Computer-based teaching of pathology at the Zagreb University School of Medicine.

AIM: To review the experience gained in transferring USA computer-based teaching system of medical school pathology to Croatia. METHODS: Computer-based teaching program of pathology developed at the University of Kansas School of Medicine, Kansas City, Kansas, USA, was transferred to the University of Zagreb School of Medicine, Zagreb, Croatia. The experimental group of 49 students was enrolled into this computer-based program. Their performance was compared with that of 195 classmates enrolled in the standard course. Objective (performance on the examinations) and subjective data (students' interviews and written evaluations of the course) were analyzed. RESULTS: The computer program was operational 5 months from the inception of the transfer. It was well received by the students, even though many initially complained that it required more effort and a continuous commitment. The major problems concerned scheduling, reflecting various requirements i mposed on students by other departments teaching in parallel with the Pathology course. Objective data gathered so far indicate that the students enrolled in the computer-based program took the first midterm examination at a significantly higher rate than the rest of the class (p<0.001), and passed the examination with significantly better grades (p<0.001). CONCLUSION: Computer-based teaching programs can be readily transferred to other countries. Full implementation of the program, however, may require significant changes in the existing curriculum in the medical school to which such a program has been transferred or considerable modifications in the program adopted for transfer. It appears that the students enrolled in the computer-based program perform better than students in the standard pathology course.

A novel oligodeoxynucleotide inhibitor of thrombin. I. In vitro metabolic stability in plasma and serum.

PURPOSE: To determine the degradation rates and pathways of GS-522, a potent oligodeoxynucleotide (GGTTGGTGTGGTTGG) inhibitor of thrombin, in serum and plasma. METHODS: A stability-indicating, anion-exchange HPLC method was developed and used to determine concentrations of GS-522 and metabolites. RESULTS: In monkey plasma at 2 microM or below, the degradation of GS-522 can be fit to a first-order exponential with a kpobs approximately 0.01 min-1. At 3 microM and above the degradation process deviates from a monoexponential decay profile. An initial fast degradation process is followed by a slower phase with an observed rate constant equal to that observed at 2 microM and below. In monkey serum, the KM and Vmax are 8.4 microM and 0.87 microM min-1, respectively. CONCLUSIONS: The kinetics are consistent with an equilibrium binding of GS-522 to prothrombin in plasma (Kd = 50 nM) which saturates at GS-522 concentrations > 2 microM. Compared to a scrambled sequence (GGTGGTGGTTGTGGT), with no defined tertiary structure, GS-522 is 4-fold more stable in serum. The metabolic profile in plasma is consistent with a 3'-exonuclease catalyzed hydrolysis of GS-522.

A novel oligodeoxynucleotide inhibitor of thrombin. II. Pharmacokinetics in the cynomolgus monkey.

PURPOSE: To determine the pharmacokinetics of GS-522, an oligodeoxynucleotide (GGTTGGTGTGGTTGG) inhibitor of thrombin, after constant infusion and bolus administration in the cynomolgus monkey. METHODS: Using a stability indicating HPLC method, the GS-522 plasma concentration versus time data were obtained after constant infusion (0.1, 0.3, 0.5 mg/kg/min) and bolus administration (11.25 and 22.5 mg/kg). Plasma data after bolus administration was fit to a three-compartment model. RESULTS: The half-lives for the alpha and beta phases were 1.4 and 5.4 min, respectively. Steady state GS-522 concentrations were reached within 10 minutes after initiation of constant infusions. Termination of infusions resulted in a rapid elimination of GS-522 with an average elimination half-life equal to 1.5 min. The Vss calculated from both the constant infusion and bolus data approximated the blood volume of the monkey. Substitution of the phosphodiester backbone at the 3' end of GS-522 with two phosphorothioate linkages did not substantially effect the elimination half-life upon termination of infusion. CONCLUSIONS: These data in conjunction with published biodistribution data suggest that oligodeoxynucleotides are rapidly cleared from plasma by tissue uptake and that little efflux back into blood takes place. Additionally, strategies designed to increase oligodeoxynucleotide resistance to exonucleases will not dramatically increase plasma half-lives.

