Cathepsin B Is Not an Intrinsic Factor Related to Asparaginase Resistance of the Acute Lymphoblastic Leukemia REH Cell Line.

L-Asparaginase (ASNase) is a biopharmaceutical used as an essential drug in the treatment of acute lymphoblastic leukemia (ALL). Yet, some cases of ALL are naturally resistant to ASNase treatment, which results in poor prognosis. The REH ALL cell line, used as a model for studying the most common subtype of ALL, is considered resistant to treatment with ASNase. Cathepsin B (CTSB) is one of the proteases involved in the regulation of in vivo ASNase serum half-life and it has also been associated with the progression and resistance to treatment of several solid tumors. Previous works have shown that, in vitro, ASNase is degraded when incubated with REH cell lysate, which is prevented by a specific CTSB inhibitor, suggesting a function of this protease in the ASNase resistance of REH cells. In this work, we utilized a combination of CRISPR/Cas9 gene targeting and enzymatic measurements to investigate the relevance of CTSB on ASNase treatment resistance in the ALL model cell line. We found that deletion of CTSB in REH ALL cells did not confer ASNase treatment sensitivity, thus suggesting that intrinsic expression of CTSB is not a mechanism that drives the resistant nature of these ALL cells to enzymes used as the first-line treatment against leukemia.

The effect of recombinant human intrinsic factor on the uptake of vitamin B12 in patients with evident vitamin B12 deficiency.

We report on the use of recombinant human intrinsic factor (rhIF) in a new vitamin B12 absorption test. Holotranscobalamin (holoTC) was measured before and 24 hours after intake of three 9-mg doses of vitamin B12 (B12) and again 24 hours after intake of the same dose of B12 together with rhIF (rhIF-B12). Nine patients with evident vitamin B12 deficiency had a significantly higher increase in holoTC after intake of rhIF-B12 than after intake of B12. Twenty-eight patients with suspected vitamin B12 deficiency showed no additional increase in holoTC after intake of rhIF-B12. We conclude that rhIF promotes B12 absorption among patients with evident vitamin B12 deficiency.

Influence of duodenal intrinsic factor on the absorption of labelled vitamin B12.

The intrinsic factor activity of powdered hog duodenal mucosa was confirmed by measurements of the absorption of labelled vitamin B 12 (Schilling-test) in 7 cases of Addisonian pernicious anaemia. In view of the fact that, according to earlier studies of the authors based on a bacteriological test, human duodenal juice also exhibits an intrinsic factor activity, it is suggested that the duodenal intrinsic factor may be involved in the pathogenesis of pernicious anaemia. The results of the Schilling-test are critically reviewed in the light of recent published evidence.

Evidence that pancreatic proteases enhance vitamin B12 absorption by acting on curde preparations of hog gastric intrinsic factor and human gastric juice.

Crude preparations of hog gastric intrinsic factor or their own previously collected gastric juices administered with labeled vitamin B12 did not enhance vitamin B12 absorption in patients with vitamin B12 malabsorption secondary to pancreatic insufficiency. However, when these sources of gastric intrinsic factor were incubated with three times crystallized preparations of insolubilized bovine trypsin or chymotrypsin, the proteolytic enzymes were removed by centrifugation, and the preparations of gastric intrinsic factor were readministered to these patients, the absorption of vitamin B12 was markedly enhanced. Studies of hog gastric intrinsic factor before and after exposure to proteolytic enzymes failed to show any difference on Sephadex chromatography or polyacrylamide gel electrophoresis or on its affinity for vitamin B12 or the ileal receptor in guinea pigs. These investigations demonstrate that: (1) gastric intrinsic factor as secreted by subjects with pancreatic insufficiency or obtained from hog pyloric mucosal extracts is ineffective in promoting vitamin B12 absorption in patients with pancreatic insufficiency, (2) incubation of crude preparations of gastric intrinsic factor with insolubilized pancreatic proteases modified these preparations of gastric intrinsic factor in an as yet undefined manner, allowing them to enhance vitamin B12 absorption, and (3) in vitro studies using gut sacs or brush border preparations do not reflect the abnormality in vitamin B12 absorption associated with pancreatic dysfunction.

[Biologic activity of various intrinsic factor preparations in Schilling's test]. / Biologische Aktivität verschiedener Intriniscfaktor-Präparationen im Schillingtest

In 16 patients with pernicious anaemia investigations concerning the biological activity of 7 different Intrinsic-factor praeparations by means of the urinary excretion test were performed. Oral longterm-pretreatment with IF and biologically inactive IF-praeparations may cause false pathologic results.

[Blocking and binding antibodies to intrinsic factor and their behaviour during oral therapy with intrinsic factor-vitamin B 12 complex (author's transl)]. / Uber blockierende und bindende Antikörper gegen Intrinsic-Factor und deren Verhalten unter peroraler Intrinsic-Factor B 12-Therapie

13 patients with pernicious anaemia and 2 patients who had been subjected to total gastrectomy were treated orally with a hog instrinsic factor preparation (IF) for 1 to 4.5 years. During this therapy 11 of the patients with pernicious anaemia and both gastrectomized patients developed blocking and binding antibodies to IF. Antibodies already present before the commencement of therapy showed an increase in titre. All patients remained in complete haematological remission. The conclusion is drawn that circulating antibodies to IF do not play any significant role in the absorption of the hog IF-B 12 complex.

Pernicious anaemia: its pathogenesis in the light of the beneficial effect of duodenal intrinsic factor.

Oral ingestion of powdered duodenal mucosa with small doses of vitamin B12 proved efficient in four cases of untreated pernicious anaemia. The erythropoietic as well as the other responses were similar as those elicited by any reliable anti-pernicious drug. It has thus been confirmed that the growth factor demonstrated earlier in human duodenal juice by means of E. coli mutant corresponds in fact to the intrinsic factor. It is suggested that besides the deficiency of gastric intrinsic factor the duodenal intrinsic factor and the immune mechanisms neutralizing it are also involved in the pathogenesis. It has been shown that the aqueous extracts of hog stomach and duodenum differ in addition to their growth factor and nitrogen contents, also in aminoacid composition.