[Neutrophil chemotactic dysfunction in multitransfused thalassemia patients]. / Disfunción quimiotáctica de los neutrófilos en pacientes talasémicos politransfundidos.

PURPOSE: To evaluate the chemotactic capability of neutrophils in thalassaemic patients under poly-transfusion regimens and in thalassaemia carriers. PATIENTS AND METHODS: Twenty-one patients in multi-transfusion regimen diagnosed in the Ricardo Gutiérrez Children Hospital were studied. Of them, 17 had thalassaemia major, 3 S/beta thalassaemia and one sickle-cell anaemia. Twenty-one normal subjects comprised a control group. Chemotaxis was evaluated by two methods, namely, migration under agarose layer and in microchemotaxis chamber under stimulation with N-formyl-methionyl-n-phenylalanine at optimal concentrations of 10(-5) M and 10(-6) M, respectively. RESULTS: In thalassaemia major patients, directed mobility of neutrophil assessed by both methods was significantly decreased with regard to the normal controls, whereas random mobility was preserved. The four patients under poly-transfusion who had not thalassaemia major showed the same neutrophil defect. On the contrary, chemotaxis and random mobility of the neutrophils from thalassaemia carriers (thalassaemia minor) were similar to those of the normal controls. CONCLUSIONS: These results suggest that the defect found in the patients might be caused by transfusion overload.

Functional and nonfunctional measles virus matrix genes from lethal human brain infections.

Subacute sclerosing panencephalitis (SSPE) is a lethal disease induced by the persistence of measles virus in the human brain. In many SSPE cases, the viral matrix (M) protein cannot be detected; in others, M proteins of the expected size are found and sequence analysis of M cDNAs has confirmed that the reading frames are intact, showing only several missense mutations. To determine whether these alterations result in nonfunctional proteins, we have replaced the M gene of an infectious full-length genomic cDNA (from vaccine strain Edmonston) with different M genes derived from four patients with SSPE. One of the SSPE M genes tested proved to be functionally competent, giving rise to a virus yielding titers similar to those of viruses containing the M gene from control lytic strains. The other three SSPE M genes were not functionally competent in the same test. In all three cases, the inactivating changes resided in the carboxyl-terminal half of the M protein, as shown by the exchange of either of the two genes halves. In summary, mutational M gene alterations, which either prevent synthesis of M protein altogether or only allow synthesis of nonfunctional M protein, have been detected by us and by others in 9 of 10 SSPE cases. The one functional M gene appears to be an exception to the rule, indicating that M gene alteration might not be an absolute requirement for disease development.

Infectious measles virus from cloned cDNA.

The study of measles virus (MV) and of negative strand RNA viruses in general has been hampered by the lack of an experimental system for genetic manipulation. Here we describe a procedure for generating infectious MV from cloned MV cDNA. First we assembled a genetically marked DNA copy of the MV genome in plasmids, under the control of phage T3 or T7 promoters, allowing production of transcripts almost identical to the MV genome or antigenome. Incubation of these linearized plasmid DNAs with the appropriate phage polymerase and only two ribonucleoside triphosphates yielded committed transcription complexes. Microinjection of these complexes into the cytoplasm of helper cells which provide the proteins necessary for MV genome encapsidation and transcription/replication, reproducibly give rise to lytic MVs. The transcripts of one of these viruses were analysed by sequencing after reverse transcription followed by DNA amplification, and found to contain the genetic tags. The described procedure permits the analysis of a negative strand RNA virus with the same genetic tools previously applicable only to positive strand RNA viruses and retroviruses.

[The monocyte-macrophage system in Hodgkin's disease]. / El sistema monocito-macrófago en la enfermedad de Hodgkin.

