Search

To determine whether growth hormone has a direct effect on skeletal tissues not mediated by somatomedins, and to better define the role of somatomedin-C/ insulin-like growth factor I (Sm-C/IGF-I) in skeletal development, bovine growth plate, and rabbit articular and growth plate chondrocytes in primary culture were evaluated under a variety of experimental conditions designed to elicit growth hormone and Sm-C/IGF-I stimulation. Under none of these conditions did bovine growth plate chondrocytes respond to either homologous bovine growth hormone or heterologous hGH. Under the same conditions, these cells were highly responsive to human Sm-C/IGF-I with respect to both 3Hthymidine and 35Ssulfate incorporation, indices of mitotic and differentiated cell functions, respectively. Similarly, both rabbit articular and growth plate chondrocytes showed enhanced incorporation of 3H thymidine and 35SJsulfate in the presence of Sm-C/IGF-I, but did not respond to either native or recombinant hGH. Cells at different stages of maturation within the bovine growth plate differed in their reaction to Sm-C/IGF-I with proliferative zone cells manifesting a greater response to the peptide than cells of the reserve zone. These results suggest that the action of Sm-C/IGF-I on growth plate and articular chondrocytes is direct and that the effect of GH on these cells is indirect. The data further suggest that within the growth plate, the transition from reserve to proliferative status is associated with an increased Sm-C/ IGF-I responsiveness, a change which may contribute to the functional differences in these cells

A fluorescent focus identification assay (FFIDA) was developed for use in experimental studies and for quantitation of the components in a tetravalent live oral rotavirus vaccine. The assay utilizes four serotype-specific neutralizing monoclonal antibodies (MAb) to detect and quantify individual rotaviruses by immunofluorescence staining of fixed virus-infected monkey kidney cells. In mixed virus infections, all four MAb, W1 (serotype 1), 1C10 (serotype 2), R1 (serotype 3), and S4 (serotype 4), specifically stain the relevant homologous serotype without exhibiting any cross-reactivity against the other serotypes. Furthermore, the test is sensitive enough to differentiate at least twofold (0.3 log) differences in virus titer. The results of testing four individual experimental vaccine lots three or more consecutive times showed that all four lots contained similar proportions of the four vaccine strains as detected by the classical plaque neutralization identification test. The rapidity and efficiency of the FFIDA are desirable attributes that make it suitable for use in studies requiring identification and quantitation of one or more of the four major rotavirus serotypes.

Tissue-type plasminogen activator (t-PA) is a mosaic protein containing several distinct structural domains attached to the serine protease catalytic unit present at its COOH terminus. To investigate structure-function relationships in t-PA, we deleted the NH2-terminal domains, finger and epidermal growth factor, by genetic engineering. The genes for the parent and mutant t-PA were expressed in a bovine papilloma virus-dependent mammalian cell system. The secreted proteins were purified to homogeneity. The mutant protein was processed to the expected size of about 60 kDa compared to approximately 68 kDa for the parent t-PA, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fibrin autography. While the mutant t-PA had amidolytic activity comparable to native t-PA, it did not bind appreciably to fibrin. Consequently, fibrin-dependent enzymic activity, i.e. plasminogen activation in the presence of soluble fibrin and fibrinolysis were lower than with native recombinant t-PA. The effect of deletion of NH2-terminal domains on the plasma half-life (t1/2) was investigated by injecting native and mutant t-PA into mice. While the majority of the t-PA disappeared initially with a t1/2 of about 2 min, mutant t-PA cleared at a much slower rate with t1/2 of about 50 min. These findings suggest that the NH2-terminal domains of t-PA not only determine its specificity for binding to fibrin but also mediate its clearance from plasma in vivo. Furthermore, the catalytic unit in t-PA seems to function autonomously.

