZV than A-symptom patients. It is concluded that HD patients have impaired immune function many years after discontinuation of therapy, but there are certain differences regarding the in vitro immunity within the patient groups.     

Cellular and Humoral Immunity in Hodgkin's Disease

Cell-mediated and humoral immunity to herpes simplex virus (HSV), cytomegalovirus (CMV) and varicella zoster virus (VZV) were examined in 37 patients with Hodgkin's disease in continuous long term remission. This group had lower blast-transformation than a matched control group to all 3 antigens. Patients originally showing B-symptoms had higher transformation to VZV than those with A-symptoms. Patients treated with irradiation only had higher transformation than those treated with either chemotherapy or a combination of chemotherapy and irradiation. There was a clear tendency towards lower transformation in patients having been in remission for 2 years or less. Phytohaemagglutinin (PHA) stimulation gave lower response in the patient group than in the control group. Patients with B-symptoms had lower response than those with A-symptoms. Interferon production was specially impaired in patients with B-symptoms. The patient group had higher CF titers against HSV and CMV while the control group had higher titers against VZV. B-symptom patients had higher titers against VZV than A-symptom patients. It is concluded that HD patients have impaired immune function many years after discontinuation of therapy, but there are certain differences regarding the in vitro immunity within the patient groups.     

Expression of the markers BDCA‐2 and BDCA‐4 and production of interferon‐α by plasmacytoid dendritic cells in systemic lupus erythematosus

Objective

To study the expression of blood dendritic cell antigen 2 (BDCA‐2) and BDCA‐4 molecules by plasmacytoid dendritic cells (PDCs) in the blood of patients with systemic lupus erythematosus (SLE), and to study PDC production of interferon‐α (IFNα) and its inhibition by anti–BDCA‐2 and anti–BDCA‐4 antibodies.

Methods

Peripheral blood mononuclear cells (PBMCs) from SLE patients (SLE PBMCs) and from healthy controls were induced to produce IFNα in vitro by SLE serum containing an endogenous IFNα‐inducing factor (SLE‐IIF) or by herpes simplex virus type 1 (HSV‐1). The frequencies and numbers of BDCA‐2–, BDCA‐3–, and BDCA‐4–expressing cells were analyzed by flow cytometry, and the effects of anti–BDCA‐2 and anti–BDCA‐4 monoclonal antibodies (mAb) on IFNα production were investigated.

Results

IFNα production by SLE PBMCs induced by SLE‐IIF or HSV‐1 was decreased compared with that of healthy control PBMCs (P = 0.002 and P = 0.0007, respectively). The proportions of BDCA‐2– and BDCA‐3–expressing cells in SLE PBMCs were reduced compared with those in PBMCs from healthy controls (P = 0.01 and P = 0.004, respectively). IFNα producers in culture, especially among SLE PBMCs, displayed reduced BDCA‐2 expression and constituted only a minority of the BDCA‐2–positive cells, at least in healthy control PBMCs (median 18%). IFNα production by both SLE and healthy control PBMCs stimulated by SLE‐IIF or HSV‐1 was markedly reduced by anti–BDCA‐2 mAb (median 81–98% inhibition). Anti–BDCA‐4 mAb only partially inhibited SLE‐IIF–induced IFNα production.

Conclusion

SLE patients had a reduced number of BDCA‐2–expressing PDCs, also termed natural IFNα‐producing cells, and their IFNα production could be inhibited by anti–BDCA‐2/4 mAb. Such mAb may be a therapeutic option for inhibiting the ongoing IFNα production in SLE patients.

     

Interleukin-2 protects neonatal mice from lethal herpes simplex virus infection: a macrophage-mediated, gamma interferon-induced mechanism

Administration of human recombinant interleukin-2 (IL-2) protected neonatal mice from a lethal herpes simplex virus (HSV) infection. Protection was not associated with viral antibody production, enhanced natural killer cell cytotoxicity, or intrinsic resistance of macrophages to viral infection. Protection was associated with increased macrophage-mediated antiviral antibody-dependent cellular cytotoxicity (ADCC). Spleen cells from IL-2-treated neonatal mice and from neonatal mice that were treated in vitro with IL-2 transferred protection to neonatal mice. These cells, by adherence, silica, and asialo GM 1 antibody treatment, were shown to be macrophages. IL-2 treatment in vitro enhanced the neonatal macrophages' ADCC function and superoxide release. Similar protection was induced by gamma interferon (IFN-gamma)-treated spleen cells. Antibody to IFN-gamma ablated both IFN-gamma- and IL-2-induced protection by adherent spleen cells. Thus, IL-2-mediated protection against murine neonatal HSV infection was affected by stimulated macrophage activity, via helper T cell-produced IFN-gamma.     

Phytohaemagglutinin induced proliferation of lymphocytes from patients with rheumatoid arthritis and iron deficiency.

