Diagnosis of haemophilia: use of an artificial factor-VIII-deficient human plasma system

An artificial clotting system deficient in factor VIII has been made from normal human plasma. Factors XII and XI are supplied as `activation product'. An eluate from Al(OH)<sub>3</sub>, which has been incubated with normal plasma, supplies factors X and IX in their `plasma' (unactivated) form with II. Factor V is provided as the supernatant after the Al(OH)₃- treated plasma has been precipitated at one-third saturation with (NH₄)₂SO₄. Fibrinogen is freed of factor VIII by freezing and thawing a lyophylized preparation and then added. Of these, activation product and the fibrinogen may be prepared in advance and stored frozen, and the eluate and supernatant may be made on the day of testing. A phospholipid source and CaCl₂-solution are also required. In use, a patient's and a control plasma are first diluted in a mixture of the eluate, supernatant, and fibrinogen solution, and clotting times are recorded after completing the system by adding the phospholipid, activation product, and calcium chloride. The clotting times from the mixtures containing the patient's and the control plasmas may then be compared.     

Changes in fibrinogen levels,platelet counts,clotting times and fibrinolytic activity in relation to thrombosis in contagious bovine pleuropneumonia

Changes in the packed cell volume, haemoglobin, fibrinogen and total protein levels, in the prothrombin, thrombin clotting and kaolin partial thromboplastin times, platelet numbers and fibrin degradation products were investigated in 7 cattle in which contagious bovine pleuropneumonia (CBPP) was induced by inoculating Mycoplasma mycoides by the endobronchial intubation method. Thrombosis of lung vessels was confirmed in all animals at necropsy.The results of the complement fixation and slide agglutination blood tests indicated that typical lesions of CBPP were present at 7 days. There was a rise in fibrinogen levels soon after inoculation and the difference between the means of the levels of the infected and control animals was significant from the 7th day. Platelet counts in the infected animals were reduced on the 2nd to the 5th day and, although a significant difference between these and the controls could not be demonstrated, this was confirmed by a compensatory rise in the severely affected animals that survived. The kaolin partial thromboplastin time test showed a significant rise in the infected animals from the 8th day, but there was no increase in the prothrombin time nor the thrombin clotting time indicating that depletion of factors in the intrinsic clotting system had commenced by the 8th day and that factors, VIII, IX, XI and XII could have been involved. Fibrin degradation products increased significantly in the infected animals from the 9th day.We conclude that thrombosis is an early event in the pathogenesis of CBPP, it contributes to the subsequent pathology of the lung lesions and platelets are involved to a substantial degree in the formation of the thrombi.     

Attenuated Junin virus infection in Callithrix jacchus

Twenty marmosets, male Callithrix jacchus, were used during this study. Fifteen of the marmosets were inoculated with 5,000 TCID50 of the attenuated XJC13 strain of Junin virus by intramuscular route and five were left as uninoculated controls. Animals were observed for a 420-day period. In order to carry out virologic, hematologic, serologic, and histologic studies the animals were bled and/or killed at different days post infection(pi). Results obtained showed that the attenuated strain produced an infection with no mortality or signs of illness. There was only a slight loss of weight at 18-40 days pi, which was soon recovered. Viremia was present from day 6 to 22, titers peaking at 4.0 log. Viral spread was limited to the lungs, spleen, lymph nodes, and bone marrow in the animal killed on day 14. No virus was found in the organs of the animal killed on day 23, and neither hematologic alterations nor pathologic lesions were seen in these monkeys except for ganglionar hypertrophy with immunoblast proliferation. Antigen was detected by immunofluorescence (IF) in lymph nodes, spleen, adrenals, lungs and brain. Neutralizing antibodies were detected from the third week onward. Protection conferred by the XJC13 strain proved effective when XJC13-inoculated monkeys were challenged with 1,000 TCID50 of the pathogenic XJ strain at days 60 or 380 pi, while normal controls died. When viral persistence was searched for on days 370, 390, and 420 pi, no infectious virus was detected, but viral antigen was seen in certain organs, which, however, lacked tissue damage.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protection of Junín virus-infected marmosets by passive administration of immune serum: association with late neurologic signs