Recent data from several studies have suggested that the non-protease domains in tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA) determine their biological specificities, including binding to fibrin clots and survival in the circulatory system (Van Zonneveld, A.-J., Veerman, H., and Pannekoek, H. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 4670-4674; Rijken, D. C., and Emeis, J. J. (1986) Biochem. J. 238, 643-646). Structural manipulations (e.g. deletions, additions, or substitutions) in these domains can thus be utilized to maximize the desired biological effects. Using recombinant DNA technology, we constructed a number of hybrid molecules from the t-PA and u-PA genes. In hybrid A, the epidermal growth factor and finger domains of t-PA (residues 1-91) were replaced by the epidermal growth factor and kringle of u-PA (residues 1-131). In hybrids B and C, the u-PA kringle (residues 50-131) was inserted either before (residue 92) or after (residue 261) the double-kringle region of t-PA. All these hybrid PAs containing three kringles were expressed in mouse fibroblast cells (C-127). The hybrid proteins were synthesized in predominantly a single-chain form with molecular weights of 70,000-80,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were enzymatically active as assayed by the fibrin-agar plate method. In vitro studies on the binding of hybrid PAs to fibrin showed that hybrid B, like t-PA, possesses affinity toward fibrin, while hybrid A shows lower binding. This suggests that the finger domain, which is not present in hybrid A, plays a role in conferring fibrin affinity to the hybrid PAs. The enzymatic activities of the hybrids were compared with that of recombinant t-PA (rt-PA) expressed in the same vector/host system and found to be similar in activity toward a chromogenic peptide substrate. In addition, plasminogen activation with all the hybrid-PAs, as with rt-PA, was stimulated by fibrin, with the order of activity being rt-PA greater than or equal to hybrid B greater than hybrid C greater than hybrid A. This study shows the feasibility of shuffling functional domain(s) of known specificity in plasminogen activators which may lead to the design of a superior thrombolytic agent.

The results of the present investigation indicate a simple approach to the development of a single-vaccine formula which may ultimately be used to confer protection against both cholera and certain types of Escherichia colidiarrheal disease in humans and domestic animals. The design of the vaccine is based on the well-documented ability of cholera antitoxin to neutralize both cholera and heat-labile E. colienterotoxins (CT and LT, respectively) and on the ability of killed E. colito enhance the immune response to cholera toxoid and, possibly, to conventional cholera vaccine as well. Evidence presented shows that a parenterally administered E. colivaccine, prepared from an LT-only enterotoxigenic strain, reproducibly elevated rabbit antitoxin responses to cholera toxoid and that such responses correlated with dramatic protection against live cholera vibrios and the homologous E. colistrain in the rabbit ligated loop model of diarrheal disease. The results show also that cholera vaccine acted to suppress the rabbits' immune response to the cholera toxoid and E. colivaccine formula, even though all three antigens combined still provided significant protection against live organism challenge. On the basis of data presently available, the vaccine formula would be composed simply of cholera toxoid and E. colivaccine, but may also include cholera vaccine. Since it has already been established that cholera toxoid and cholera vaccine are each safe for human use, additional vaccine development would require investigation of the safety of E. colivaccine, alone and in combination with the other components.

The addition of an ultrafiltration step to the purification procedures previously described for cholera toxin (Rappaport et al., (1974) permitted the preparation of highly purified antigenic toxoids essentially free of somatic antigen(s). The purity of such toxoids is established: (i) by the absence of more than about one part limulus amebocyte lysate (LAL)-positive endotoxin per 10(5) parts toxoid and (ii) by the inability of the toxoids to elicit a significant rise in rabbit vibriocidal antibody. The antigenicity of the toxoids is demonstrated by their ability to produce the same high levels of rabbit serum antitoxin as are produced by comparable toxoids containing small amounts of somatic antigen. The results also indicate that amounts of somatic antigen of the order of less than or equal to 1 mug/100 mug of toxoid do not exert an adjuvant effect on the toxoid, at least with respect to circulating antitoxin. Other data show that, where present, the ability of somatic antigen to elicit vibriocidal antibody is influenced by the immunization schedule employed and that a correlation exists between the LAL-determined endotoxin content of the toxoids and their ability to stimulate vibriocidal antibody. Somatic antigen-free toxoids, purified and tested by the refinements herein described, were prepared for use in the National Institutes of Health sponsored field trials, and data pertaining to their purity and antigenic properties are presented.

