IgG subclasses of FVIII inhibitors in an argentine cohort of severe hemophilia A patients: Analysis by flow cytometry.

INTRODUCTION: FVIII inhibitors consist of a polyclonal population of antibodies. Previous studies have demonstrated different distribution of IgG subclasses. IgG4 was associated to high level of FVIII inhibitors and failure of immune tolerance induction (ITI) treatment. This study monitored the relative distribution of IgG subclasses of anti-FVIII in patients with severe hemophilia A (SHA). METHODS: Anti-FVIII antibodies were measured employing an immunomethod, developed in our laboratory, that combines flow cytometry (FC) with microspheres coupled (FVIII-m) or not (Control-m) to FVIII. Seventy-five patients with SHA were studied, 17 without inhibitors (Group I); 58 with inhibitor history, 13 low responders: (LR: Group II), and 45 high responders (HR: Group III). Eight patients undergoing ITI were also included. RESULTS: We found anti-FVIII antibodies in 11 of 27 patients (40%) without inhibitors and in 45 of 48 with inhibitors at the moment of the study. IgG4 was predominant only in the Group III: P=0.02 in patients with low level of inhibitors and P=0.0001 with high titer of inhibitors. Longitudinal analysis performed on patients undergoing ITI showed a gradual decrease of IgG4 values that was associated to improvement of clinical parameters during treatment. CONCLUSION: We suggest the use of the FC method to supplement functional traditional assays and to help to improve the management of patients with SHA.

Correlation between cyclophosphamide-induced viral susceptibility and depletion of Junin virus-induced suppressor populations.

In contrast to lymphocytic choriomeningitis virus, another arenavirus, Junin virus (JV), the etiologic agent of Argentine hemorrhagic fever, when inoculated into suckling mice, induces lethal meningoencephalitis characterized by a delayed-type hypersensitivity (DTH)-like immune response. However, the adult BALB/c mouse is resistant to infection and no DTH reaction can be seen. This different viral sensitivity may be related to the development of an antigen non-specific DTH-suppressor cell pathway at work in the adult mouse. When the resistant mice are treated with cyclophosphamide (Cy) (50 mg/kg each dose) given at days -1,+1,+4 (zero: infection day), animals become susceptible and develop DTH reaction in brain that leads to death. We analyze the influence of the timing of Cy administration on the suppressor system developing after infection. It was found that Cy depletes the previously described JV-induced suppressor populations (Tsv) but a new suppressor cell (Tsv*) is disclosed bearing the Thy 1+ Ly1+2- phenotype which is unable to depress DTH in Cy-treated animals. With only two doses of Cy corresponding to days -1 and +1, the target of Tsv* cells is depleted but the third dose is still required to achieve full depletion of Tsv cells which are able to employ the Cy-resistant antigen-specific suppressor cells as targets. Since the Cy treatment is able to deplete the Tsv population together with the target of Tsv* cells, animals became unable to regulate lethal DTH reaction. Thus, a cellular explanation for an empirically established Cy schedule able to abrogate the adult mouse resistance to JV is proposed.

Junin virus-induced non-specific suppressor cells interact with unrelated antigen-specific suppressor cells.

Junin virus (JV) infection of adult (resistant) BALB/c mice induces antigen non-specific delayed-type hypersensitivity (DTH) suppressor T cells, termed Tsv, bearing the Thy-1+, Ly-1+2- phenotype. These cells may be related to survival to infection since DTH reaction is associated with lethal meningoencephalitis. Employing several xenogeneic red blood cell (RBC) sensitization schedules to induce different cell subpopulations, we have attempted to establish the target of JV-induced suppressor cells (Tsv). The target of Tsv cells was actually included in the antigen-specific suppressor cell compartment, as demonstrated for the RBC system. Tsv cells were able to trigger suppressor cells to act without loss of their specificity. The presence of two sets of sheep RBC-induced DTH suppressor cells bearing the Ly-1+2- and Ly-1-2+ phenotypes was disclosed in low (10(6))-dose sensitized mice. Both sets were simultaneously required by Tsv to achieve DTH suppression. In contrast, in high (10(8] SRBC-dose sensitized animals treated with cyclophosphamide (doses of 50 mg/kg), a single Ly-1-2+ suppressor cell was required.

Contrasuppressor cells induced by Junin virus.

