Small animal jet injection technique results in enhanced immunogenicity of hantavirus DNA vaccines

DNA vaccine evaluation in small animals is hampered by low immunogenicity when the vaccines are delivered using a needle and syringe. To overcome this technical hurdle we tested the possibility that a device developed for human intradermal medicine delivery might be adapted to successfully deliver a DNA vaccine to small animals. Disposable syringe jet injection (DSJI) does not currently exist for small animals. However, a commercialized, human intradermal device used to to administer medicines to the human dermis in a 0.1 mL volume was evaluated in Syrian hamsters. Here, we found that hantavirus DNA vaccines administered to hamsters using DSJI were substantially more immunogenic than the same vaccines delivered by needle/syringe or particle mediated epidermal delivery (gene gun) vaccination. By adjusting how the device was used we could deliver vaccine to either subcutaneous tissues, or through the skin into the muscle. RNA and/or antigen expression was detected in epidermal, subepidermal and fibroblast cells. We directly compared six optimized and non-optimized hantavirus DNA vaccines in hamsters. Optimization, including codon-usage and mRNA stability, did not necessarily result in increased immunogenicity for all vaccines tested; however, optimization of the Andes virus (ANDV) DNA vaccine protected vaccinated hamsters from lethal disease. This is the first time active vaccination with an ANDV DNA vaccine has shown protective efficacy in the hamster model. The adaptation of a human intradermal jet injection device for use as a method of subcutaneous and intramuscular jet injection of DNA vaccines will advance the development of nucleic acid based medical countermeasures for diseases modeled in hamsters.     

Tumor cell adhesion to frozen lymph node sections — a correlate of lymphatic metastasis in breast carcinoma models of human and rat origin

Summary The role of tumor cell adhesion in lymphatic metastasis of breast cancer was investigatedin vitro using a rat mammary carcinoma model of four cell lines with different metastatic phenotypes, two human breast cancer cell lines, and cryostast sections of normal rat or human lymph nodes, respectively. A positive correlation was found between the adhesion levels obtained with three metastatic rat mammary cell lines (TMT-081 > MT-100M & TMT-50) and a non-metastatic line MT-W9B, the latter being 3–4 fold less adhesive to the lymph node sections than the metastatic tumors. This selective adhesion was specific, as it was not found with cryostat sections of rat liver and brain. Enzyme assays indicated that cell surface glycoproteins bearing terminal -galactoside residues were involved in the adhesion of the rat tumors.Adhesion of the human breast carcinoma cells Hs578T to sections of human lymph nodes was significantly higher than that of the normal breast epithelial cell line Hs578Bst, and comparable to adhesion of a second breast carcinoma line, MCF-7. Moreover, Hs578T cells isolated from regional lymph nodes of tumor-bearing nude mice were significantly more adhesive to human lymph node sections than the parental line.Adhesion of both human and rat tumors could be partially blocked by the addition of the synthetic peptide GRGDSPK and by antibodies directed to the 1 chain of integrin, suggesting that an integrin receptor may played a role in the adhesion. The results suggest that tumor cell adhesion to cryostat sections of lymph nodes is a correlate of the malignant phenotype in mammary tumors of diverse origins, and could be used to delineate the adhesion factors mediating lymphatic metastasis.     

Full-Face Laser Resurfacing and Rhytidectomy

The Ultrapulse CO₂ laser (Coherent Inc., Palo Alto, CA, USA) was used in 239 patients, from March 1996 to July 1998, for full-face laser resurfacing. In 106 (43%) of these patients rhytidectomy was performed in the same procedure. All patients submitted to laser resurfacing were prepared for 1 to 2 months beforehand with retinoic acid and hydroquinone. The procedures were done under local anesthesia controlled by an anesthesiologist. A clear film dressing impregnated with silicone gel (Silon TSR; Bio-Med Sciences, Bethlehem, PA, USA) was used for 6 to 7 days and complete healing was observed in 7 to 10 days. Complications were exclusively dermatologic, without relation to surgery. No necrosis of the cutaneous flap was observed. Skin biopsies of 10 consecutive patients undergoing the combined procedures revealed no vascular impairment to the dermis. The patients were able to resume their activities 2 weeks after the procedure.     

Vanishing Psammoma Bodies in the Anterior Pituitary of the Human Newborn: An Immunohistochemical and Histometric Study

Adenohypophyses of human newborns contain characteristic psammoma bodies. Their numbers are maximal within 2 weeks of the neonatal period and diminish thereafter. They are very rare in infant pituitaries, seeming to disappear by shrinkage in that there is a significant direct correlation between their number and size. The bodies were found to contain a high concentration of endogenous peroxidase, thus suggesting that the enzyme may be responsible for their disappearance. A statistical majority of psammoma bodies were located within follicular lumens. By immunohistochemistry, the follicular epithelium surrounding psammoma bodies showed immunoreactivity for various pituitary hormones. Light microscopy demonstrated that adenohypophysial cells surrounding psammoma bodies contain randomly, scattered granules or globules exhibiting peroxidase activity. Extrusion of such granules into follicular lumens may play a role in the genesis of the concretions. The conspicuous lamellar nature of the calcified psammoma bodies suggests that waves of calcium deposition occur during their morphogenesis. Despite histologic similarities, the histochemical characteristics of this type of psammoma body differ from those in other organs as well as from the calcification encountered in prolactin (PRL)-producing pituitary adenomas.     