A spectrum in the pathology of toxoplasmosis in patients with acquired immunodeficiency syndrome.

We describe a variety of toxoplasmic lesions in seven patients with the acquired immunodeficiency syndrome. The first patient had multiple small-intestinal ulcers associated with Toxoplasma tachyzoites and high antibody titers; he died of disseminated histoplasmosis. The second patient, who died of tuberculosis, also had an inactive chronic Toxoplasma infection, with tissue cysts in the brain that were associated with glial nodules. A third patient died of Toxoplasma encephalitis, manifested by multiple foci of necrosis associated with Toxoplasma tachyzoites, cysts, and hypertrophic arteritis. A fourth patient had been treated for toxoplasmic encephalitis with co-trimoxozol (trimethoprim-sulfamethoxazole combination) for 3 to 4 days and showed degenerating tachyzoites associated with necrotic areas. A fifth patient, treated for toxoplasmic encephalitis with co-trimoxazol for 14 days, had necrotic lesions associated with Toxoplasma antigen and a few cysts. A sixth patient with encephalitis and Toxoplasma tachyzoites and young cysts in the biopsy showed healed brain lesions after 22 days of treatment. A seventh patient, diagnosed radiologically and serologically with Toxoplasma encephalitis, was treated for 7 months; his ring-enhancing lesions subsided, and he died of a central nervous system lymphoma. Toxoplasma could not be isolated from the brain, although toxoplasmic DNA was detected in the brain and heart by polymerase chain reaction. The pathogenesis of the range of these lesions, their diagnosis, and the possibility of terminating Toxoplasma infection by prolonged chemotherapy are discussed.

Oral bioavailability of the antiretroviral agent 9-(2-phosphonylmethoxyethyl)adenine (PMEA) from three formulations of the prodrug bis(pivaloyloxymethyl)-PMEA in fasted male cynomolgus monkeys.

The bioavailability of PMEA from three oral formulations of the prodrug bis(POM)-PMEA has been evaluated in fasted male cynomolgus monkeys. The formulations examined included a hydroxypropyl-beta-cyclodextrin (HPBCD) complex, a PEG based cosolvent solution, and an aqeous suspension. Oral formulations containing 3H-bis(POM)-PMEA were compared to intravenous 3H-PMEA at 10.9 mg-eq/kg in a crossover study in four monkeys, with a 7 day washout period. No intact bis(POM)-PMEA or monoester were detected in plasma. Bioavailabilities of PMEA from the prodrug were 24.7 +/- 6.5%, 27.3 +/- 12.3% and 22.2 +/- 15.6% for the HPBCD complex, PEG solution and aqueous suspension, respectively. The oral bioavailability of PMEA from bis(POM)-PMEA was not limited by dissolution rate of the prodrug. Data for the PEG cosolvent solution and suspension indicate that the prodrug could potentially be formulated as a soft gelatin capsule or a tablet.

Immunization of cats with tissue cysts, bradyzoites, and tachyzoites of the T-263 strain of Toxoplasma gondii.

Previous studies have demonstrated that oral administration to cats of tissue cysts of the oocyst-negative mutant strain of Toxoplasma gondii, T-263, induces immunity to oocyst shedding following challenge. Experiments were designed to compare the levels of protection induced by T. gondii T-263 when tissue cysts, bradyzoites released from tissue cysts, and tachyzoites were administered to cats. In 1 experiment, groups of cats received 2 oral doses of intact tissue cysts or released bradyzoites of T. gondii T-263 and were challenged 47 days later with the oocyst-producing strain of T. gondii T-265. All cats seroconverted following immunization and none of them shed oocysts following challenge. In a second experiment, groups of cats received tachyzoites of T. gondii T-263 as a single oral dose and either 1 or 2 intraduodenal doses; they were challenged 60 days after the last vaccination. All cats seroconverted following immunization. Following challenge, all cats shed oocysts except for 2 of 7 cats that received 2 intraduodenal doses of tachyzoites. Thus, orally administered bradyzoites of T. gondii T-263, either contained in intact tissue cysts or liberated from cysts, induced immunity to oocyst shedding. In contrast, tachyzoites did not completely protect against oocyst shedding, even when delivered directly to the duodenum and despite the development of high antibody titers.