Argentine hemorrhagic fever (Junín virus) is a human viral disease for which immune therapy proves effective, though a late neurologic syndrome is occasionally associated with the treatment. We attempted to determine in the infected marmoset Callithrix jacchus whether immune therapy leads to protection and/or CNS damage. Fifteen C jacchus were inoculated with 10(3) tissue culture infectious dose 50% (TCID50) of the XJ strain of Junín virus. On day 6 post infection (pi), 12 primates were treated with homologous immune serum. Animals were observed daily; and hematologic, serologic, virologic, and histologic studies were performed. All primates, both treated and controls, presented leukopenia, thrombocytopenia, anemia, and weight loss from day 14 pi onward. The three control animals died on days 22, 25, and 32 pi. Among the 12 treated monkeys, 3 died on days 21, 22, and 29. Hematologic values returned to normal during the second month; initial weight was recovered by the fourth month. Three out of the nine survivors showed neurologic alterations of various degrees, with hind-limb paralysis in the most severe case. Among treated monkeys, viremia and viral titers in the lungs, kidney, and lymph nodes were lower than in controls. Neutralizing antibodies were present in high titers in all treated marmosets, except in the one presenting paralysis in which values were minimal and viral persistence was detected in CNS. In conclusion, immune serum treatment of Junín virus-infected marmosets was found to reduce mortality from 100% to 25%. Viremia and viral titers in organs were lowered, and late neurologic signs appeared in 30% of treated survivors.     

Cross-protection in nonhuman primates against Argentine hemorrhagic fever.

The susceptibility of the marmoset Callithrix jacchus to Tacaribe virus infection was investigated to perform cross-protection studies between Junin and Tacaribe viruses. Five marmosets inoculated with Tacaribe virus failed to show any signs of disease, any alterations in erythrocyte, leukocyte, reticulocyte, and platelet counts or any changes in hematocrit or hemoglobin values. No Tacaribe virus could be recovered from blood at any time postinfection. Anti-Tacaribe neutralizing antibodies appeared 3 weeks postinfection. The five Tacaribe-infected marmosets and four noninfected controls were challenged with the pathogenic strain of Junin virus on day 60 post-Tacaribe infection. The former group showed no signs of disease, no viremia, and no challenge virus replication, whereas the control group exhibited the typical symptoms of Argentine hemorrhagic fever, high viremia, and viral titers in organs. Soon after challenge, the Tacaribe-protected marmosets synthesized neutralizing antibodies against Junin virus. These results indicate that the marmoset C. jacchus can be considered an experimental model for protection studies with arenaviruses and that the Tacaribe virus could be considered as a potential vaccine against Junin virus.     

Factor VIII activity as measured by an amidolytic assay compared with a one-stage clotting assay

Factor VIII amidolytic activity was measured by a commercially available method and compared with clotting activity measured in a one-stage assay. Parallel assays with both methods were used for plasma samples from 40 blood donors with normal or high Factor VIII levels, 22 patients with hemophilia before or after treatment with Factor VIII concentrates, 10 patients with chronic liver disease, and 10 normal subjects with high Factor VIII levels after treatment with desmopressin. The results obtained with the amidolytic assay were highly correlated (r = 0.97) with those obtained in the one-stage clotting assay. There were no significant differences in the results with the two methods in any of the patient groups, although in two cases of mild hemophilia the amidolytic assay gave lower values than the clotting assay. The reproducibility of the amidolytic assay (coefficient of variation = 6%) was better than that of the clotting assay (12%) at both normal and low levels of Factor VIII.     

Tacaribe virus infection of guinea-pig

Summary Guinea-pigs were inoculated with live Tacaribe virus by the intramuscular route. For a period of 48 days after inoculation infectious virus was isolated from lymph nodes, but not consistently. Virus is present in serum on the 7th day and in liver on the 13th day.There is no immunity against challenge with Junin virus in the first 3 days. Partial resistance appears after 7 days and complete immunity afterwards.The complement fixing antibodies against Tacaribe and Junin viruses are detected on the 13th day. The antibody titer is higher against the homologous antigen.The mechanism of the immunity against Junin virus infection after Tacaribe virus inoculation is discussed.This paper has been supported in part by a grant of NIH from U.S. No. E 4753 y por la Comisión de Lucha contra la Fiebre Hemorrágica Argentina del Ministerio de Asistencia Social y Salud Pública de la República Argentina.     