Intracerebral inoculation of Junin virus (JV) in all susceptible mouse models available induces a lethal meningoencephalitis compatible with a delayed-type hypersensitivity (DTH) immune response. In contrast, adult BALB/c mice prove resistant to infection and no DTH response is seen. JV inoculation in adult BALB/c mice induces DTH suppression to unrelated antigens such as sheep red blood cells. (SRBC). This suppression is mediated by JV-induced T cells (Tsv), which are operative from 1 to 24 days post-infection (p.i.), and seems to be related to adult mouse survival. The presence of JV-induced contrasuppressor cells (CS) bearing the Thy-1+, Ly 1+2- phenotype, able to abrogate Tsv cells-mediated suppression, is described here. Thus, CS cells may be involved in the mechanism by which mice avoid over-exposure to Tsv-mediated DTH suppression. Such CS cells were found in the spleen of inoculated animals and may also be induced by transferring JV-free Tsv cells to 'naive' recipients, in which JV inoculation then induces morbidity.

Junin virus-induced suppressor cells are effective only on the efferent limb of the delayed-type hypersensitivity response.

Junin virus (JV), the etiological agent of Argentinian hemorrhagic fever, induces high mortality in the suckling BALB/c mouse, which correlates with delayed-type hypersensitivity (DTH)-like immune response. In contrast, the adult mouse is resistant to infection, and no DTH response can be detected. An antigen-nonspecific DTH suppressor T-cell activity induced by JV has been described that may be related to adult mouse survival. In this report, we present evidence supporting the inability of such cells, identified here as bearing the L3T4 marker, to affect the normal establishment of a DTH reaction. In the same form, virus-induced suppressor cells were unable to alter the expression of DTH effector cells in the absence of antigen-specific DTH suppressor cells. Besides, we found that a single high dose (200 mg/kg) of cyclophosphamide before or after JV inoculation was unable to alter the activity of virus-induced suppressor cells.

Susceptible adult murine model for Junin virus.

The adult mouse model had been considered resistant to Junin virus (JV) infection. However, we found that C3H/HeJ murine strain proved highly susceptible up to 5 months of age to intracerebral inoculation with the prototype XJ JV strain, showing neurological signs and 80-90% mortality within 13 days. Neutralizing antibodies (Nt Ab) were absent, but low immunofluorescent Ab levels (1:5) were detected as from day +7. The virus could only be rescued by coculture of brain samples with Vero cells. Histopathological findings were consistent with the suckling mouse model and with a delayed-type hypersensitivity reaction. XJ inoculation by extraneural routes failed to cause disease, however, it induced Nt Abs. Ic infection with XJCL3 strain of JV attenuated for man and guinea pig, but not for mouse, induced high Nt Ab levels but not mortality. In either case, mice resisted ic XJ challenge. Thus, C3H/HeJ is the first adult mouse model susceptible to JV.

Suppressor T-cell population induced by Junin virus in adult mice.

Intracerebral (i.c.) Junin virus (JV) infection of adult BALB/c mice is characterized by the absence of morbidity and a low mortality (barely 8-10%). In contrast, the suckling mouse model exhibits almost 100% mortality following central nervous system (CNS) alterations consistent with a delayed-type hypersensitivity (DTH)-like immune response. Besides, JV infection of adult (resistant) mice leads to immunosuppression of DTH to unrelated antigens. Here we present evidence demonstrating that such suppression is mediated by JV-induced cells present in spleen from 24 hr to 24 days post-infection, bearing the Thy-1+, Ly-1+2- phenotype and reactive to an unrelated antigen such as sheep red blood cells (SRBC). No evidence of suppressor factors was found. A relatively low number of total splenic cells (5 x 10(6) cells/mouse) was enough to transfer suppression. Therefore, this cell population may be involved in adult mouse survival to JV infection.

Junin virus-induced delayed-type hypersensitivity suppression in adult mice.

Junin virus (JV) infection of suckling mice leads to lethal meningoencephalitis consistent with a delayed-type hypersensitivity (DTH)-like immune response. In contrast, there are no central nervous system (CNS) alterations, and high antibody titers are induced in resistant adult mice. As a possible explanation, JV infection in adult mice may provoke DTH depression. Thus in this work we study the alterations induced by JV in the immune response of adult mice by using sheep red blood cells (SRBC) as an unrelated indicator antigen. JV infection was found to abrogate DTH significantly, regardless of SRBC priming time, virus strain attenuation, and viral route of inoculation. The effect proved viral dose-dependent and required live and infectious virus. However, the humoral response to SRBC, as determined by splenic "plaque-forming cell" count was found higher than controls. These results are consistent with adult mouse response to JV infection. In contrast to the guinea pig model, there is no destruction of immunocompetent cells. T-cell percentage in the spleen was high, suggesting involvement with DTH-suppressive action. We suggest that the immune response to SRBC in adult infected mice may be used for understanding the mechanisms involved in resistance.