Peritoneal coccidioidomycosis associated with human immunodeficiency virus infection.

Peritoneal coccidioidomycosis is extremely rare. This report describes a patient infected with the human immunodeficiency virus who presented with unexplained ascites and was found to have peritoneal coccidioidomycosis. The ascites had a low serum-ascites albumin gradient, and laparoscopy showed peritoneal implants that grew Coccidioides immitis. This case is unique in several ways; this is the first case in which a patient's acquired immunodeficiency syndrome-defining illness was peritoneal coccidioidomycosis, and the serum-ascites albumin gradient determination as well as laparoscopy provided information critical to the diagnosis. This patient's dramatic response to systemic antifungal therapy, as evidenced by resolution of ascites and constitutional symptoms, underscores the importance of timely diagnosis and prompt therapy. In summary, this report reviews the previous cases of coccidioidal peritonitis and reports the first case in which localized peritoneal coccidioidomycosis was the acquired immunodeficiency syndrome-defining illness in a human immunodeficiency virus-infected patient.

Toxoplasmosis.

Toxoplasmosis is a common infection of animals and man, yet remains a rare disease. The disease appears in the offspring of mothers first infected during pregnancy and by relapse of chronic infection in immunodeficient animals and man. Toxoplasmosis can be prevented by the simplest of hygienic measures, such as handwashing. Further control can be achieved by widespread education of the public about the dangers of toxoplasmosis and the methods of prevention. Education of veterinary and zoo workers would reduce transmission in an environment where it is most likely to occur because of high concentrations of cats and cat feces. Toxoplasmosis of sheep and pigs on farms may be amenable to vaccination with the ts-4 strain of Toxoplasma. Finally, the new T-263 toxoplasmosis vaccine for cats offers the possibility of drastically reducing environmental contamination with oocysts, especially on farms. The reduction in numbers of viable oocysts in the environment would eventually reduce the infection of bird and rodent intermediate hosts, and ultimately, the infection of humans.

Tumor necrosis factor alpha mRNA and protein are present in human placental and uterine cells at early and late stages of gestation.

Tumor necrosis factor alpha (TNF-alpha), a polypeptide that regulates cellular growth and modulates the synthesis of various cell surface and secreted molecules, has been identified in the pregnant uterus. To determine which specific cells transcribed and translated this gene, extraembryonic fetal tissues (placenta and membranes) and uterine tissue from early and late stages of gestation were analyzed for TNF-alpha mRNA by in situ hybridization using biotinylated antisense and sense TNF-alpha probes, and for immunoreactive TNF-alpha using two monoclonal antibodies. Tumor necrosis factor-alpha transcripts and protein were identified in both extraembryonic and maternal cells. In first-trimester placental villi, TNF-alpha mRNA was present in syncytiotrophoblast but was low to absent in cytotrophoblast and villous stromal cells. Decidual and epithelial cells in maternal tissues contained TNF-alpha transcripts. In term placentas, both syncytiotrophoblast and villous stromal cells contained TNF-alpha mRNA, and transcripts were present in maternal cells in the decidua adjacent to the extraplacental membranes. In both first-trimester and term tissues, coincident expression of TNF-alpha mRNA and immunoreactive TNF-alpha was demonstrated. The results of this study show that TNF-alpha is synthesized by cells in both extraembryonic membranes and maternal tissues during human gestation and that transcription in specific types of cells is influenced by gestational age. These observations are consistent with a major role for TNF-alpha in the dynamic developmental events of human pregnancy.

