Biomedical subjects
Publications and source records attributed to S Radulovic.
The activities of doxycycline, rifampin, chloramphenicol, and erythromycin against ELB rickettsiae (Rickettsia azadi) were determined by dye uptake and plaque assays. Plaque formation in Vero cells was inhibited by 0.12 microgram of doxycycline per ml. The data presented demonstrate the susceptibility of ELB rickettsiae to commonly used antibiotics for the treatment of rickettsial diseases.
ELB rickettsiae from cat flea homogenates were recovered in tissue culture cells following sequential passage through laboratory rats and the yolk sacs of embryonated chicken eggs. Seven days after inoculation of ELB from the infected yolk sacs, Vero cells and L929 cells were observed to contain intracellular bacteria as demonstrated by Diff Quik and indirect immunofluorescence assay staining. The rickettsial and ELB identity of the cultured agent was confirmed by PCR detection of the 16S rRNA and citrate synthase genes and PCR-restriction fragment length polymorphism analysis of the 17-kDa conserved rickettsial antigen gene. The ELB rickettsiae induced plaques in Vero cells on day 11 postinfection. Rat anti-ELB serum reacted at 1:4,096 to cultured ELB and had lower reactivity to Rickettsia typhi Wilmington (1:1,024), Rickettsia akari Kaplan (1:512), and Rickettsia australis JC (1:64). Spotted fever group polyclonal sera also exhibited lower reactivity to ELB than to the homologous antigen. Coomassie blue-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles of the ELB isolate and two R. typhi strains were identical.
Spotted fever rickettsiosis in Israel has been considered as possibly somewhat more severe than boutonneuse fever, from which it also differs in having a very low proportion of cases with a tick-inoculation site eschar. This investigation was undertaken to determine whether the Israeli spotted fever group (SFG) rickettsiae differed sufficiently from Rickettsia conorii to be considered as a distinct species. Strains of Rickettsia conorii from Morocco and South Africa, four SGF rickettsial isolates from Israel, one from Russia, and one from Zimbabwe were compared by microimmunofluorescence serotyping, Western immunoblotting, monoclonal antibody reactivity, and polymerase chain reaction amplification of the repeat domain of the rickettsial outer membrane protein A (rOmpA). All are strains and isolates of R. conorii, yet there is considerable molecular and antigenic diversity of both rOmpA and rickettsial outer membrane protein B (rOmpB) among them. The rOmpA gene of the Israeli isolates and the Astrakhan strain from Russia is estimated to encode 15 rOmpA repeat units as compared with 10 for the South African strain and six for the strains from Morocco and Zimbabwe. The Israeli SFG rickettsial strains appear to be R. conorii, a species with substantial antigenic and genetic diversity. The Israeli strains appear to fall within the limit previously described for the genetic and antigenic diversity of R. conorii.
In this study, we investigated the effects of luteinizing hormone-releasing hormone (LH-RH) agonist [D-Trp6]LH-RH, LH-RH antagonist [Ac-D-Nal(2)1,D-Phe(pCl)2,D-Pal(3)3,D-Cit6,D-Ala10]LH-RH (SB-75), and estradiol on the growth of human epithelial ovarian cancer cell line OV-1063. Cells were cultured under estrogen-deprived conditions. Estradiol inhibited cell proliferation, as measured by cell number at 10(-9)-10(-7) M and [3H]thymidine incorporation into DNA at 10(-13)-10(-8) M. Both LH-RH analogs inhibited cell growth dose dependently in the range 10(-8)-10(-5) M, but SB-75 induced a greater growth inhibition than [D-Trp6]LH-RH. In OV-1063 cells, 125I-labeled [D-Trp6]LH-RH was bound to one class of specific, saturable binding sites with high affinity (Kd = 1.4 +/- 0.3 nM) and low capacity (4000 binding sites per cell). 125I-labeled [D-Trp6]LH-RH could be displaced by unlabeled [D-Trp6]LH-RH and SB-75, suggesting that both analogs are bound to the same receptor on OV-1063 cells. Ligand binding was dependent on time and temperature. Receptor internalization assay showed that the ligand-receptor complex was internalized at 37 degrees C, which indicates the presence of biologically active LH-RH receptors on OV-1063 cells. These results suggest that estradiol and LH-RH analogs can suppress the growth of OV-1063 human epithelial ovarian cancer cells by a direct action and that the inhibitory effect of LH-RH analogs is mediated through the high-affinity LH-RH receptors.