Maturation of suckling rat response to SRBC.

The immune response of the Buffalo/Sim rat to heterologous sheep red blood cells (SRBC) was studied here. The earliest time of response to 10(9) SRBC, the most suitable inoculation route and the behavior to challenge were determined. The intraperitoneal (ip) proved more effective than the subcutaneous (sc) route, since serum agglutinins became detectable in low titers in animals inoculated at 6-7 days of life by the former route and at 12 days by the latter. No splenic plaque-forming cells (PFC) were found in rats immunized ip at 2 days of age, and strong inhibition developed on challenge at day 14 post-inoculation (pi) (agglutinin titers at day 7 pi: 0.71 +/- 0.47 vs 4.6 +/- 0.51 in unprimed controls; PFC/10(7) cells at day 5: 122.21 +/- 36.17 vs 3,977.38 +/- 777.5 in unprimed controls). Serum agglutinin formation was also decreased, though to a lesser degree, when: a) animals were challenged at 30 or 60 days of age; b) both priming and challenge took place by sc route; or c) antigen dose was lowered to 10(7) or 10(5) SRBC. Mechanisms interpreting observed behavior are discussed.

Brain inflammatory exudate in Junin virus-infected rats: its characterization by the immunoperoxidase (PAP) technique.

Morphologic changes in cyclophosphamide (CY)-suppressed vs. control non-suppressed new-born rats infected i.c. with XJC13 strain of Junin virus were compared and the cells involved in CNS lesions were identified by the PAP technique. Fifty per cent of the control rats exhibited widespread cerebral necrosis vs. only 15% of the immunosuppressed animals. The first cells to reach Junin virus-infected CNS in controls were T lymphocytes, which destroyed viral antigen-laden target neurons and astrocytes. B lymphocytes and macrophages, presumably attracted by viral antigen and/or by lymphokines, made their appearance a day or two later. Activated macrophages phagocytosed necrotic cells and perhaps exerted a cytotoxic effect upon target neural cells, whereas the actual role of B lymphocytes requires further explanation. In CY-treated rats, cerebral lesions were smaller and the cellular exudate, though similar, proved much scantier than in controls. A similar extent of cerebellar necrosis was observed in both groups.

Protection conferred against Junin virus infection in rats.

2-day-old Wistar rats intracerebrally infected with the XJC13 strain of virus exhibited a 5% survival rate which rose to 71% after immune serum treatment. Brain viral titers were relatively unaffected by this treatment. Histologic studies showed necrosis in the cerebellum and brain cortex with mononuclear cell infiltration in both treated and nontreated groups. Beginning on day 16 postinfection, however, intracerebral perivascular gliosis foci and mild meningeal congestion were minimal in the treated animals. These findings imply that passively transferred humoral immunity leads to prompt recovery in this experimental model.

Action of antithymocyte serum on Junin virus infection in rats.

Two-day-old rats resistant to intracerebral (i.c.) infection with XJ strain of Junin virus (JV), were rendered sensitive to JV by treatment with antithymocyte serum (ATS). The mortality reached 80%, the virus titres in brain were higher and the serum neutralizing antibodies dropped but brain lesions were absent throughout. The same host was susceptible to XJCl3 strain infection, which induced lethal encephalitis manifested by severe necrotic foci in cerebellum. ATS treatment conferred significant protection against this strain; the mortality was 63%, viral titres in brain remained unchanged but the lesions were mild as compared with non-treated animals. It seems likely that the XJ strain allowed the 2-day-old rat to develop serum antibodies against JV, while the XJCl3 strain unleashed an immunopathologic response.

[Infection in rats by the intraperitoneal route with Junín virus]. / Infección en ratas por vía intraperitoneal con virus Junín.

The susceptibility of the rat, infected at various ages by intraperitoneal route with the XJ prototype strain of Junin virus was studied two day-old animals showed maximum mortality, being the most suitable dose 10(3) LD50. Antiviral humoral response at 35 days pi was tested in survivors of all ages. Highest neutralizing antibody titers were found in those infected up to 4 days of age. This host behaves quite differently when infected by intracerebral route with the same XJ strain.

