Induction of tolerance to factor VIII in a child with a high-titer inhibitor: in vitro and in vivo observations

High dose factor VIII concentrate was infused over 12 months in a 3-year-old child with hemophilia A and a high-titer inhibitor. This regimen was successful in producing clinical tolerance to the factor VIII and was associated with a fall of inhibitor titer from 85 BU to less than 1 BU. Subsequently, a rise in inhibitor to peak at 3.5 BU correlated with a decrease in infusion dose. In vitro studies using a microdroplet assay for immunoglobulin synthesis in the presence of varying concentrations of factor VIII was carried out prior to the clinical trial. This system indicated that the patient's peripheral blood mononuclear cells were stimulated to release IgG by 0.01 U/ml factor VIII coagulant activity and inhibited at 0.9 U/ml FVIIIc. Lymphocyte subsets were monitored during the course of therapy. The ratio of Leu-3a+/Leu-2a+ (helper/suppressor) varied considerably, fluctuating between a peak of 4.4 and low point of 0.6, with the initial pretrial ratio being 1.2.     

Acquired von Willebrand's syndrome with lupus-like serology

We describe a 12-year-old boy with acquired von Willebrand's syndrome, who also had various autoantibodies. He presented with recent hemorrhagic symptoms and a prolonged bleeding time. Hemostatic studies revealed severely reduced levels of factor VIII procoagulant activity (VIII:C), von Willebrand's factor (vWF) antigen (vWF:Ag), and ristocetin cofactor activity (RCoF). An inhibitor that could be detected in the patient's plasma moderately decreased the levels of vWF:Ag in normal plasma, but did not interfere with the measurement of VIII:C or RCoF. Following the infusion of cryoprecipitate, half-lives of VIII:C, vWF:Ag, and RCoF were markedly reduced. 1-Deamino-8-D-arginine vasopressin infusion induced normalization of the prolonged bleeding time and caused a marked increase in VIII:C, vWF:Ag, and RCoF. Prior to treatment, there was a uniform reduction of all the multimers of plasma vWF in sodium dodecyl sulfate agarose gel electrophoresis. Following prednisone therapy, clinical and hemostatic findings were improved, and the multimeric patterns of vWF were normalized. These findings suggest that the low levels of all three parameters of factor VIII and all the multimers of plasma vWF in the patient are caused by rapid elimination of factor VIII complex from the circulation.     

Persistently circulating C3 nephritic factor (C3 NeF)-stabilized alternative pathway C3 convertase (C3 CoF) in serum of an 11-year-old girl with meningococcal septicemia--simultaneous occurrence with free C3 NeF

Hemolytic complement was found to be absent in the serum of an 11-yr-old girl (R.N.) with meningococcal septicemia. C1, C4, and C2 were slightly decreased, C3 was absent, C5-C9 within the normal range. B levels immunochemically and electrophoretic mobility of B were normal. C3d was greater than 1000% of a pooled EDTA-plasma standard indicating hypercatabolism of C3. On incubation of the patient's serum with normal human serum activation of C3 occurred even in the presence of 0.04 M EDTA. The amount of C3b generated was, however, greater without any chelating agent or in Mg-EGTA. On gel filtration of the serum two protein containing peaks were found to be responsible for activation of C3: the IgG containing peak was able to activate C3 in normal human serum without chelating agents and in Mg-EGTA but not in the presence of EDTA. The IgM-containing peak activated the third component of complement even in the presence of EDTA. The factor responsible for this phenomenon was termed C3 converting factor (C3 CoF). The IgG fraction of the patients serum caused activation of C3 in Mg-EGTA. However, in the presence of EDTA no activation of C3 could be induced even if physiological concentrations of the patients IgG were added to normal human EDTA-plasma. Thus the activity of the patient's IgG did not differ from typical C3 nephritic factor. The decay of C2 in EAC42 intermediates in the presence of the patient's IgG was uninfluenced indicating that it did not carry autoantibody activity against the classical pathway convertase C4b,2a, an activity recently termed NFc.(ABSTRACT TRUNCATED AT 250 WORDS)     

Coagulation factors in the premature infant born after about 32 weeks of gestation