The response of peripheral blood lymphocytes to stimulation by mitogens such as phytohaemagglutinin (PHA) is commonly depressed in both rheumatoid arthritis and iron deficiency, and as many rheumatoid patients are anaemic with evidence of abnormal iron metabolism it is possible that the same mechanism underlies the observed suppression in both conditions. In the present study the mitogenic response to PHA of lymphocytes from three rheumatoid patients, who were also iron deficient, and two healthy controls has been shown to be significantly less in iron deficient than iron containing media (p less than 0.001). In addition, iron deficient sera from these patients reduced the PHA induced proliferation of lymphocytes from a normal subject (p less than 0.01), an effect which was prevented by prior addition of iron to these serum samples. In iron containing media lymphocytes from five patients and two controls showed no difference in their response to PHA for both the minimum mitogen concentration which enhanced transformation and the peak [3H]thymidine uptake; but patients' lymphocytes showed significantly less response to PHA concentrations of 5 and 10 mg/l (p less than 0.02), resulting in a reduction in the area under the dose response curves up to 20 mg/l (p less than 0.05). These findings show both that iron deficient sera can impair PHA induced lymphocyte transformation and that lymphocytes from iron deficient rheumatoid patients have impaired responsiveness to PHA. Iron is known to be required intracellularly for the enzyme ribonucleotide reductase, which is important for DNA synthesis, and reduced activity of this enzyme could explain these observed effects.     

Double-faced cell-mediated immunity in β-thalassemia major: stimulated phenotype versus suppressed activity

In this study, the immunologic abnormalities of Iranian β-thalassemia major patients were characterized, considering their clinical parameters including splenectomy status and iron overload. Serum samples and peripheral blood mononuclear cells were collected from 28 patients and 30 age- and sex-matched healthy individuals. Patients with thalassemia showed significantly increased absolute lymphocyte counts compared with the control group. An increased number of activated T cells and higher levels of serum neopterin were also observed in thalassemia patients, which suggest chronic stimulation of immune system. On the contrary, T-cell proliferation and interleukin 2 (IL-2), interferon gamma (IFN-γ), and IL-4 production were suppressed in patients compared to controls. Patients with high serum ferritin levels produced significantly less IFN-γ and IL-2, indicating the immunosuppressive effect of iron overload in β-thalassemia patients. The serum levels of tumor necrosis factor alpha and absolute counts and percentages of B and T cells were higher in splenectomized patients; however, serum levels of neopterin significantly decreased in splenectomized patients compared to the non-splenectomized group. Taken together, T lymphocytes express activated phenotype in polytransfused β-thalassemia major patients, while T cell proliferation and effector function are significantly suppressed. Multiple blood transfusion and continuous immune stimulation could be responsible for making such a double-faced immune response.     

Humoral and cellular responses to Pneumocystis carinii, CMV, and herpes simplex in patients with AIDS and in controls

The titers of IgG and IgA to Pneumocystis carinii in 36 AIDS patients did not differ significantly from those in 31 controls. Only 2/15 patients (13%) with P. carinii pneumonia (PCP) had titers of IgM antibodies greater than or equal to 5, which is significantly less frequent than in 32 controls (62%) and in 21 AIDS patients without PCP (43%). The risk of PCP was 5 times higher in patients without IgM antibodies to P. carinii than in patients who had these antibodies. A significantly higher percentage of those without PCP (57%) showed increasing titers of IgM antibodies to P. carinii in the second of paired samples taken about 6 months apart, compared with whose with PCP (9%; p = 0.05). All patients had high titers of antibodies to CMV and HSV and normal total concentrations of immunoglobulins. None of the patients responded in lymphocyte transformation to P. carinii, CMV, or HSV antigens. There is no obvious explanation to the selective lack of IgM antibodies to P. carinii in patients with PCP. Lack of IgM antibodies may be a marker for an immunodeficiency to P. carinii.     

Deficient IFNα production in hairy cell leukemia

Summary Since the application of low doses of IFN-alpha is necessary to maintain remissions in Hairy Cell Leukemia (HCL) it is of interest whether peripheral blood mononuclear cells (MNC) of HCL patients can be induced in vitro to produce IFN-alpha. 9 patients suffering from advanced HCL were included in the study. The diagnoses were confirmed by characteristic findings in peripheral blood and bone marrow biopsies. For IFN treatment we initially used natural IFN-alpha (Bioferon) and switched later to recombinant IFN-alpha₂ (Boehringer). MNC of 5 patients before IFN therapy and of 6 patients during IFN therapy (2–47 weeks) were induced by phythemagglutinin (PHA), Corynebacterium parvum (C.p.), and sendai virus (SV). PHA is known to induce IFN-gamma. Both, C.p. and SV induced IFN-alpha but no IFN-gamma in MNC of healthy controls and of IFN treated breast cancer patients. In HCL patients normal antiviral activities could be induced by PHA. Zero or only low antiviral activities could be induced in MNC from 9 patients tested on 22 occasions. It is concluded that MNC from patients with advanced HCL can be induced to produce IFN-gamma but no IFN-alpha. Since IFN-alpha but not IFN-gamma is produced by monocytes it is likely that reduced numbers of monocytes which were found in our HCL patients before and during IFN treatment account for the described deficiency of IFN-alpha production.