Infección en ratas por vía intraperitoneal con virus Junín. / [Infection in rats by the intraperitoneal route with Junín virus]

The susceptibility of the rat, infected at various ages by intraperitoneal route with the XJ prototype strain of Junin virus was studied two day-old animals showed maximum mortality, being the most suitable dose 10(3) LD50. Antiviral humoral response at 35 days pi was tested in survivors of all ages. Highest neutralizing antibody titers were found in those infected up to 4 days of age. This host behaves quite differently when infected by intracerebral route with the same XJ strain.

Infección en ratas por vía intraperitoneal con virus Junín / [Infection in rats by the intraperitoneal route with Junín virus]

The susceptibility of the rat, infected at various ages by intraperitoneal route with the XJ prototype strain of Junin virus was studied two day-old animals showed maximum mortality, being the most suitable dose 10(3) LD50. Antiviral humoral response at 35 days pi was tested in survivors of all ages. Highest neutralizing antibody titers were found in those infected up to 4 days of age. This host behaves quite differently when infected by intracerebral route with the same XJ strain.

New attenuation marker for junin virus based on immunologic responses of guinea pigs.

A new attenuation marker to distinguish a virulent strain (XJJV) from an attenuated strain (XJC13JV or XJOJV) of Junin virus by means of the humoral and cellular responses to unrelated antigens was studied in guinea pigs. Strain XJJV suppressed the humoral immune response, as shown by the lower titers of precipitating antibody to ovalbumin. The concomitant decrease in serum complement level contributed to a milder Arthus cutaneous reactivity. In contrast, the attenuated strains did not decrease the humoral response. The pathogenic strain suppressed cell-mediated immunity, as demonstrated by decreased contact sensitivity to 2,4-dinitro-1-fluorobenzene and by depression of delayed skin reactions to tuberculin purified protein derivative. When attenuated strains were used, such suppressive effects were not observed. For virulent strain XJJV, virus replication in lymphoid organs and immunosuppressive effects were correlated. These findings provide a further means to differentiate between virulent and attenuated strains of Junin virus for the purpose of vaccine control of Argentine hemorrhagic fever.

[Effect of cyclophosphamide on experimental infection of rats with 2 strains of the Junin virus]. / Acción de la ciclofosfamida en la infección experimental de la rata con dos cepas de virus Junín.

The course of viral infection in rats of several ages after intracerebral inoculation with two strains of Junin virus, as well as the effect of an immunosuppressor was studied. The survival rate in 2-day-old rats was 95%, which fell to 45% in cyclophosphamide-treated infected animals (Figure 1a). However, barely 5% of these rats inoculated with the XJCl3 strain survived, while the cyclophosphamide suppressive treatment increased the rate to 36% (Figure 1b). This contrasting behaviour suggested that the XJ strain produces a subclinical infection in 2-day-old rats and the host immune mechanisms are responsible for recovery from the viral infection. Adequate immunosuppression converts sublethal experimental infections into lethal infection, accompanied by persistent viral replication in the target organ (brain) and suppression of anti-viral antibodies (Figure 2a). On the other hand, the inoculation of 2-day-old rats with the XJCl3 Junin virus may give rise to an immunopathology avoidable by CY treatment. In 10-day-old rats both strains of Junin virus caused a direct pathology, not modified by CY treatment (Table 3). This treatment failed to change susceptibility or virus concentration in the brain, but specific antibodies were considerably reduced. In the case of 26-day-old rats, there was total resistance to viral infection which remained unchanged after CY treatment.

Argentine hemorrhagic fever: a biologic marker.

Mortality rates and viral replication in blood and brains of Wistar rats between 6 h and 26 days of age inoculated with two strains of Junin virus of different virulence were compared. Viral growth curves in brains showed no differences between strains. However, differences in mortality rates were significant among rats between 1 and 3 days of age. When the intracerebral (i.c.) route was used, high mortality rates were induced by the attenuated XJC13 strain and low mortality rates were induced by the pathogenic XJ strain. On the other hand, when the intraperitoneal or subcutaneous route was used, mortality rates were reversed: low for the attenuated strain and high for the pathogenic one. The use of different doses of each virus and the application of various routes of inoculation in 2-day-old rats showed that 10(3) TCID50 by the i.c. route resulted in the greatest difference in mortality rates.