In plasma samples from 10 premature infants born after about 32 weeks of gestation, a number of coagulation factors have been determined. For 9 infants, who were healthy, mean values are given: fibrinogen-antigen, 311 mg/dl; factor II, +/- 0.46 U/ml; factor V, 0.80 U/ml; factor VII, 0.59 U/ml; factor VIII coagulant activity, 0.93 U/ml; factor VIII-related antigen, 1.66 U/ml; procoagulant factor VIII antigen, 1.15 U/ml; factor IX coagulant activity, 0.41 U/ml; factor IX antigen, 0.42 U/ml; factor X coagulant activity, 0.52 U/ml; factor X antigen, 0.61 U/ml, and antithrombin III-antigen (AT-III), 0.43 U/ml. In 5 infants a second sample was taken a week after the first one; for most values there was no significant rise, except for factor II and AT-III. The values we found in this group of premature infants are within the range of those reported in earlier literature. They are higher than the ones we found in early fetal samples and most of them are similar to those we found in early fetal samples and most of them are similar to those we found in the cord blood of full-term newborns.     

Factor VIII inhibitors in patients suffering from severe haemophilia A: problems of "very low" responders]

Without recognition of any inhibitor until now, low concentrations of factor VIII inhibitors (< 1 Bethesda unit (BU)/mL plasma) can be occasionally measured in patients with severe haemophilia A. The existence of so-called "very low" responders is assessed contradictorily due to a methodically caused inhibitor increase. Plasma from 10 patients with severe haemophilia A was incubated with human plasma or animal plasma from pig, cattle, or cat and assayed for factor VIII inhibitors. No signs of inactivation could be detected in five specimen (0 BU/mL plasma). However, measurable signs of factor VIII inactivation (< 1 BU/mL plasma) did occur in the other five. Therefore, the existence of yet not defined unknown inhibitory substances in certain haemophilic plasmas must be assumed. They are directed against human factor VIII as well as partly against animal factor VIII. These "very low" inhibitors are not identical with factor VIII antibodies of "low" and "high" responding haemophiliacs. The clinical importance of "very low" inhibitors is insignificant because they do not tend to increase after exposure to factor VIII. In fact, a effect of factor VIII therapy is the neutralization of this kind of inhibitors.     

Analysis of von Willebrand factor in platelets of patients with various forms of von Willebrand disease: Is there a clinical relevance?

Besides the investigation of coagulation factor VIII:c and von Willebrand factor in plasma, vWF antigen and vWF collagen-binding activity in platelets of 24 patients with various forms of von Willebrand disease were analysed. No platelet vWF:Ag or vWF:CBA was detectable in type 3 patients (n = 4). In contrast 6 out of 7 patients with type 2 vWD had normal or increased vWF levels. Two type 1 patients (out of n = 13) with low von Willebrand factor in platelets had no increased bleeding tendency. In two other individuals with normal amounts of von Willebrand factor in platelets and low plasmatic vWF and factor VIII:c, more frequent bleeding episodes reflecting the low plasmatic levels were observed in a long-term follow-up. Conclusion In our patients, bleeding history corresponded to plasmatic levels of FVIII:c and vWF.     

Plasma somatomedin activity in an infant with Beckwith—Wiedemann syndrome

Plasma growth hormone levels and somatomedin activity were determined in a child with Beckwith-Wiedemann syndrome at birth and at 8 mth of age. Birthweight and length were above the 97th centile. Somatomedin activity in the cord plasma was elevated (2.8 U/ml) compared with controls (0.15-1.3 U/ml; n=15). Growth hormone was also high (76 ng/ml compared with control group range of 5.5–42.1 ng/ml, n=26). At 8 mth of age both somatomedin activity and plasma growth hormone had fallen to normal levels and weight and length were on the 75th centile. It is suggested that the high somatomedin activity may have been a contributing factor in the excessive fetal growth of this child.     

A study of the plasma kinin-generating system in children with the minimal lesion, idiopathic nephrotic syndrome.

