Biosynthesis of a ubiouitin-related peptide in rat brain and in human and mouse pituitary tumors

A biosynthetic labeled peptide structurally related to the thymic peptide ubiquitin was first identified fortuitously in bovine pars intermedia cells in regard to its partial NH₂ terminal amino acid sequence (Met 1, Leu 8, 15 and Lys 6, 11, 27, 29, 33) after a protein segment data bank search. A peptide with the same behavior on carboxymethylcellulose chromatography and polyacrylamide gel electrophoresis has been purified after labeling experiments in two areas of rat brain, hypothalamus and striatum, and in a mouse and a human ACTH-secreting pituitary tumors. It represents about 1 to 10% of the total labeled proteins in the various experiments. Its identity with the above mentioned bovine pituitary peptide was confirmed by microsequence analysis with respect to Met 1, Lys 6, 11 in hypothalmus, Met 1 in striatum, and Lys 6, 11, 27, 29, 33 in the two pituitary tumors. The availability of standard purified ubiquitin allowed us to show that labeled and cold peptides have the same electrophoretic mobility and elution volume on Sephadex G-50 chromatography this further confirms their identity. Possible interests of such a biosynthetic characterization of a ubiquitin-related peptide are discussed, particularly in view of the structural relationship of ubiquitin to the non histone component of nuclear protein A-24, and as a test of tissue viability and biosynthetic efficiency in our in vitro biosynthetic systems.     

Intralesional lipid-complexed cytokine/superantigen immunogene therapy for spontaneous canine tumors

These studies sought to determine the gene expression and short-term effects of intralesional lipid-complexed immunogene therapy with constructs encoding Staphylococcus aureus enterotoxin A and canine interleukin-2 (L-SEA/cIL-2) in dogs with tumors of various histotypes, and then to assess the safety and efficacy of repeated L-SEA/cIL-2 injections in dogs with spontaneous soft tissue sarcomas (STS). In the first study, pet dogs with a variety of tumors received a single intralesional injection of L-SEA/cIL-2, and surgical excision was performed 48 h later. In the second study, dogs with histologically confirmed STS were treated weekly for a maximum of 12 weeks with escalating doses of L-SEA/cIL-2. Tumors were then surgically excised and assessed histologically and immunohistochemically. Overall, treatments were well tolerated, with no dose-limiting toxicities encountered. At 48 h, in the single injection study, plasmid DNA was detected in 14 of 16 tumor samples, and plasmid-specific mRNA was detected in 3 of 14. In the multiple injection study, the overall response rate in dogs with STS was 25%, consisting of 3 complete responses (CR) and 1 partial response (PR). Diffuse lymphoplasmacytic inflammation was observed in all tumors from patients experiencing CR or PR, whereas these changes were not evident in tumors from nonresponders. The infiltrate was composed primarily of CD3(+) cells at 48 h from the single-injection study, and was composed of both CD3(+) and CD79a(+) cells at 12 weeks in responding dogs from the multiple-injection study. In conclusion, these studies suggests that intralesional L-SEA/cIL-2 immunotherapy is well tolerated, results in detectable transgene expression in canine tumors, and has antitumor activity in dogs with spontaneous STS.     

Intralesional injection of interferon gamma affects reactive proliferation of astrocytes in the neonatal rat brain. A preliminary study of the age-dependent effect

Following a mechanical lesion of the left cerebral hemisphere, newborn male rats received a single injection of recombinant rat interferon gamma (IFN gamma) into the lesion cavity at doses of 5, 50 and 500 U. One or two days after the injury the rats were injected with 3H-thymidine. Brain sections were immunostained for glial fibrillary acidic protein (GFAP), subjected to autoradiography and examined microscopically to record proliferating GFAP-immunopositive (GFAP+) astrocytes labeled with 3H-thymidine. Following the intermediate 50 U dose of IFN gamma, numbers of GFAP+ astrocytes and of their mitoses on day 1 after injury were significantly higher than in controls. Nevertheless, the astrocyte labeling index remained at the control level. Injections of the minimal 5 U or the maximal 500 U doses of IFN gamma had no effect on that day. On day 2, however, each of the three doses evoked a statistically significant but dose-independent reduction of the labeling index without similar changes in total number of GFAP+ astrocytes or in numbers of their divisions. The changes appear to indicate a non-linear relation between the intensity of reactive behaviour of astrocytes and the amount of IFN gamma injected into the lesion area. On the basis of previous publications, IFN gamma effects on the astrocyte reactivity are discussed as being age-dependent.     