Tumor necrosis factor-alpha mRNA and protein in rat uterine and placental cells.

The pregnant uterus contains TNF-alpha, a potent cytokine with pleotrophic effects. The uterus also contains numerous macrophages, which are well described sources of TNF-alpha. In order to determine if uterine TNF-alpha originated with these macrophages, patterns of macrophage tissue distribution and population densities were first established in rat uterine tissues from early, mid, and late stages of gestation by immunohistology. The potential of these and other uterine and placental cells to synthesize TNF-alpha was then tested by in situ hybridization with the use of biotinylated antisense and sense RNA probes. Although TNF-alpha mRNA was present during all stages of pregnancy, hybridization signals were highest in gestation day 15 tissues. The predominant TNF-alpha mRNA-containing cells were uterine epithelium, decidual cells, and placental trophoblast cells; these cells also contained immunoreactive TNF. Transcription of the TNF gene in the uterus and placenta was also documented by Northern blotting experiments, which showed that the transcript sizes for uterine, placental, and macrophage TNF mRNA were similar. Although stromal cells that were located in macrophage-rich uterine compartments (myometrium, metrial gland) contained TNF-alpha mRNA, the cells did not contain high levels of immunoreactive TNF-alpha. Thus, cells other than macrophages are likely to be the major contributors of TNF-alpha during uncomplicated pregnancy in the rat.

Prospective vaccine prepared from a new mutant of Toxoplasma gondii for use in cats.

Kittens are the principal disseminators of Toxoplasma gondii. They can shed greater than 10(8) oocysts in the feces after initial infection with bradyzoites in tissue cysts. Thereafter, most kittens develop protective immunity and do not shed oocysts again if they are reinfected. Bradyzoites of a T gondii mutant, designated T-263, were used to vaccinate kittens. Their use did not result in oocyst shedding, but successfully prevented 84% (31/37) of the kittens from shedding oocysts when challenge exposed with a normal isolate of T gondii. Vaccination of outdoor-roaming cats and kittens would be a useful public health measure to prevent transmission of toxoplasmosis near homes, on farms, and in zoos. It is anticipated that several years will be required for a lyophilized bradyzoite vaccine to be ready for licensing and possible commercial availability.

Human papillomavirus and carcinoma of the urinary bladder.

The human papillomaviruses have been strongly associated with anogenital cancers. Sporadic reports have linked papillomavirus infection to bladder neoplasms. We analyzed 27 normal and malignant bladder tissue samples for the presence of human papillomavirus by in situ DNA hybridization and the polymerase chain reaction. Only one invasive, keratinizing squamous cell carcinoma was found to contain human papillomavirus DNA. This occurred in a 61-yr-old female who was immunocompetent and had no previous evidence of papillomavirus-associated disease. This case represents the first invasive carcinoma of the bladder associated with papillomavirus infection.

Analysis of HLA-G mRNA in human placental and extraplacental membrane cells by in situ hybridization.

Trophoblast cells arising from the implanted blastocyst form the fetal component of the maternal-fetal interface throughout pregnancy. Previous in situ hybridization studies have shown that some subpopulations of these cells, cytotrophoblast cells within and exterior to placental villi, contain class I HLA mRNA. Those studies, performed under moderate conditions of stringency, did not determine which member(s) of the class I HLA gene family was transcribed. In this study, in situ hybridization experiments were conducted under conditions of high stringency using biotinylated antisense and sense RNA probes specific for HLA-G and HLA-E. HLA-G mRNA was identified in first trimester cytotrophoblast cells and in term chorionic membrane cytotrophoblast cells, but was low to undetectable in syncytiotrophoblast of both early and late gestation placentas. Placental villous mesenchymal cells in first trimester but not term placentas contained HLA-G transcripts. HLA-E mRNA was clearly identified only in small round cells present in first trimester decidua and term membranes. These experiments provide the first direct evidence for transcription of the HLA-G gene by cytotrophoblast cells in situ. Expression of this nonpolymorphic gene in place of HLA-A,B,C by trophoblast cells exposed to maternal blood and tissues may allow the juxtaposition of genetically disparate cells required for human pregnancy.