BACKGROUND: The results of several clinical trials using various luteinizing hormone-releasing hormone agonists for treatment of advanced breast cancer are encouraging. However, only about 30% of breast cancers are estrogen-dependent and can be treated by hormonal manipulation. New therapeutic approaches combining estrogen ablation therapy with other compounds must be explored. Various studies suggest that bombesin or gastrin-releasing peptide acts as an autocrine growth factor and may play a role in the initiation and progression of some cancers, including that of the breast. METHODS: Female athymic nude mice bearing xenografts of the MCF-7 MIII human breast cancer cell line were treated for 7 weeks with bombesin/gastrin-releasing peptide antagonist (D-Tpi6, Leu13 psi[CH2NH]-Leu14) bombesin(6-14) (RC-3095) injected subcutaneously daily at a dose of 20 micrograms and luteinizing hormone-releasing hormone antagonist SB-75 (Cetrorelix) administered biweekly in the form of microgranules releasing 45 micrograms/day. RESULTS: After 2 weeks of treatment, a significant inhibition of tumor volume was observed in the groups treated with RC-3095 alone or in combination with SB-75 but not in those treated with SB-75 as a single agent. After 7 weeks, tumor growth as measured by tumor volume and percentage changes in tumor volume and tumor weight was greatly inhibited in all of the treated groups. Uterine and ovarian weights were reduced and serum luteinizing hormone levels decreased by administration of SB-75 alone or in combination with RC-3095. Histologically, a significant decrease in argyrophilic nucleolar organizer region count in tumor cell nuclei was observed in all of the treated groups, indicating a lower proliferation of these cells. High-affinity binding sites for bombesin were detected in cultured MCF-7 MIII cells. Chronic treatment with RC-3095 caused a significant down-regulation of epidermal growth factor receptors in tumor cell membranes, which might be related to tumor inhibition. In studies in vitro, SB-75 inhibited proliferation of MCF-7 cells in culture but not proliferation of MCF-7 MIII cells. CONCLUSIONS: Because previously we demonstrated that RC-3095 inhibits the proliferation of MCF-7 MIII cells in vitro, it appears that the major antitumoral effect of RC-3095 on the MCF-7 MIII cancer line is direct, whereas that of SB-75 is indirect, and that it is mediated by suppression of the pituitary-gonadal axis. In view of its immediate and powerful inhibitory effect on MCF-7 MIII tumors, bombesin/gastrin-releasing peptide antagonist RC-3095 might be considered as a possible new agent for the treatment of breast cancer.
In a mountainous area in the Dinaric Beech-Fir Forest of southern Slovenia, summer nests of the European fat dormouse (Glis glis) were collected. From these dormouse nests, 180 Monopsyllus sciurorum sciurorum fleas were examined by polymerase chain reaction with primers for the Rickettsia citrate synthase gene. Samples from one nest yielded the expected 381 base pair DNA product. The origin of the DNA product was identified as Rickettsia typhi by AluI restriction fragment length polymorphism analysis. Inoculation of the triturated positive fleas into Vero cell culture resulted in the cultivation of a rickettsia which reacted with polyclonal and species-specific monoclonal antibodies for R. typhi. The widespread distribution of this sylvatic flea species in nearly all of Europe as well as in the Middle East and its presence on other mammalian and avian hosts suggests that R. typhi might exist in unrecognised enzootic cycles. Further investigations are needed to determine the extent of these cycles in Europe and the potential occurrence of human infections.
A soldier developed characteristic manifestations of boutonneuse fever shortly after leaving Somalia. Rickettsial DNA was detected in a biopsy sample of the tache noire by a polymerase chain reaction (PCR) in which primers derived from the 190-kD antigen gene of Rickettsia rickettsii were used. The source of this DNA was identified as Rickettsia conorii by restriction fragment length polymorphism (RFLP) analysis of the PCR product. R. conorii was also isolated from the skin biopsy specimen. The patient did not develop a significant increase in specific antibodies, as assessed by indirect fluorescent antibody testing, until several weeks after the onset of symptoms. This case demonstrates that the PCR/RFLP technique can be used for the direct identification of rickettsiae from clinical specimens. To our knowledge, this is the first confirmed case of R. conorii infection in Somalia.