Levamin and cerebrolysin as immunostimulants]

The experiments on mice have shown that the in vitro treatment of mouse bone marrow cells with levamin or cerebrolysin (amino acids mixture) increased the number of Thy-1 positive cells and stimulated the in vivo immune response to SRBC. Levamin proved more active. At the same time levamin and cerebrolysin had no effect on the immune response to thymus-independent Vi-antigen.     

Effects of injection routes of growing tumors PSK or OK-432 on antiproliferative activity of mouse serum

Summary The present study examined the effects of various treatments on the antiproliferative activity of mouse serum. Its activity was estimated against the growth of EL4 tumor cells and L929 cells and against splenic blastogenesis in culture. The activity varied among mouse strains tested and among individuals in any strain. However, normal outbred NIH Swiss mice showed the highest activity among the strains and the least variation among individuals. The activity of serum from NIH Swiss mice constantly decreased 7 or 14 days after an injection of 10⁶ Ehrlich or sarcoma 180 tumor cells subcutaneously in the right-hind footpad, intradermally in the right side of the chest or into the palm. Other routes, such as intraperitoneal, intravenous in the tail vein, subcutaneous in the right side of the chest and intramuscular in the left thigh, however, hardly affected the activity. The activity also decreased 7 days after an injection into the footpad of a biological response modifier such as PSK or OK-432. The antiproliferative activity of mouse serum seems to depend on macrophages but not natural killer-cell activity, because treatment with silica but not anti-(asialo-GM1) antibody totally reduced the activity. The active fraction was heat-stable (100° C, 30 min) and its molecular mass was 127–140 kDa.     

The use of immunostimulants in fish larval aquaculture

The production of fish larvae is often hampered by high mortality rates, and it is believed that most of this economic loss due to infectious diseases is ca. 10% in Western European aquaculture sector. The development of strategies to control the pathogen load and immuno-prophylactic measures must be addressed further to realise the economic "potential" production of marine fish larvae and thus improve the overall production of adult fish. The innate defence includes both humoral and cellular defence mechanisms such as the complement system and the processes played by granulocytes and macrophages. A set of different substances such as beta-glucans, bacterial products, and plant constituents may directly initiate activation of the innate defence mechanisms acting on receptors and triggering intracellular gene activation that may result in production of anti-microbial molecules. These immunostimulants are often obtained from bacterial sources, brown or red algae and terrestrial fungi are also exploited as source of novel potentiating substances. The use of immunostimulants, as dietary supplements, can improve the innate defence of animals providing resistance to pathogens during periods of high stress, such as grading, reproduction, sea transfer and vaccination. The immunomodulation of larval fish has been proposed as a potential method for improving larval survival by increasing the innate responses of the developing animals until its adaptive immune response is sufficiently developed to mount an effective response to the pathogen. To this end it has been proposed that the delivery of immunostimulants as a dietary supplement to larval fish could be of considerable benefit in boosting the animals innate defences with little detriment to the developing animal. Conversely, there is a school of thought that raises the concern of immunomodulating a neotanous animal before its immune system is fully formed as this may adversely affect the development of a normal immune response.     

Evaluation of time and dose in treating mammary adenocarcinoma with immunostimulants

Summary BCG, C. parvum, and reovirus were used as immunostimulants in treating murine mammary adenocarcinoma (A-10) after tumor burden had been minimized with BCNU. Immunostimulants were administered at different times with respect to chemotherapy. Different doses were used to determine the optimal response as measured by survival. BCG induced the best response when 6.67×10⁵ organisms were given 2 days after chemotherapy. The optimal response with C. parvum was observed after a dose of 0.35 mg was given 1 or 2 days after chemotherapy. Similarly, reovirus produced the best response when 10¹⁰ plaque-forming units were given 2 days after chemotherapy. These data are consistent with previous findings and support the notion that immunostimulants require an appropriate lymphoid substrate in order to induce an adequate anti-tumor response.Tge abbreviations used are: BCNU: 1,3-bis-(2-chloroethyl)-1-nitrosourea Saline: 0.9% NaCl solution; BCG: Bacillus Calmette-Guerin; C. parvum: Corynebacterium parvum; pfu: plaque-forming unitsThis study was supported, in part, by Contract No. N01-CB-43864 and Grant No. CA 14460 from The National Cancer Institute     

Analysis of the effect of immunostimulants on the macrophages of mouse peritoneal exudates by using mathematical simulation and planning methods

Using methods of mathematical modelling and planning an analysis was made of changes in 5-nucleotidase activity in murine peritoneal macrophages after intraperitoneal administration of immunostimulants of different chemical structure and biological origin. The changes in 5-nucleotidase activity after the administration of immunostimulants were shown to exhibit a similar linear pattern.