Development of a von Willebrand-like syndrome after prolonged use of hydroxyethyl starch

A 29-year-old female with no prior history of bleeding tendencies had gingival bleeding develop after 10 days of treatment with hydroxyethyl starch (HES), 1,000 mL/day. Laboratory studies revealed a marked reduction in Factor VIII coagulant activity (VIII:C), von Willebrand's factor antigen (vWF:Agn), and (VIII R:RCo), resembling severe type I von Willebrand's disease. The HES was stopped and the bleeding subsided. The levels of Factor VIII:C, vWF:Agn, and Factor VIII R:RCo gradually increased to normal over the following 17 days.     

Therapeutic effect of the antiviral agent ribavirin in Junín virus infection of primates

In order to assess the effect of the antiviral Ribavirin on the course of Junín virus infection in Callithrix jacchus, seven inoculated monkeys were treated with 15 mg of the drug, twice a day, starting 6 days after infection when all animals were viremic. The three untreated controls showed typical signs of Junín virus infection at 14 days pi and their mean time of death was 18 days. In contrast, no signs of illness were detected in Ribavirin-treated animals until 24 days pi, when marmosets showed signs of neurological involvement: 5 of these animals died (mean day of death: 36) but the two remaining treated monkeys improved and survived infection without sequelae. The comparison of survival rates (0% vs 28%) and the delay of the mean day of death observed indicates that the Ribavirin treatment used has therapeutic effect on Junín virus infection in vivo.     

Paradoxical reconstitution of complement activity following plasma transfusion of an individual with deficiency of the seventh component of complement.

A subject deficient in the seventh component of complement (C7) was plasmapheresed with 660 ml C7-sufficient plasma. The expected reconstitution of C7 activity, followed by exponential decay, was not observed. During day 1, serum haemolytic C7 and total haemolytic complement were undetectable and C7 levels were very low by C7 ELISA. However, low levels of circulating fluid phase terminal complement complex (TCC) were detected. On day 2 about microgram C7/ml serum was detected and this rose to 6 micrograms/ml by day 17. Functional complement activity was also present. At day 28 the serum C7 and total haemolytic complement had dropped to pretransfusion levels. A low level of C5b6 was present in pretransfusion serum and this increased markedly immediately following transfusion when the patient's serum also acquired C7 consuming activity. Throughout the study low levels of anti-C7 antibodies were present but there was no evidence that antibody was directly responsible for the C7 consumption. Nevertheless antibody-antigen interactions could have generated circulating C5b6. C5b6 has been shown previously to have the capacity to inhibit C7 activity in vitro. Investigations of the C7 circulating on days 2-17 demonstrated normal molecular weight, functionally active C7. The donor sera and the recirculating C7 allotyped C7-1 by isoelectric focusing; however, the recirculating C7 showed additional weak bands with C7 functional activity, suggesting a possible genetic or acquired abnormality. Although the disappearance of C7 immediately post-transfusion may be explained by the presence of C5b6, there is no satisfactory explanation for the rising C7 levels on days 2-17 and we cannot exclude temporary C7 secretion by the patient.     

Prostanoids during acute sarcocystiosis in growing pigs

The stable metabolites of thromboxane A₂, prostaglandin E₂, and prostaglandin F₂ (TxB₂, PgEM, and PgFM, respectively) were measured in the blood plasma of nine castrated male pigs, each inoculated with 2×10⁵ sporocysts ofSarcocystis miescheriana (group A), and in that of nine non-infected controls (group B). all infected pigs developed mild disease, the clinical signs being most severe between days 14 and 17 post infection (p.i.). In the infected pigs of group A, the TxB₂ plasma levels increased with the onset of the acute phase of illness (12 day p.i), reaching peak values at day 14 p.i. The mean TxB₂ values were significantly higher in the infected pigs from day 12 p.i. until the termination of the experiment on day 21 p.i. The PgEM values increased steadily in the infected pigs from day 12 p.i. until day 21 p.i. but remained relatively constant in the control pigs during the same period. In contrast, PgFM values remained low in the infected pigs throughout the experiment, and no significant differences between infected and non-infected pigs could be found. We conclude that the elevated TxB₂ and PgEM values reflect a major involvement of prostanoids in the pathogenesis of sarcocystiosis.Supported by the German Research Council (DFG)     

Argentine hemorrhagic fever. Alterations of the complement system and anti-Junin-virus humoral response.