Hodgkin's disease (HD) is considered as a tumor of the lymph nodes histologically characterized by a variety of cell types, resembling a nonspecific inflammatory reaction. The Reed-Sternberg cells present in the granuloma are considered neoplastic due to cytogenetic alterations, tissue culture properties and heterotransplantability. They originate from a macrophage-derived interdigitating reticulum cell. The lymph node is an immunologic organ and its alterations reveal qualitative and/or quantitative defects of the immune system. These are observed in HD at very early stages even with a minimum of lymph node involvement. Considering HD as a neoplasm of the monocyte-macrophage system, our objective was to investigate the functional capability of peripheral blood monocytes transformed into macrophages in vitro. The phagocytic and lytic activities were evaluated by the generation of toxic oxygen metabolites as due to an excessive production of PGE-2. This defect could be corrected by cyclo-oxygenase inhibitors. The defect was present at very early stages of HD and persisted even during prolonged continuous complete remissions. We also found a defect in the ingestion of candida which could not be modified by drug treatment, indicating the existence of a global dysfunction of the phagocyte. Presently, more than 90% of HD patients respond to specific therapy and remain in prolonged remission, being considered "cured". This fact may contribute to the diminished number of reports in relation to the biology of the monocyte-macrophage system in this disease.

[Immunologic studies in thalassemia major]. / Estudios inmunológicos en talasemia mayor.

It is accepted that the immune alterations in patients with thalassemia major (TM) are secondary to the continuous transfusion-related antigenic stimulation together with iron overload. We evaluated the immune status of TM patients and found quantitative alterations in the distribution of peripheral blood lymphocyte subpopulations as well as functional alterations in natural killer (NK) cytotoxicity, B-cell differentiation, T-cell immunoregulation and phagocyte functional activities. TM patients, 10 years old or younger, have a lymphocyte profile and phagocytic activity similar to normal controls. Non-splenectomized thalassemic patients, older than 10, present lymphocytosis due to an increase in B lymphocytes and with splenectomy the T-CD8+ lymphocytes increase. With respect to phagocytes, the capacity to ingest candida is preserved while the candidacidal activity and the generation of toxic oxygen metabolites during the respiratory burst are diminished, and are inversely proportional with age and serum ferritin concentration, that is, older in age and higher in iron overload, more profound are the phagocyte dysfunctions. The altered B-cell function, the dysfunction of T immunoregulatory cells and the defective NK activity observed in TM patients were independent of the age of the patients and they were observed even in children younger than 10 years old and in general are attributed to blood transfusions. Moreover, there are some alterations that thalassemic carriers can express such as a defect at the level of NK and at B-cell function regulations, suggesting a possible genetic origin. Although complex, TM constitutes a human model that allows the dissection of specific immune defects, involving multiple factors, and can provide a better comprehension of how this complex immunoregulatory system works.

Dysfunction of the monocyte-macrophage system in the idiopathic minimal change nephrotic syndrome.

We studied the function of phagocytes and the distribution of lymphocyte subpopulations in 23 patients with Idiopathic Minimal Change Nephrotic Syndrome. All the patients were in relapse at the time of the study. The latter was performed before specific therapy was started. Our control group consisted of 26 normal children who were studied while undergoing routine analysis prior to plastic surgery. Polymorphonuclear leukocytes from the patients showed no alterations in their ability to ingest and to kill candidas. On the contrary, peripheral blood monocytes had a normal phagocytic function with a decreased candidacidal activity when compared to normal controls (p less than 0.001). No correlation was found between serum immunoglobulin levels and the monocyte lytic function. The absolute number of B lymphocytes was significantly increased (p less than 0.05), whereas the absolute number of total lymphocytes, T lymphocytes and T4+ and T8+ cell subsets did not differ from those of the age-matched normal controls. Natural killer cells were functionally normal.