Nude mice bearing xenografts of HT-29 human colon cancer cell line were treated for 4 weeks with somatostatin analog (RC-160), bombesin/gastrin releasing peptide (GRP) antagonists (RC-3095 and RC-3440). In three separate experiments somatostatin analog RC-160 (50 micrograms/day) released from microgranules significantly reduced tumor growth. Bombesin/GRP antagonists, RC-3095 and RC-3440 injected subcutaneously (s.c.) twice daily at a dose of 10 micrograms had the greatest and consistently significant inhibitory effect on tumor growth. RC-3095 given once daily s.c. at a dose of 20 micrograms was less effective. RC-3095 also inhibited metastatic tumor growth after intrasplenic injection of HT-29 cells in nude mice. Specific binding sites of somatostatin, bombesin and epidermal growth factor (EGF) were detected on intact HT-29 cells or on the membranes from HT-29 tumor xenografts. The inhibitory effects of bombesin antagonists on tumor growth were consistently linked with a significant down-regulation of EGF receptors. Bombesin/GRP antagonists and somatostatin analogs could be considered for the development of new hormonal therapies for colon cancer.
To evaluate the prevalence of spotted fever group rickettsiae along the Adriatic Coast of Croatia, 832 ticks were examined by hemolymph test, direct immunofluorescence, antigen-capture enzyme immunoassay, and polymerase chain reaction. Very good agreement was observed among direct immunofluorescence, polymerase chain reaction, and antigen-capture enzyme immunoassay. Twelve ticks that were positive by hemolymph test and negative by both direct immunofluorescence and polymerase chain reaction presumably do not represent spotted fever group rickettsiae. By direct immunofluorescence, spotted fever group rickettsiae were present in 12% of Rhipicephalus bursa, 10.6% of Rh. sanguineus, and 7.8% of Dermacentor marginatus. From the 98 ticks containing rickettsia-like organisms by hemolymph test, seven spotted fever group rickettsial isolates were established in cell culture. Four isolates were identified as Rickettsia conorii. The antigen-capture enzyme immunoassay, which utilizes a monoclonal antibody to antigens of the 135-kD surface protein shared among many members of the spotted fever group, is recommended for primary screening of tick samples because it is reliable and yet less labor-intensive than the hemolymph and direct immunofluorescence tests. Although the polymerase chain reaction is too expensive for use as a screening method, it is recommended for confirmation of positive screening results. In addition to the technologic advance of the antigen-capture enzyme immunoassay, this study documented by contemporary methods that R. conorii is present along the eastern Adriatic Coast not only in the classic vector, Rh. sanguineus, but also in Rh. bursa and D. marginatus.
The effects of hybrid cytotoxic LH-RH analogs, produced by linking anthraquinone or methotrexate to carrier LH-RH agonist [D-Lys6]LH-RH, were evaluated in Copenhagen-Fisher F1 rats bearing Dunning R-3327H prostate adenocarcinoma. The two cytotoxic LH-RH analogs T-98 [(D-Lys6)LH-RH coupled to glutaryl-2-(hydroxymethyl)anthraquinone (G-HMAQ)], and AJ-04 [(D-Lys6)LH-RH linked to methotrexate (MTX)], carrier [D-Lys6]LH-RH, or the free cytotoxic compounds MTX and G-HMAQ were administered from Alzet Osmotic minipumps for 7-8 weeks. The cytotoxic LH-RH analogs caused somewhat greater tumor growth inhibition than the carrier peptide, while anthraquinone or methotrexate alone, administered in equimolar doses, were ineffective. The inhibition of androgen sensitive organs (testes, ventral prostates, and seminal vesicles) was pronounced with both carrier and cytotoxic analogs, showing the latter to be fully hormonally active in suppressing the pituitary-gonadal axis. Histological changes were also evaluated. The inhibition of mitosis and the frequency of apoptosis were higher in tumors treated with AJ-04, T-98, [D-Lys6]LH-RH, or by castration than in those of controls. Serum hormone levels were lowered by both carrier peptide and cytotoxic analogs, LH being substantially depressed, and testosterone not detectable. These results and other findings indicate that LH-RH analogs containing cytotoxic radicals anthraquinone or methotrexate retain their hormonal activity after administration in vivo, and can effectively inhibit tumor growth. Extensive further studies are required on this new class of compounds, but apparent binding of cytotoxic LH-RH analogs to tumors such as prostate cancer, which have receptors for LH-RH, could greatly reduce peripheral toxicity of chemotherapeutic agents. This approach, based on targeted chemotherapy, might be of practical therapeutic importance for the management of advanced prostate cancers, which eventually relapse after palliative hormonal therapy.