We investigated immunologic mechanisms and the role of complement in the pathogenesis of Argentine hemorrhagic fever, a disease caused by the Junin virus, a member of the arenavirus group. Total serum complement activity was reduced to 68 per cent of control values in patients with severe or moderate disease (P less than 0.001). C2, C3 and C5 values were also low (12 to 60 per cent) during the early acute period of the disease. However, serum C4 content was increased to 160 per cent of the control values in the same patients. Total complement activity returned to normal with clinical and laboratory recovery, at the time of detection of antibodies against Junin virus. C1q reactive material was found in four of 19 cases and no relation to the evolution of the disease could be established. These results suggest that immune complexes are not important in the pathogenesis of Argentine hemorrhagic fever, but that activation of the complement system has a role.     

Imbalance Between Th17 Cells and Regulatory T Cells During Monophasic Experimental Autoimmune Uveitis

The aim of this study is to explore the dynamic changes in IL-17-expressing T cells (Th17)/Treg expression in monophasic experimental autoimmune uveitis (mEAU). mEAU was induced in Lewis rats with IRBP_1177–1191 peptide and evaluated clinically and pathologically on days 9, 13, 18, 23, 28, 35, and 48. Lymphocytes isolated from inguinal lymph nodes were subjected to flow cytometry to analyze the frequency of Th17/Treg cells. The levels of cytokines (IL-17, IL-6, IL-10, transforming growth factor (TGF)-β) in serum were detected by enzyme-linked immunosorbent assay (ELISA). Real-time quantitative PCR (RT-PCR) was used for measuring the levels of IL-17, IL-6, TGF-β, and Foxp3. Clinical and histopathologic assessment showed that mEAU began on day 9, peaked on day 13, and decreased to normal on day 18. The frequency of Th17 cells increased obviously on day 9, peaking on day 13, while the frequency of Treg cells increased on day 13, peaked on day 18, and remained at a high level until day 48. In the serum, the levels of IL-17 and IL-6 peaked on day 9 and gradually decreased to normal on day 28. The level of TGF-β increased on day 9, peaked on day 13, and decreased to normal on day 35. Meanwhile, the level of IL-10 increased on day 9 and stayed at a high level until day 48. Additionally, the above results were further confirmed by RT-PCR. The imbalance between Th17 and Treg cells contributes to the onset and progression of mEAU, and a compartmental imbalance of Treg over Th17 exists in the recovery phase of mEAU.     

Hepatitis C virus (HCV)-induced IgG-IgM rheumatoid factor (RF) complex may be the main causal factor for cold-dependent activation of complement in patients with rheumatic disease

A low serum complement level is commonly found in patients with rheumatic diseases. We evaluated 170 patients with such diseases to determine their serum levels of CH₅₀, C3 and C4 protein. Persistent hypocomplementaemia was found in 19 of those patients, particularly in those with systemic lupus erythematosus (SLE). Cold-dependent activation of complement (CDAC) was demonstrated in nine of the 19 (47.4%), and six of the nine patients demonstrated infection with HCV (66.7%). The nine patients that exhibited CDAC had nearly normal haemolytic complement activity when the sera were separated either at 37°C or in EDTA-treated plasma. Conversely, it markedly decreased, even to the point of being immeasurable, when the sera were separated at 4–21°C. No significant deficiency in C3 and C4 protein levels was found in these patients. Clinical parameters other than levels of anti-HCV antibody, transaminase, and RF were not influenced by CDAC. In an attempt to isolate the causal factor for CDAC, we isolated IgG fractions from the CDAC patients by using a protein G column, in which case precipitates were collected from the eluates. The precipitates were mixed with normal serum and incubated at 4–21°C for 18 h. A decrease in the level of CH₅₀ in normal serum was observed, which predominated (P<0.001) when precipitates from HCV-infected patients were used. This indicated CDAC was possibly interrelated to the precipitates of such patients. This precipitate was proved to contain IgM besides IgG. It is therefore possible that an HCV-related IgG complex or an IgG-IgM RF complex may be formed at low temperature and be involved in activating the complement system in vitro.     

Effect of oral contraceptives on factor VIII clotting activity and factor VIII related antigen in normal women.