Although the precise etiologic incitant of the minimal lesion idiopathic nephrotic syndrome of childhood is not known, it is likely that a host mechanism mediates the permeability alterations of the glomerular capillary wall resulting in massive proteinuria. As a first step in examining the possibility that local kinin release may account for the proteinuria in this disorder, two parameters of the plasma kinin-generating system, plasma prekallikrein and kallikrein inhibitor, were assayed during 27 nephrotic episodes in 21 corticosteroid-responsive children. Plasma kallikrein was assayed by means of its esterase activity on a synthetic arginine ester substrate, N-alpha-tosyl-L-arginine methyl ester (TAMe), after activation of Hageman factor by kaolin. This activity, after subtraction of spontaneous arginine esterase activity (i.e., TAMe esterase activity measured in plasma not exposed to kaolin) is derived from prekallikrein. Plasma prekallikrein activity in 11 normal children was 99.6 +/- 2.9 mumol TAMe hydrolyzed/ml plasma/hr (mean +/- SEM). Kallikrein inhibitor was quantified in arbitrary units. Kallifrein inhibitor activity in 11 normal children was 0.94 +/- 0.04 units. During the overt nephrotic syndrome, before initiation of intensive daily corticosteroid treatment, mean values were: prekallikrein, 58.5 +/- 7.24 mumol/ml/hr; and kallikrein inhibitor, 0.35 +/- 0.06 units. After corticosteroid-induced remission occurred, mean values were: plasma prekallikrein, 118.6 +/- 3.2 mumol/ml/hr; and kallikrein inhitor, 0.78 +/- 0.03 mumol/ml/hr. Both parameters were again assayed in 14 of the 21 children after complete cessation of corticosteroid treatment. Plasma prekallikrein was normal, 99.6 +/- 4.8 mumol/ml/hr; but kallikrein inhibitor was still somewhat depressed, 0.84 +/- 0.03 units. A subset of 9 patients had marked depression of plasma prekallikrein to levels less than 20 mumol/ml/hr and essentially undetectable inhibitor activity. Serum alpha-2 macroglobulin was elevated in nephrotic patients: mean value during relapse, 862 +/- 29 mg/100 ml; during corticosteroid-maintaining remission, 615 +/- 29 mg/100 ml. After cessation of corticosteroids, mean serum level was 481 +/- 20 mg/100 ml. The proportional reduction of plasma prekallikrein and kallikrein inhibitor suggested that an enzyme-inhibitor complex formed in vivo, perhaps at a local site of activation in proximity to the glomerular basement membrane. These data suggest that the plasma kinin-generating system may be the host effector mechanism subserving the increased glomerular capillary permeability in the minimal lesion nephrotic syndrome of childhood.     

IMMUNORADIOMETRIC ASSAY OF INHIBITORS OF ANTIHAEMOPHILIC FACTOR A

ABSTRACT. An immunoradiometric assay (IRMA) for determination of antibodies against f. VIII:C in haemophilia A was developed. The assay was based on competitive binding of radiolabelled anti-VIII:C and antibodies in the test material to immobilized VIII:C. Fifteen haemophiliacs with known inhibitors were investigated with the new method and with a conventional neutralization test. In 3cases the inhibitors were detected only with the IRMA and in the other 12 there was good agreement between the inhibitor levels found with the two methods. It was also possible to demonstrate the antibodies in three non-haemophilic patients with acquired inhibitors. The IRMA, which can detect the antibodies down to a concentration of 0.02 inhibitor units per ml, is more sensitive than conventional neutralization tests and is thus of practical importance in the investigation of patients with low inhibitor titres.     

Activity of blood coagulation Factor XIII as a prognostic indicator in patients with Henoch-Schönlein purpura

Determination of coagulation Factor XIII (F XIII)-related parameters in 21 patients with Henoch-Schönlein purpura documented a significant decrease of F XIII activity as well as of the F XIII-related antigenic determinants. Subgroup analysis with regard to the clinical symptoms showed an even further decrease of these parameters in patients with gastrointestinal complications. Stimulated by these findings a substitution therapy with a F XIII concentrate was initiated in those patients whose F XIII activity in plasma remained low and who developed severe abdominal pain accompanied by persisting gastrointestinal bleeding. This therapeutic approach not only corrected the laboratory data, but more important led to a cessation of pain and bleeding. A rapid decrease of F XIII levels after transfusion below 40 U/ml was indicative of relapse of abdominal symptoms, while increasing values were associated with the recovery of the patients. In conclusion: F XIII activity determinations appear to have a predictive value in patients with Henoch-Schönlein purpura, and the administration of F XIII concentrates may contribute to the improvement of gastrointestinal complications.Abbreviations F XIII Factor XIII - SHP Henoch-Schönlein purpura - XIII-act Factor XIII activity - MCA monochloroacetic acid - XIII-A Factor XIII subunit A antigen - XIII-S Factor XIII subunit S antigen - F VIII R:AG Factor VIII related antigen - FDP Fibrin degradation products - F VIII:C Factor VIII coagulant activity     