Binding capacities and apparent dissociation constants of receptors for luteinizing-hormone-releasing hormone (LHRH) were investigated in estrogen-independent MXT mammary cancers of untreated mice and after in vivo treatment with agonistic or antagonistic analogs of LHRH containing cytotoxic radicals: AJ-04 (agonist [D-Lys6]LHRH linked to methotrexate), T-98-([D-Lys6]LHRH coupled to glutaryl-2-(hydroxmethyl)anthraquinone (G-HMAQ)) and T-121/B (LHRH antagonist T-147 containing two residues of G-HMAQ), which induced tumor growth inhibition. The effects were compared to LHRH agonist [D-Trp6]LHRH and carriers [D-Lys6]LHRH, LHRH antagonist T-147, as well as to methotrexate, G-HMAQ and surgical bilateral overiectomy. Analysis of the binding data revealed that in control tumors the interaction of 125I-[D-TRP6]LHRH was consistent with the presence of one class of saturable, specific, noncooperative, high-affinity and low-capacity binding sites. Chronic treatment of mice bearing MXT tumors with LHRH analogs AJ-04 and T-121/B carrying cytotoxic radicals, but not with T-98 produced significant down-regulation of membrane receptors for LHRH. The largest decrease in dissociation binding constant and Bmax of receptors for LHRH was also found in animals treated with T-121/B. Specific, high affinity binding of 125I-labelled epidermal growth factor (EGF) was detected in the membranes from control and treated MXT tumors. Treatment with cytotoxic LHRH analogs, AJ-04, T-98 and especially with T-121/B, reduced maximal binding capacity of EGF receptors. Our results indicate that LHRH analogs carrying cytotoxic radicals retain their hormonal activity and inhibit tumor growth while inducing down-regulation of LHRH receptors. In addition, probably both components of the cytotoxic LHRH analog, peptide carriers and cytotoxic radicals, reduce the binding capacity of EGF receptors, which might be useful in the treatment of breast cancer.
Reactions of Al3+ (50-500 mumol l-1) with intact blood plasma and its low relative molecular mass ultrafiltrate (< 5 kDa) have been studied by proton nuclear magnetic resonance spectroscopy. Binding to citrate was detected and was reversed by addition of desferrioxamine. The use of combined exponential and sine-bell functions for the resolution enhancement of spectra of plasma is illustrated.
All currently available assays for antibodies to the etiologic agents of spotted fever group rickettsioses detect reactivity with antigens of lipopolysaccharides and the major cell wall proteins, which contain epitopes that are shared among many spotted fever group rickettsiae. The hypothesis of this study was that a monoclonal antibody to a species-specific epitope of Rickettsia rickettsii would be blocked from binding to the rickettsial surface if the rickettsiae were incubated previously with serum containing species-specific antibodies to the epitope. In an EIA, binding of monoclonal antibody 5C10-F3 to R. rickettsii was blocked by convalescent sera from patients with Rocky Mountain spotted fever but not from those with Mediterranean spotted fever, endemic typhus, or Q fever or normal subjects. This assay should be useful in determining the origin of the high seroprevalence of spotted fever rickettsial antibodies in certain geographic regions and establishing a species-specific diagnosis in patients with undetermined rickettsial exposure.