The factor VIII clotting activity (VIIIc1 and factor VIII related antigen (VIIIRAg) were determined repeatedly in 24 pairs of age-matched normal women, one of each pair being on oral contraceptives. No significant differences in either parameter or in the VIIIc/VIIIRAg ration were found between the two groups ,although the mean factor VIII clotting activity and VIIIc/VIIIRAg ratios for women on oral contraceptives were very slightly higher than for those not on oral contraceptives.     

Cytolysis of Junin infected target cells by immune guinea pig spleen cells

Spleen cells from guinea pigs infected with an attenuated strain of Junin virus (the causative agent of Argentine hemorrhagic fever) specifically lysed virus-infected syngeneic target cells in vitro. This activity was detected as early as 6 days after infection, reached a maximum on days 10-13, and persisted at lower levels, at least through day 30. Monoclonal antibody to guinea pig T cells had no effect on the activity. After B or T cell enrichment techniques, the cytolysis was found with the B cell fraction. Aggregated IgG blocked the cytolysis. These characteristics suggested lytic activity was mediated at least in part by an antibody-dependent cell-mediated cytotoxicity (ADCC) mechanism. Although some cytolysis could be detected by using exogenously added antiserum and normal spleen effector cells, such reconstruction showed less efficient killing than when spleen cells from Junin-infected guinea pigs were used. This apparent discrepancy was resolved when spleen cells from these infected animals exhibited enhanced activity in non-viral ADCC systems as well. The cytotoxic activity by spleen cells against Junin-infected targets was detected only with non-fatal Junin infections. Cytolysis could not be measured in spleen cell suspensions from guinea pigs lethally infected with Junin virus; i.e. adults infected with a virulent strain of Junin and baby guinea pigs or immunosuppressed adult animals infected with an attenuated strain. However, spleen cells from both the immunosuppressed, infected adults and the adult guinea pigs infected with a virulent strain of Junin were able to mediate cytotoxicity in a nonviral system (antibody-sensitized Vero cells). The development of spleen cell cytotoxicity by Junin-infected guinea pigs against Junin-infected target cells correlated with whether the infection was resolved or was lethal.     

Complement activation patterns in atypical haemolytic uraemic syndrome during acute phase and in remission

Atypical haemolytic uraemic syndrome (aHUS) is associated with (genetic) alterations in alternative complement pathway. Nevertheless, comprehensive evidence that the complement system in aHUS patients is more prone to activation is still lacking. Therefore, we performed a thorough analysis of complement activation in acute phase and in remission of this disease. Complement activation patterns of the aHUS patients in acute phase and in remission were compared to those of healthy controls. Background levels of complement activation products C3b/c, C3bBbP and terminal complement complex (TCC) were measured using enzyme‐linked immunosorbent assay (ELISA) in ethylenediamine tetraacetic acid (EDTA) plasma. In vitro‐triggered complement activation in serum samples was studied using zymosan‐coating and pathway‐specific assay. Furthermore, efficiencies of the C3b/c, C3bBbP and TCC generation in fluid phase during spontaneous activation were analysed. Patients with acute aHUS showed elevated levels of C3b/c (P < 0·01), C3bBbP (P < 0·0001) and TCC (P < 0·0001) in EDTA plasma, while values of patients in remission were normal, compared to those of healthy controls. Using data from a single aHUS patient with complement factor B mutation we illustrated normalization of complement activation during aHUS recovery. Serum samples from patients in remission showed normal in vitro patterns of complement activation and demonstrated normal kinetics of complement activation in the fluid phase. Our data indicate that while aHUS patients have clearly activated complement in acute phase of the disease, this is not the case in remission of aHUS. This knowledge provides important insight into complement regulation in aHUS and may have an impact on monitoring of these patients, particularly when using complement inhibition therapy.     