Suppression of factor VIII inhibitor in children with hemophilia A

In 1977 Brackmann for the first time reported an induction of tolerance in hemophiliacs with inhibitors using a protocol requiring administration of very high daily doses of factor VIII and activated prothrombin complex concentrates. Since the costs of such a protocol are enormous, we report our experiences in 3 hemophiliac children with inhibitors against factor VIII treated with much lower doses of factor VIII concentrate only for induction of tolerance. Two "high responder" and one "low responder" initially for only a short period received factor VIII concentrate in doses comparable to the Brackmann protocol. After that period patients were treated with 25--30 units factor VIII concentrate/kg/day, doses that are comparable to conventional prophylactic treatment. In one high responder the inhibitor disappeared completely after 22 months, in the other high responder the inhibitor titer decreased from 133 BU to 4.4 BU. In the low responder also a significant decrease of the inhibitor titer was observed. At this time bleedings in all three patients can be controlled by conventional doses of factor VIII concentrate.     

Gingival bleeding, epistaxis and haematoma three days after gastroenteritis: the haemorrhagic lupus anticoagulant syndrome

A 3 year and 9 month-old girl presented with gingival bleeding, epistaxis, and multiple haematomas 3 days after an acute episode of gastroenteritis. Prothrombin time and activated partial thromboplastin time were prolonged with reduced clotting activity of factor II (<10%), VIII (<1%), IX (3%), XII (10%) and evidence of a high titre inhibitor. Prothrombin (factor II) level was below the detection limit, both in a functional and immunological assay. It did not increase after administration of vitamin K or fresh frozen plasma. Further studies revealed presence of a strong lupus anticoagulant and a specific IgG antibody against prothrombin. Factor VIII antigen levels also were reduced (31%), but to a lesser extent than functionally determined factor VIII (<1%). Blood coagulation normalised following clinical recovery 6 weeks after admission. The pathophysiology of this acquired inhibitor phenomenon (accelerated clearance of complexes of clotting factors and phospholipids) is discussed. Conclusion The haemorrhagic lupus anticoagulant syndrome (acquired hypoprothrombinaemia lupus anticoagulant syndrome) is a rare presentation of acquired bleeding diathesis in childhood. Since most cases in post-infectious children are asymptomatic, it might be underdiagnosed. In children with newly appearing bleeding symptoms or unclear prolonged prothrombin time or activated partial thromboplastin time, one has to consider this syndrome which could lead to relevant bleeding. Received: 23 March 2000 / Accepted: 8 August 2000     

Umbilical artery catheterization in newborns. III. Thrombosis--a study of some predisposing factors

Thrombosis following umbilical artery catheterization is a relatively frequent complication. Low fibrinolytic activity in the vessel walls, high factor VIII and low antithrombin III (AT III, Heparin cofactor activity) in blood are factors known to favour the formation of thrombosis. In 30 newborns who died and in 2 foetuses the fibrinolytic activity determined in the aorta and the femoral vessels was in the normal 'adult' range except for a few very immature infants and the foetuses. The five cases with arterial thrombosis were not associated with low fibrinolytic activity. The various factor VIII activities (VIII:C, VIIIR:Ag, and VIIIR:RCF) ant AT III were studied in 30 sick newborns and in 20 healthy newborns. The sick exhibited increased levels of various factor VIII activities (VIIIR:Ag and VIIIR:RCF mainly) and markedly reduced levels of AT III. The high factor VIII activities and the low AT III found will add to the existing risk of thrombosis due to the presence of a foreign material. AT III substitution is suggested as a possible prophylactic.     

Diphtheria, tetanus and pertussis antibodies in 10-year-old children before and after a booster dose of three toxoids: implications for the timing of a booster dose