El sistema monocito-macrófago en la enfermedad de Hodgkin. / [The monocyte-macrophage system in Hodgkins disease]

Hodgkins disease (HD) is considered as a tumor of the lymph nodes histologically characterized by a variety of cell types, resembling a nonspecific inflammatory reaction. The Reed-Sternberg cells present in the granuloma are considered neoplastic due to cytogenetic alterations, tissue culture properties and heterotransplantability. They originate from a macrophage-derived interdigitating reticulum cell. The lymph node is an immunologic organ and its alterations reveal qualitative and/or quantitative defects of the immune system. These are observed in HD at very early stages even with a minimum of lymph node involvement. Considering HD as a neoplasm of the monocyte-macrophage system, our objective was to investigate the functional capability of peripheral blood monocytes transformed into macrophages in vitro. The phagocytic and lytic activities were evaluated by the generation of toxic oxygen metabolites as due to an excessive production of PGE-2. This defect could be corrected by cyclo-oxygenase inhibitors. The defect was present at very early stages of HD and persisted even during prolonged continuous complete remissions. We also found a defect in the ingestion of candida which could not be modified by drug treatment, indicating the existence of a global dysfunction of the phagocyte. Presently, more than 90

of HD patients respond to specific therapy and remain in prolonged remission, being considered [quot ]cured[quot ]. This fact may contribute to the diminished number of reports in relation to the biology of the monocyte-macrophage system in this disease.

Estudios inmunológicos en talasemia mayor. / [Immunologic studies in thalassemia major]

It is accepted that the immune alterations in patients with thalassemia major (TM) are secondary to the continuous transfusion-related antigenic stimulation together with iron overload. We evaluated the immune status of TM patients and found quantitative alterations in the distribution of peripheral blood lymphocyte subpopulations as well as functional alterations in natural killer (NK) cytotoxicity, B-cell differentiation, T-cell immunoregulation and phagocyte functional activities. TM patients, 10 years old or younger, have a lymphocyte profile and phagocytic activity similar to normal controls. Non-splenectomized thalassemic patients, older than 10, present lymphocytosis due to an increase in B lymphocytes and with splenectomy the T-CD8+ lymphocytes increase. With respect to phagocytes, the capacity to ingest candida is preserved while the candidacidal activity and the generation of toxic oxygen metabolites during the respiratory burst are diminished, and are inversely proportional with age and serum ferritin concentration, that is, older in age and higher in iron overload, more profound are the phagocyte dysfunctions. The altered B-cell function, the dysfunction of T immunoregulatory cells and the defective NK activity observed in TM patients were independent of the age of the patients and they were observed even in children younger than 10 years old and in general are attributed to blood transfusions. Moreover, there are some alterations that thalassemic carriers can express such as a defect at the level of NK and at B-cell function regulations, suggesting a possible genetic origin. Although complex, TM constitutes a human model that allows the dissection of specific immune defects, involving multiple factors, and can provide a better comprehension of how this complex immunoregulatory system works.

El sistema monocito-macrófago en la enfermedad de Hodgkin / The monocyte-macrophage system in Hodgkins disease

La enfermedad de Hodgkin (EH) se caracteriza por la aparición de un tumor en los ganglios linfáticos constituido por una gran variedad de células que se asemejan a una reacción inflamatoria inespecífica. Las células de Reed-Sternberg (R-S) presentes en la granuloma tienen características citogenéticas, de cultivo y de heterotransplantabilidad que parecen neoplásicas. Su origen más probable es en las células interdigitantes ganglionares derivadas de los macrófagos. Al ser el ganglio linfático un órgõo inmunológico, sus alteraciones se manifestan en defectos de la respuesta inmune, los cuales pueden deberse tanto a la expresión de la calidad de las mismas. En la EH la alteración de la respuesta inmune se observa en estadíos precoces, aún con una mínima extensión de compromiso ganglionar, lo que sugiere más una lesión cualitativa que cuantitativa. Teniendo en cuenta que el origen más probable de esta extraña neoplasia es la célula de R-S y que ésta deriva de los macrófagos, investigamos la capacidad funcional de los monocitos sanguíneos transformados in vitro en macrófagos. Se estudió su capacidad fagocítica y lítica a través de la generación de productos tóxicos del oxígeneo, medidos por quimioluminiscencia y citomorfologia. Se encontró un defecto en la generación de productos tóxicos del oxígeno, que se debía a un exceso en la producción de PGE2 y era corregido por inhibidores de la síntesis de prostaglandinas (ciclooxigenasas); este defecto aparece precozmente en la EH y continúa... (AU)