Prostaglandins of the E type specifically inhibited the production of interleukin 2 (IL-2) by normal human lymphocytes, whereas PG synthetase inhibitors such as indomethacin and fentiazac raised IL-2 production above normal levels. Removal of adherent cells from mononuclear cell populations also resulted in enhanced IL-2 production. The resultant nonadherent cell population lost sensitivity to the enhancement effect of PG synthetase inhibitors, suggesting that a PGE-producing adherent cell plays a major role in the regulation of IL-2.

In a large group of 74 patients irradiated with 2400 rad for acute lymphoblastic leukemia an unusually high frequency of complete GH deficiency was observed (40 %). Only 11 out of 46 prepubertal children had growth retardation and seven children received hGH treatment. On the contrary 11 other prepubertal cases had normal growth rates in spite of lack of response to AITT. Plasma SmC values were correlated with growth rates but were discrepant with GH responses to AITT in somecases. Because of this high frequency of GH dysregulation further growth at time of puberty should be carefully documented.

A model is proposed in which stimulation of cortical cytoplasm occurs near the distal ends of astral rays. Levels of stimulation sufficient to cause furrowing occur only in equatorial zones between asters. The model can account for positioning of furrows in very large cells (fertilized eggs of amphibians, birds, and fish) and in cells with several mitotic apparatuses (insects). Finally, the model correctly predicts the positioning and occurrence of furrowing in two experiments in which cellular shape was manipulated into either an hourglass or a cylindrical form before division. These results are consistent with equatorial stimulation theories in which mitotic asters differentially stimulate the future furrow region (equatorial cortex). The results are not consistent with models requiring differential stimulation of nonfurrowing, polar regions of the cell.

Extract: In one case studied from the neonatal period through a follow-up period at almost five years of age, a salt-losing syndrome could be related to a defect in 18-OH-dehydrogenase.In the intial course of the disease, treatment with desoxycorticosterone acetate controlled the salt-losing tendency. At that time, the rate of aldosterone secretion was less than 10 µg/day and that of urinary tetrahydroaldosterone was less than µg/day. In contrast, the rate of corticosterone secretion was 5400 µg/day and the F:B ratio was 0.8. Urinary 18 hydroxytetrahydro A (18 OH-THA) was increased to 128 µg/24 h. There was no cortisol insufficiency; secretion rate was 20 mg m2/day and plasma ACTH 0.24 mU/100 ml plasma. At 20 months of age, treatment was stopped because of hypernatremia. From that time on, the child was clinically normal and received no salt supplement.The hormonal results were as follows: At 22 months of age, the secretion rate of aldosterone was less than 5 µg/day and of corticosterone was 8600µg/day on the sixth day of a well-tolerated salt suppression test. 18 OH-THA was 116µg/day; the F:B ratio for urinary metabolites was low, 0.9.At 3 years and 7 months of age, excretion of urinary tetrahydroaldosterone was 10 µg/day, 18 OH-THA was in the normal range, and the F:B ratio remained low, 0.6 (normal salt intake).At 4 years and six months of age, secretion rate was 19 µg/day and the 18 hydrocorticosterone secretion rate was 470 µg/day (ratio 18 OH-B: aldosterone 24.7). Plasma renin activity was 88.8 ng/l/min.The brother of the propositus was observed when 6 years of age. Urinary tetrahydrosterone was 27 µg/day, 18 OH-THA was 75/µg/day, and the urinary F:B ratio was 0.7 while receiving a normal salt intake.The spontaneous clinical improvement of subjects with the salt-losing syndrome is possibly related to the appearance of a certain amount of aldosterone, an increase in secretion of corticosterone, and modification in salt content of a normal diet during this period.Speculation: A salt-losing syndrome due to 18-OH-dehydrogenase deficiency, probably familial, improved spontaneously. Long-term studies showed persistence of the biosynthetic defect. The data presented raise questions concerning clinical recovery that could be correlated with steroid administration and, possibly, with modifications in sodium homeostasis at that period of life.

In one case studied from the neonatal period through a follow-up period at almost five years of age, a salt-losing syndrome could be related to a defect in 18-OH-dehydrogenase.