Detection of class I MHC mRNA in subpopulations of first trimester cytotrophoblast cells by in situ hybridization.

Class I MHC mRNA has been identified in both villous and extravillous cytotrophoblast cells in first trimester placentas by in situ hybridization. In this report, we expand those observations to additional morphologically and anatomically distinct subpopulations of trophoblast cells in early placentas using the same experimental approach. In the transition zone of first trimester placental villi, where cytotrophoblast cells are proliferating to form new villi or to migrate into adjacent tissue, both cytotrophoblast cells beneath the uninterrupted syncytium and the proliferating cytotrophoblast cells contained class I mRNA whereas a layer of cytotrophoblast cells proximal to the villus core did not contain class I mRNA. In the placental bed, migrating cytotrophoblast cells contained high levels of class I mRNA as determined by the intensity of staining. In contrast, multinucleated giant trophoblast cells contained little specific message. Alterations in levels of class I mRNA seem therefore to be associated with trophoblast proliferation, migration and differentiation.

Comparison of polymerase chain reaction, DNA hybridization, and histology with viral culture to detect cytomegalovirus in immunosuppressed patients.

Formalin-fixed, paraffin-embedded lung tissue from 34 autopsies and eight open-lung biopsies of bone marrow transplant recipients was analyzed for cytomegalovirus (CMV) infection. The cases were studied by the polymerase chain reaction (PCR), in situ DNA hybridization, and histologic examination. The results were compared with viral culture for CMV taken at the time of biopsy or autopsy. In the autopsy series, hybridization and histology identified CMV in 15% of the cases, whereas PCR detected CMV in 24% of the cases. In the open-lung biopsy cases, both PCR and hybridization were found to be equivalent to culture in detecting CMV. Histology was less sensitive, with the molecular biology methods detecting CMV in 50% of the lung biopsies while histologic examination identified only 25%. Specificity was high (100%) since CMV was not detected in any culture-negative case by either PCR or hybridization. However, PCR, hybridization, and histology failed to identify CMV in three known culture-positive autopsy cases. Overall, PCR and hybridization were found to be more sensitive than histology, and PCR was more sensitive than hybridization for the detection of CMV. The advantage of high sensitivity and specificity combined with more rapid diagnosis (24 to 48 h) compared with viral culture (average, 16 days in this study) makes the molecular biology methods useful adjuncts to histology for detection of CMV in formalin-fixed, paraffin-embedded tissue.

Modified deoxyoligonucleotides stable to exonuclease degradation in serum.

Unmodified deoxyoligonucleotides are rapidly degraded in serum-containing medium. Utilizing internally labelled deoxyoligonucleotides the deoxyribonuclease profile for fetal calf serum and human serum was determined. It was found that the predominate nuclease activity in both systems was 3' exonuclease. Deoxyoligonucleotides are protected from exonuclease degradation in sera and cell media by simple terminal modifications that maintain high binding activity for the complementary DNA sequence.

Identifying acoustic scattering sources in normal renal parenchyma from the anisotropy in acoustic properties.

Acoustical and histological properties of dog kidney parenchyma are examined in vitro to determine sources of acoustic scattering in the normal kidney. The speed of sound, attenuation, backscatter, effective scatterer size and scattering strength were measured within the frequency range 1-15 MHz and at eight angles of incidence with respect to the predominant nephron orientation. Significant angular dependence, or anisotropy, was observed in backscatter coefficient and scattering strength estimates; attenuation was found to be weakly anisotropic. All three parameters, each measured at 19 degrees C, exhibited values that were maximum for perpendicular incidence and minimum for parallel incidence. Speed of sound and scatterer size estimates were observed to be independent of scanning angle. Comparisons between these data for renal cortex and histological observations suggest that the glomerulus is the principal scatterer at low frequencies, and renal tubules and blood vessels at high frequencies.