Estudios inmunológicos en talasemia mayor / Immunologic studies in thalassemia major

Los pacientes con talasemia mayor (TM) presentan defectos inmunes secundarios la estimulación antigénica y sobrecarga de hierro que resultan de su contínuo tratamiento con alteraciones cuantitativas en las subpoblaciones linfocitarias sanguíneas, como defectos funcionales en la citotoxicidad natural (NE), en la diferenciación B, en la inmunorregulación por células T, y en la actividad efectora de los fagocitos. Los pacientes con TM de hasta 10 años de edad tienen una distribución linfocitaria similar a la de controles normales y lo mismo ocurre con la función de sus fagocitos. Los pacientes de más de 10 años presentan una linfocitosis, con aumento de linfocitos B y en los pacientes esplenectomizados, también de los linfocitos T-CD8 positivos. El defecto en los fagocitos consiste en una disminución en la generación de metabolitos tóxicos del O2 durante el estallido respiratorio, con menor capacidad candidicida, pero con capacidad fagocitica normal. Este defecto es proporcional a la edad y a la concentración de ferritina sérica o sea que a mayor edad y/o sobrecarga de hierro, mayor defecto en los fagocitos. Las disfunciones B, T y NK eran independientes de la edad de los pacientes, observándose incluso en pacientes de menos de años, aunque se las atribuye a las transfusiones de sangre. Algunos defectos se pueden encontrar incluso en los portadores de TM, específicamente en la función B y NK, lo que sugiere un componente genético. La TM constituye un modelo humano que se pese a su... (AU)

El sistema monocito-macrófago en la enfermedad de Hodgkin / The monocyte-macrophage system in Hodgkin's disease

La enfermedad de Hodgkin (EH) se caracteriza por la aparición de un tumor en los ganglios linfáticos constituido por una gran variedad de células que se asemejan a una reacción inflamatoria inespecífica. Las células de Reed-Sternberg (R-S) presentes en la granuloma tienen características citogenéticas, de cultivo y de heterotransplantabilidad que parecen neoplásicas. Su origen más probable es en las células interdigitantes ganglionares derivadas de los macrófagos. Al ser el ganglio linfático un órgäo inmunológico, sus alteraciones se manifestan en defectos de la respuesta inmune, los cuales pueden deberse tanto a la expresión de la calidad de las mismas. En la EH la alteración de la respuesta inmune se observa en estadíos precoces, aún con una mínima extensión de compromiso ganglionar, lo que sugiere más una lesión cualitativa que cuantitativa. Teniendo en cuenta que el origen más probable de esta extraña neoplasia es la célula de R-S y que ésta deriva de los macrófagos, investigamos la capacidad funcional de los monocitos sanguíneos transformados in vitro en macrófagos. Se estudió su capacidad fagocítica y lítica a través de la generación de productos tóxicos del oxígeneo, medidos por quimioluminiscencia y citomorfologia. Se encontró un defecto en la generación de productos tóxicos del oxígeno, que se debía a un exceso en la producción de PGE2 y era corregido por inhibidores de la síntesis de prostaglandinas (ciclooxigenasas); este defecto aparece precozmente en la EH y continúa...

Estudios inmunológicos en talasemia mayor / Immunologic studies in thalassemia major