Nude mice bearing xenografts of the MIA PaCa-2 human pancreatic cancer cell line were treated with sustained-release formulations (microcapsules) of luteinizing hormone releasing hormone (LH-RH) agonist [D-Trp6]-LH-RH, somatostatin analogue RC-160 (D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Trp-NH2), or combination of both analogues. Other groups of mice received daily subcutaneous injections of LH-RH antagonist SB-75 [Ac-D-Nal(2)',D- Phe(4Cl)2,D-Pal(3)3,D-Cit6,D-Ala10-LH-RH] or bombesin antagonist RC-3095. At necropsy, in mice given microcapsules releasing 25 micrograms/day of [D-Trp6]-LH-RH, tumor weight and volume were decreased, but not significantly, as compared with control mice. Microcapsules of RC-160, releasing 25 micrograms/day, significantly reduced tumor volume, percentage change in tumor volume, and tumor weight. Combination of RC-160 and [D-Trp6]-LH-RH inhibited tumor growth to a somewhat greater extent than RC-160 alone. Bombesin antagonist RC-3095, at a dose of 25 micrograms/day, did not influence the growth of tumors. In mice receiving 100 micrograms/day of antagonist SB-75, there was a significant decrease in tumor weight and volume and a significant reduction in the weight of ovaries and uteri. Specific binding of [125I]RC-160 and [125I][D-Trp6]-LH-RH, but not [125I]Tyr4-bombesin, was found on MIA PaCa-2 cells in culture. [D-Trp6]-LH-RH, SB-75, and RC-160 inhibited the growth of MIA PaCa-2 cells in vitro. Neither bombesin nor RC-3095 influenced the growth of MIA PaCa-2 cells in cultures. The results indicate that the LH-RH antagonist SB-75 could be tried for treatment of pancreatic cancer. Our findings confirm the efficacy of somatostatin analogue RC-160 in inhibiting the growth of pancreatic cancers and suggest that the combination of RC-160 and agonist [D-Trp6]-LH-RH might possibly increase the therapeutic response.
A seroepidemiological survey in coastal Croatia detected antibodies reactive with Rickettsia conorii in 4.2% of sera by immunofluorescence assay and in 5.0% of sera by enzyme immunoassay. Western immunoblotting demonstrated antibodies to the 120-kDa surface protein in all 20 positive serum samples examined and to rickettsial lipopolysaccharide in 3 of these serum samples. Humans in this area are clearly being exposed to spotted fever rickettsiae.
BACKGROUND: Many breast cancers are estrogen independent, and even in patients who initially respond to estrogen suppression therapy, the regression is often temporary. We have recently shown that antagonists of bombesin and gastrin-releasing peptide, including RC-3095, inhibit the growth of pancreatic, colonic, and prostatic cancers in experimental animals. This effect was associated with a substantial decrease in epidermal growth factor (EGF) receptor levels in pancreatic and colon cancers. PURPOSE: In view of these findings, we investigated the effects of our synthetic bombesin and gastrin-releasing peptide receptor antagonist D-Tpi6,Leu13 psi (CH2NH)-Leu14 bombesin(6-14) (RC-3095) on the growth of hormone-dependent and hormone-independent MXT mouse mammary cancers in vivo. METHODS: Female (C57BL x DBA/2)F1 mice bearing estrogen-dependent or estrogen-independent MXT mammary carcinomas were treated with small doses (20 micrograms/d) of RC-3095 administered from osmotic minipumps. Separate groups of mice with estrogen-independent tumors received RC-3095, bombesin, or gastrin-releasing peptide(14-27) at 20 micrograms/d. We determined tumor volume and weight, mitotic index, apoptosis (programmed cell death), and argyrophilic nucleolar organizer regions, an indicator of tumor cell proliferation. Levels of receptors for EGF and bombesin were measured in tumor membrane fractions. RESULTS: Growth of both estrogen-dependent and estrogen-independent MXT breast cancers was significantly inhibited by RC-3095. Bombesin or gastrin-releasing peptide had no effect on the growth of estrogen-independent tumors. Inhibition of tumor cell proliferation was indicated by a 45%-65% reduction in tumor volume, a 35%-58% reduction in tumor weight, and statistically significant decreases in argyrophilic nucleolar organizer region counts after treatment with RC-3095. In estrogen-independent cancers, tumor inhibition was associated with a decrease in the capacity of EGF receptors from 0.21 +/- 0.016 pmol/mg membrane protein in controls to 0.03 +/- 0.003 pmol/mg membrane protein in the RC-3095-treated group. CONCLUSIONS: This is the first demonstration of inhibitory effects of bombesin and gastrin-releasing peptide antagonists on the growth of breast cancers in vivo. IMPLICATIONS: These findings suggest that bombesin antagonists should be considered for breast cancer therapy.