In an open study, 502 10-year-old children, who had received primary vaccination against diphtheria and tetanus in infancy and had varying histories of pertussis disease and vaccination, were vaccinated with diphtheria-tetanus vaccine (DT) alone or with the addition of 20 µg or 40 g of pertussis toxoid. Diphtheria toxin neutralising antibodies, pertussis toxin IgG and tetanus toxoid IgG antibodies were measured before and 1 month after the booster. All toxoids were highly immunogenic. In pertussis toxoid recipients, median levels of pertussis toxin IgG increased to 16.5 U/ml (DTaP20) and to 36 U/ml (DTaP40) in children with non-detectable (<1 U/ml) antibodies before vaccination and to >400 U/ml in children (both DTaP20 and DTaP40) with detectable antibodies before vaccination. A total of 60 children (12%) with non-detectable (<0.01 IU/ml) diphtheria antibodies and 36 children (7%) with non-detectable (<0.01 IU/ml) tetanus antibodies before the booster had lower median antibody concentrations post-vaccination than children with detectable antibodies before the booster (diphtheria: 5.12 vs. 20.48 IU/ml; tetanus: 4.0 vs. 10.0 IU/ml). There were no differences in diphtheria and tetanus antibodies after vaccination between children who did and did not receive pertussis toxoid. Conclusion:10-year-old children with non-detectable diphtheria and tetanus antibodies before the booster had lower post-vaccination antibodies than those with detectable antibodies before the booster indicating a poor immunological memory. Addition of pertussis toxoid to diphtheria-tetanus vaccine did not affect the antibody responses to diphtheria and tetanus toxoids when the three toxoids were combined as a booster. Even though immunity to diphtheria and tetanus was only estimated by surrogate markers (serum antitoxin antibodies) the results indicate that a lower age for the booster dose of diphtheria-tetanus vaccine or diphtheria-tetanus acellular pertussis vaccine should be considered.     

The clinical relevance of factor VIII: C and factor VII R: Ag determination in newborns

Higher levels of factor VIII: C and factor VIII R: Ag were found in healthy newborns (n=60) as compared to adults. This could be explained as a stress reaction due to birth and the adaptation to extrauterine life. A further stress factor is disease. The highest values for factor VIII R: Ag were found in ill (n=32) and in severely ill newborns (n=21). The large ranges of factor VIII: C and of the ratio of factor VIII: C/VIII R: Ag in healthy newborns can be explained by an increased turnover of coagulation factors. Diseases in the newborn period lead to an increase of this process, resulting in even larger ranges of factor VIII: C and of the ratio of factor VIII: C/VIII R: Ag in ill and extremely ill newborns. Consumption of factor VIII: C with a low ratio of factor VIII: C/VIII R: Ag predominates in extremely ill newborns. The ratio of factor VIII: C/VIII R: Ag is more valuable than factor VIII: C for diagnosis of DIC in newborns.A diagnosis of hemophilia and von Willebrand's disease cannot be established with certainty in severely ill newborns. Stress and DIC may influence the characteristic changes of laboratory parameters.Supported by Wissenschaftsministerium des Landes Nordrhein-Westfalen     

Factor VIII levels in children with thrombosis

BACKGROUND: A number of coagulation defects have been implicated as risk factors in thrombo-embolic disease. Of these, high levels of clotting factor VIII have been shown to be associated with a five- to six-fold increased risk of thrombosis, compared to levels < 100 IU/dL in adults. The objective of this study was to investigate the prevalence of elevated plasma levels of factor VIII in a pediatric population with thrombo-embolism (TE). METHODS: Forty-two children (17 female, 25 male) with TE and 165 healthy controls without familial history of thrombosis or stroke were included in the present study. Doppler ultrasonography with or without angiography, computed tomography, magnetic resonance imaging or echocardiography was utilized to establish the diagnosis. One-stage clotting assay with factor VIII-deficient plasma for measurement of factor VIII and immunoturbidometric assay for von Willebrand factor (vWF) levels were utilized. All measurements were performed in duplicate. Plasma levels of factor VIII were assessed in parents of nine patients to establish whether high levels of factor VIII were genetically determined. RESULTS: The median age at onset of TE was 7 years (range 0-17 years). Among patients with TE compared to controls, the prevalence of high factor VIII levels was 59.5% versus 12.1% (odds ratio 10.6, 95% CI: 4.9-23.1). The prevalence of high factor VIII levels was detected in at least one of nine families. CONCLUSION: The data in the present study provide evidence that elevated plasma factor VIII levels are associated with increased risk of thrombosis in children: thus, plasma concentration of factor VIII should be measured in all children with TE.     

Lymphocyte subset ratios and factor VIII usage in haemophilia.

Type and quantity of replacement treatment, together with haematological and immunological parameters were determined in 37 boys with severe haemophilia A and 41 children with other bleeding disorders. The quantity of factor VIII concentrate given to boys with severe haemophilia A (mean U/year) showed a significant inverse correlation with total white cell counts, lymphocyte counts, platelet counts, and the ratio of monoclonal antibody defined T lymphocyte subsets, T4 and T8 (T4:T8). Of the boys with severe haemophilia A, 49% had inversed T4:T8 ratios and 24% had thrombocytopenia. Treatment with high dose factor VIII concentrate (more than 25 000 U/year) was associated with low platelet counts, low lymphocyte counts, low T4:T8 ratios, and hypergammaglobulinaemia. In addition, six patients with severe haemophilia A and factor VIII inhibitors had inversed T4:T8 ratios. Patients treated exclusively with cryoprecipitate or prothrombin complex concentrates had normal T4:T8 ratios and platelet counts. The severity of the haematological and immunological abnormalities observed seems to be associated with high usage of factor VIII concentrates. Similar abnormalities have been described in patients with the acquired immune deficiency syndrome (AIDS). Prospective study of haemophiliacs is required to assess long term sequelae of factor concentrate usage, including the possible development of AIDS.     