Los pacientes con talasemia mayor (TM) presentan defectos inmunes secundarios la estimulación antigénica y sobrecarga de hierro que resultan de su contínuo tratamiento con alteraciones cuantitativas en las subpoblaciones linfocitarias sanguíneas, como defectos funcionales en la citotoxicidad natural (NE), en la diferenciación B, en la inmunorregulación por células T, y en la actividad efectora de los fagocitos. Los pacientes con TM de hasta 10 años de edad tienen una distribución linfocitaria similar a la de controles normales y lo mismo ocurre con la función de sus fagocitos. Los pacientes de más de 10 años presentan una linfocitosis, con aumento de linfocitos B y en los pacientes esplenectomizados, también de los linfocitos T-CD8 positivos. El defecto en los fagocitos consiste en una disminución en la generación de metabolitos tóxicos del O2 durante el estallido respiratorio, con menor capacidad candidicida, pero con capacidad fagocitica normal. Este defecto es proporcional a la edad y a la concentración de ferritina sérica o sea que a mayor edad y/o sobrecarga de hierro, mayor defecto en los fagocitos. Las disfunciones B, T y NK eran independientes de la edad de los pacientes, observándose incluso en pacientes de menos de años, aunque se las atribuye a las transfusiones de sangre. Algunos defectos se pueden encontrar incluso en los portadores de TM, específicamente en la función B y NK, lo que sugiere un componente genético. La TM constituye un modelo humano que se pese a su...

Dysfunction of monocytes in Hodgkin's disease by excessive production of PGE-2 in long-term remission patients.

The candidacidal activity and the production of oxygen radicals by monocytes were investigated in untreated and long-term remission patients with Hodgkin's disease (HD). Both groups showed a decreased candidacidal function of monocytes with a chemiluminescence (CL) response significantly lower and delayed with respect to normal controls. Indomethacin at 1 microgram/ml corrected the monocyte deficiency increasing the CL response to normal values and normalizing the kinetics in the untreated patients. However, in patients in remission, the peak was delayed and followed by a significant increase in the production of oxygen radicals compared with untreated patients. A direct linear correlation was found between the percentages of lysed Candida and maximum CL peak of stimulated monocytes. When prostaglandin E2 (PGE-2) levels, measured in supernatants of cultured mononuclear cells, were plotted against the percentages of killed Candida, an inverse linear correlation was found. Therefore, monocytes from HD patients have a dysfunction in the generation of oxygen radicals and a decreased candidacidal activity associated with excessive production of PGE-2. Indomethacin can correct the oxidative metabolism in the untreated patients while in apparently "cured" patients the disorder persists.

Cyclophosphamide augments ADCC by increasing the expression of Fc-receptors.

Previous reports demonstrated that cyclophosphamide (Cy) enhances two Fc gamma receptor (Fc gamma R) mediated functions: antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis. In this paper we examine the mechanisms whereby Cy modifies the cytotoxic capacity of mouse splenocytes. The results indicate that the observed augmentation of ADCC could not be attributed to a higher proportion of macrophages and/or polymorphonuclear leukocytes (PMN), but rather to an enhanced activity per effector cell. Binding studies showed that this augmentation was associated with an increased number, but not an increased avidity of Fc gamma R sites. The possibility that the enhanced Fc gamma R expression by Cy may result in the alteration of other Fc gamma R-mediated functions is discussed.

Comparison of Candida killing activity measured by chemiluminescence and cytomorphological methods in human phagocytes.

Phagocyte function can be assayed by many laboratory tests including a cytomorphological method that uses Candida cells as target. The aim of this study was to correlate this technique with the production of toxic oxygen metabolites, measured by chemiluminescence (CL). The biological function of polymorphonuclear (PMN) cells and monocytes from the blood of 24 normal subjects and 25 patients with immunodeficiency diseases were studied. CL was measured using opsonized zymosan as the stimulating agent and, for the evaluation of Candida killing activity, C. pseudotropicalis and C. albicans were used as targets. A linear correlation between CL and lytic activity was observed with both PMN and monocytes from normal subjects and patients (r = 0.563 to 0.955; P less than 0.05 to less than 0.001). Our results indicate that the production of toxic oxygen metabolites, as measured by CL is closely related to the killing of Candida by PMN and monocytes.

Effect of alpha interferon on the altered T-B-cell immunoregulation in patients with thalassemia major.