This study was performed to evaluate the efficacy and duration of action of a new bombesin antagonist D-Tpi6,Leu13 psi (CH2NH)Leu14-bombesin (6-14) (RC-3095), given by different routes of administration, in suppressing gastrin releasing-peptide (GRP(14-27))-stimulated gastrin release in rats. First, we showed that GRP(14-27) itself was highly active when administered by different routes. GRP(14-27), given to rats at a dose of 25 micrograms/100 g b.w. significantly increased serum gastrin levels 3 and 6 min after intravenous and for more than 30 min after subcutaneous administration or pulmonary inhalation. RC-3095 was then injected subcutaneously, intravenously and also delivered by pulmonary inhalation at a dose of 10 micrograms/100 g b.w. in each case to seven male rats 2, 30, 60 or 120 min prior to i.v. administration of 5 micrograms GRP(14-27). RC-3095 administered 2 min prior to GRP(14-27) decreased the gastrin response to GRP(14-27), measured as area under the curve, by 81% in the intravenously injected group and 64% in the pulmonary inhalation group in the first 6 min. When GRP(14-27), was given 30 min after administration of RC-3095, the gastrin response was decreased by 52% in the subcutaneous group, 49% in the pulmonary inhalation group and 11% in the intravenous group during the first 6 min. RC-3095 delivered subcutaneously or by pulmonary inhalation 1 h before GRP(14-27) was also able to significantly inhibit gastrin release. Analysis of the data revealed that the bioavailability of RC-3095 given by the pulmonary inhalation route was about 69% of the s.c. route.(ABSTRACT TRUNCATED AT 250 WORDS)
The function of the pituitary-gonadal axis in normal (immunocompetent) and nude (immunocompromised) mice, like that of other species, can be suppressed by luteinizing hormone-releasing hormone (LH-RH) agonists and antagonists administered by continuous release systems and, therefore, nude mice provide a valuable model for investigation of the effects of LH-RH analogues on growth of xenografts of human cancers. To extend our findings further, we treated male nude mice bearing xenografts of human prostate adenocarcinoma PC-82, for 42 days, with sustained release formulations (microcapsules or microgranules) of the agonist [D-Trp6]LH-RH, the antagonist [Ac-D-Nal(2)1,D-Phe(4Cl)2,D-Pal(3)3,D-Cit6,D-Ala10]LH- RH (SB-75), or the somatostatin analogue D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Trp-NH2 (RC-160). At necropsy, in mice given microcapsules releasing 25 micrograms/day of [D-Trp6]-LH-RH, tumor weight and volume were significantly decreased, compared with control mice, and weights of testes, ventral prostate, and seminal vesicles were also reduced in this group. In mice which received microgranules liberating 50 micrograms/day of antagonist SB-75, there was a greater decrease in tumor weight and volume than that produced by the agonist and a significant reduction in the weight of the testes and accessory sex organs. Histological parameters also demonstrated significant tumor inhibition, with the best results being obtained by treatment with the antagonist SB-75. The tumor inhibition induced by SB-75 was demonstrated to be due to decreased cellular proliferation, with enhanced cellular death (i.e., apoptosis) of the PC-82 cells. Microcapsules releasing 50 micrograms/day of RC-160 decreased tumor weight and volume by 23% and 28%, respectively, but this reduction was not significant. Serum levels of testosterone were decreased by 90% in mice given the LH-RH agonist and by 94% in response to the antagonist SB-75. Serum levels of prostate-specific antigen were significantly lower in mice treated with LH-RH analogues, with the antagonist SB-75 causing a greater reduction. A ratio of prostate-specific antigen to tumor weight suggests that levels of serum prostate-specific antigen may be correlated with tumor mass. Using sensitive multipoint micromethods, one class of binding sites for LH-RH, with a dissociation constant of 7.8 +/- 1.2 nM and a maximal binding capacity of 126.4 +/- 23.1 fmol/mg protein, was found in the control tumors. Tumors from mice treated with either LH-RH agonist or antagonist, but not somatostatin analogue RC-160, showed a significant reduction in maximal binding capacity for LH-RH, compared to control tumors.(ABSTRACT TRUNCATED AT 400 WORDS)