Haemophilia A in two premature infants

The incidence of prematurity in Germany is about 10% and premature infants with haemophilia A (OMIM 306700) are in fact very rare. We report two new cases, one born in the 28th gestational week, weighing 1200 g with a factor VIII (FVIII) level of 0.03 IU/ml treated with bolus injections of plasma derived FVIII concentrate (pdFVIII), and one born at week 30, weighing only 710 g with a factor level of <0.01 IU/ml and treated with recombinant FVIII concentrate (rFVIII). Recovery of FVIII was 96% in case 1 and 120% in case 2, FVIII half-life was 6 h and 8 h, respectively. During FVIII substitution, neither bleeding, thrombosis nor inhibitor development were noted in both infants. Conclusion:Immediate and frequent factor VIII substitution appears to be safe and effective for prophylaxis and treatment in premature haemophilic neonates.     

Risk of bleeding and inhibitor development after circumcision of previously untreated or minimally treated severe hemophilia a children

Background: Surgery and intensive factor VIII (FVIII) replacement may be risk factors for development of inhibitors. Objective: To evaluate time and rate of inhibitor development postcircumcision over 12-month period, and to assess bleeding of children with severe hemophilia A after low-dose FVIII replacement and local hemostasis. Patients and Methods: Sixty-one previously untreated patients (PUPs) or minimally treated patients (MTPs) with severe hemophilia A less than 36 months were enrolled; 25 underwent circumcision during the 18-month enrollment period, and 36 matched patients were not circumcised. All patients were treated on demand with plasma-derived FVIII, and all were inhibitor negative at the time of enrollment. Intron 22 inversion was analyzed. A potent hemostatic agent (gelatin sponge) was applied on the site of surgery, and then dressed with gauze. Two doses of FVIII concentrate (25?U/kg) were given, 1?hour before circumcision and 1?hour before removal of dressing. The inhibitor was determined every 8 exposure days (EDs). Results: None of the patients had bleeding or infection, except one who had minimal transient bleeding 8?days after surgery, and was treated easily by a single dose of FVIII (50?U/kg). After a median of 16 EDs, high-titer inhibitors developed in seven patients: three patients in the circumcised group (12%) in contrast to four patients (11.1%) in the noncircumcised group. Conclusion: Two doses factor concentrate and gelatin sponge application were generally enough to prevent bleeding after circumcision of severe hemophilia A. Circumcision and low-dose FVIII protocol were not an additional risk for development of high-titer inhibitor.     

Milk-borne insulin with trypsin inhibitor in milk induces pancreatic amylase development at the onset of weaning in rats

BACKGROUND: The physiologic significance of milk-borne hormones and growth factors for internal organs of suckling animals is poorly understood. In this study the significance of milk-borne insulin was evaluated, as well as its combination with trypsin inhibitor, and its role in the development of pancreatic digestive capacity at the time of weaning was investigated. METHODS: Experiments were performed using insulin-deficient milk formula (standard formula), insulin (20 ng/ml) formula, or insulin with trypsin inhibitor (1 U/ml) formula by a rat artificial-rearing technique. RESULTS: In 17-day-old rats administered standard formula, the plasma insulin level was as low as that in 14-day-old rats. When insulin-trypsin inhibitor formula was administered to rat pups, the plasma insulin level was significantly higher than those in rats given standard or insulin formula. In rats artificially reared on standard formula, the usual developmental increases in pancreatic amylase activity and plasma insulin concentration at the beginning of weaning did not occur. Insulin formula elevated the pups' plasma insulin concentration and amylase activity at the onset of weaning but not to the levels observed in mother-reared rats. In rats reared on insulin-trypsin inhibitor formula, the developmental increases in the plasma insulin concentration and amylase activity observed in mother-reared rats were induced. CONCLUSIONS: The present study demonstrates the necessity of milk-borne insulin for the development of pancreatic amylase during the weaning period.