We studied the ability of B-lymphocytes to differentiate to immunoglobulin-producing cells in 14 patients with thalassemia major, 11 parents of the patients, and 86 normal subjects. In comparison with the parents of the patients and with the normal individuals, the patients were found to have 1) an increased number of cells spontaneously secreting immunoglobulin; 2) a decreased number of plaque-forming cells (PFC) when induced with pokeweed mitogen (PWM) and a deficient helper T-cell function for the induction of B-cell differentiation; 3) an increase in the number of PFC after the addition of alpha interferon (100 IU/ml) to the cell cultures with PWM. Previous incubation of non-T-cells with alpha interferon for 1 hr and subsequent coculture with T-cells from the same patients or from normal subjects in the presence of PWM increased the number of PFC. On the contrary, previous incubation of T-cells with alpha interferon followed by coculture with normal non-T-cells or cells from the same patient did not modify the number of PFC. These results suggest that patients with thalassemia major possess hyperreactive spontaneous B-cell responses and that these B-cells are unable to differentiate to immunoglobulin-secreting cells in the presence of PWM. This would be due to a T-helper-cell deficiency with an inability to produce lymphokines (interferon being one of them), while the B-cell function seems to remain intrinsically intact.

Early defect of phagocytic cell function in subjects at risk for acquired immunodeficiency syndrome.

We studied the functions of peripheral blood monocytes and polymorphonuclear cells in 15 apparently healthy homosexual men, eight homosexual or bisexual subjects with unexplained generalized lymphadenopathies (pre-AIDS), four homosexual men with acquired immunodeficiency syndrome (AIDS), and 15 heterosexual men. In comparison with normal controls, the homosexual groups studied presented a decreased monocyte candidacidal activity for Candida pseudotropicalis that gradually deteriorates as the clinical symptoms progress towards AIDS. The monocyte phagocytic function was retained. Although the phagocytic and candidacidal activities of the polymorphonuclear cells did not differ from those of the normal controls, the candidacidal activity in some of the cases studied was unusually enhanced, indicating that the cells were in an activated state. In addition, only two of nine sera tested from asymptomatic homosexual males were positive for antibodies to HTLV-III/LAV, while six out of eight pre-AIDS and both of the two AIDS patients tested had antibodies to AIDS-associated retrovirus. We suggest that in AIDS the phagocytic system is already involved, together with B and T lymphocyte abnormalities, during the early events of the syndrome, even without the detection of AIDS-associated retrovirus antibodies.

Impairment of natural killer (NK) function in carriers and patients with thalassemia major.

To evaluate natural killer cytotoxicity (NKC) and its regulation by interferon (IFN) in thalassemia major (TM), we studied 19 patients, 10 carriers (parents of 10 different patients) and 35 normal subjects (children and adults) as controls. We have found a diminished NKC, as well as a decrease in the percentages of cells bearing IgGFc receptors in TM patients, whereas they were normal in TM carriers. Although alpha-IFN enhanced NKC in TM patients and carriers, Con A (an IFN inducer) had no enhancing effect on NKC from either patients or carriers. These agents did not modify the expression of IgGFc receptors in cells from TM patients, carriers or normal controls. Our results indicate that TM patients have a defect in NK cell maturation or activation, which is reversed by alpha-IFN. The failure of TM patients and carriers to respond to Con A suggests a defect in NKC regulation which might be related to a genetic origin.

Progressive dysfunction of monocytes associated with iron overload and age in patients with thalassemia major.

We evaluated phagocytic and lytic activities of peripheral blood monocytes (PBMo) from patients with thalassemia major (ThP) using C pseudotropicalis as the target. PBMo from ThP showed decreased lytic activity (P less than .001), whereas the phagocytic activity did not differ from that of the controls. Significant inverse correlations were found between lytic activity of PBMo and age of patients (r2 = .47; P less than .01) and also between lytic activity and serum ferritin levels (r2 = .65; P less than .001). No association was found between lytic activity and other variables (blood transfusion regimens, therapy with desferrioxamine, liver damage, and the presence of sHBAg). Splenectomy showed no positive effect on PBMo functions from ThP. Our results suggest that PBMo from ThP have an intracellular defect in their microbicidal mechanisms associated with iron overload. This cell dysfunction could be responsible, at least in part, for the increased susceptibility to infections reported in ThP.