Involvement of T helper type 17 and regulatory T cell activity in Citrobacter rodentium invasion and inflammatory damage

Citrobacter rodentium is a murine pathogen that transiently colonizes the lumen of the large intestine. C. rodentium induces colitis, but the relative importance and temporal induction of the T helper type 17 (Th17) and regulatory T cell (T_reg) pathways in protection from the infection and inflammation have not been assessed. Our aim was to investigate the key immunological signalling events associated with successful clearance of C. rodentium. Mice were challenged with luminescent-tagged C. rodentium and killed at days 3 (early infection), 10 (peak infection) and 21 (late infection) post-infection. Bioluminescent imaging and bacterial culture determined levels of C. rodentium. Distal colon mRNA expression of interleukin (IL)-17, IL-6, IL-1β, tumour necrosis factor (TNF)-α, forkhead box P3 (FoxP3) and ghrelin were assessed using real-time polymerase chain reaction. Results were compared with age-matched non-infected mice. Low levels of C. rodentium were found at day 3, high levels at day 10, with clearance from the majority of the mice by day 21. In the distal colon, there was up-regulation of TNF-α and FoxP3 throughout the study and increases in IL-6 and IL-17 during the peak and late stages of infection. Ghrelin expression was increased at the peak and late stages of infection. This study has characterized changes to the T helper cell pathways, following the course of C. rodentium infection in mice. There were significant immunological changes, with up-regulation of the Th17 and T_reg pathways in the distal colon and an increase in ghrelin expression compared with non-infected control mice. These changes may play a role in the pathology and clearance of C. rodentium.     

Murine visceral leishmaniasis: IgM and polyclonal B‐cell activation lead to disease exacerbation

In visceral leishmaniasis, the draining LN (DLN) is the initial site for colonization and establishment of infection after intradermal transmission by the sand fly vector; however, little is known about the developing immune response within this site. Using an intradermal infection model, which allows for parasite visceralization, we have examined the ongoing immune responses in the DLN of BALB/c mice infected with Leishmania infantum. Although not unexpected, at early times post‐infection there is a marked B‐cell expansion in the DLN, which persists throughout infection. However, the characteristics of this response were of interest; as early as day 7 post‐infection, polyclonal antibodies (TNP, OVA, chromatin) were observed and the levels appeared comparable to the specific anti‐leishmania response. Although B‐cell‐deficient J_hD BALB/c mice are relatively resistant to infection, neither B‐cell‐derived IL‐10 nor B‐cell antigen presentation appear to be primarily responsible for the elevated parasitemia. However, passive transfer and reconstitution of J_hD BALB/c with secretory immunoglobulins, (IgM or IgG; specific or non‐specific immune complexes) results in increased susceptibility to L. infantum infection. Further, J_hD BALB/c mice transgenetically reconstituted to secrete IgM demonstrated exacerbated disease in comparison to WT BALB/c mice as early as 2 days post‐infection. Evidence suggests that complement activation (generation of C5a) and signaling via the C5a receptor (CD88) is related to the disease exacerbation caused by IgM rather than cytokine levels (IL‐10 or IFN‐γ). Overall these studies indicate that polyclonal B‐cell activation, which is known to be associated with human visceral leishmaniasis, is an early and intrinsic characteristic of disease and may represent a target for therapeutic intervention.     

Suppression of mouse complement activity by contaminants of technical grade pentachlorophenol

Pentachlorophenol (PCP) is an antimicrobial agent used chiefly for the preservation of wood. Subchronic oral exposure (14 days) to Technical Grade PCP significantly inhibited the functional activity of female B₆C₃F₁ mouse complement when measured in a microtiter hemolytic assay. When evaluated one day following the final exposure the highest administered dose (100 mg/kg) significantly suppressed the Classical complement pathway, the Spontaneous C1 autoactivation pathway, the Alternate pathway and the level of complement component, C3. Reconstitution studies using C5-deficient serum also demonstrated deleterious effects on this complment component. The Classical pathway was the most sensitive to Technical Grade PCP effects. Animals treated with 100 mg/kg Technical Grade PCP had CH 50 levels 30% of vehicle controls. Animals treated for 14 days and allowed a 15 day recovery period had CH 50 values 36% of control and animals which recovered for 30 days had only 52% of the complement activity of control animals. C3 recovery studies also demonstrated continued suppression on days 15 and 30 post-final exposure. Doses of 10 and 30 mg/kg did not produce the marked effects observed with the highest dose; however, a dose-dependent trend was observed for all responses. Animals treated with 100 mg/kg of EC-7, a PCP preparation with reduced amounts of contaminating dioxins and dibenzofurans, did not demonstrate detrimental effects on the complement system.To whom correspondence should be addressed