Effect of intravenous infusion of growth hormone-releasing hormone on the morphology of rat pituitary somatotrophs

The effect of GRH infusion on rat adenohypophysial morphology was studied by light microscopy, immunocytochemistry, in situ hybridization, and electron microscopy. Synthetic rat GRH was intravenously administered by osmotic minipumps at 14.4, 72, 360 and 720 μg/ day/rat for 1 week. In one group treated for 1 week with a daily dose of 720 μg GRH, the rats were killed 7 days after withdrawal of GRH. Control rats in which GRH was replaced by excipient, or those that received no treatment, were included as well. GRH infusion with daily doses of 360 and 720 μg resulted in a significant increase in pituitary weight and weaker GH immunoreactivity compared with other groups. Ultrastructurally, the somatotrophs were increased in size and became sparsely granulated, and the organelles involved in hormone sythesis were very prominent. The intensity of the GH mRNA signal did not differ from control animals, suggesting the desensitization of somatotrophs to GRH. The highest GRH dose induced an increased number of nuclei immunoreactive for proliferation cell nuclear antigen (PCNA). One week after GRH withdrawal, shrinkage of cytoplasm, involution of RER and Golgi complex, and a decrease of cell attachment sites indicated the reversibility of changes induced by GRH. In conclusion, GRH infusion induced, within days, hypertrophy and proliferation of somatotrophs with ultrastructural features of highly stimulated, sparsely granulated cells. Morphological changes were reversible.Endocr Pathol 4:131–139, 1993.     

The circadian rhythm of atrial natriuretic peptide,vasoactive intestinal peptide,beta-endorphin and cortisol in healthy young and elderly subjects

Atrial natriuretic peptide, vasoactive intestinal peptide, beta-endorphin and cortisol are humoral variables characterized by a 24-h periodicity. We evaluated the circadian rhythm of these peptides and hormones in healthy subjects who were young (between 20–25 years) or elderly (between 65–75 years). All were on controlled diets. Blood samples were collected six times during a 24-h period (at 06.00, 08.00, 12.00, 18.00, 20.00 and 24.00 h) beginning 8-h after start of recumbency. The time-related data were analysed by the Cosinor method in order to validate the circadian rhythm and to quantify rhythmometric parameters which included the midline estimate of rhythm (mesor). In contrast to the young subjects, Cosinor analysis failed to reveal a significant circadian rhythm in elderly subjects, for plasma cortisol. In elderly subjects oscillation (mesor) of atrial nutriuretic peptide was higher, while that of vasoactive intestinal peptide and beta—endorphins was lower. The results suggest changes in the physiological secretion of these three peptides in healthy elderly subjects.     

Factor-XII congenital deficiency. A new family study

This report describes two people in a family with Hageman trait (homozygotes) (Factor XII = 0.06%). In addition eight family members were studied to evaluate the inheritance of this congenital deficiency. A study of the Kallikrein-Kininogen system induced by the fragments of Factor XII was carried out. It is concluded that the inheritance is as described by Veltkamp and that the Kallikrein release from the prekallikreinogen (Fletcher factor) "in vitro" is related to the amount of Factor XII procoagulant protein.     

NEOPLASTIC DISEASE AND DELETION 22q11.2: A MULTICENTRIC STUDY AND REPORT OF TWO CASES

Deletion 22q11.2 is a chromosomal abnormality detected in young patients with clinical manifestations of the DiGeorge/velocardiofacial syndrome. Conotruncal heart defects are also associated with del22q11.2. An association of these cardiac malformations with neoplasias has been observed. Our series includes two cases of malignancies, a hepatoblastoma and a renal-cell carcinoma, arising in children with complex cardiac malformations. The aim of the study was to determine if the deletion at 22q11.2 was present and could be responsible for both pathological processes. Del22q11.2 was identified in both cases. Comparative genomic hybridization revealed terminal gains on chromosomes 1q and Xq and terminal loss on 1p in the hepatoblastoma, and gains in 1p, 12q, 16p, 20q, 22q, and whole chromosome 19 and loss of Xq in the renal-cell carcinoma. Our results confirm a common genetic basis for cardiac malformations, and del22q11.2 presents a risk factor for the development of pediatric tumours.     

The pathology of late recurrence of testicular germ cell tumors

A total of 91 men had histologically documented late recurrences of testicular germ cell tumors characterized by a complete response to treatment with a subsequent disease-free interval of at least 2 years and no evidence of a second primary lesion. Ninety percent of the patients for whom information was available received chemotherapy shortly after their initial diagnosis of testicular germ cell tumors; most of the other patients were known to have stage I disease initially. Overall, 60% of patients had teratoma in their late recurrences, including 20 patients (22%) in whom teratoma was the only element. Thus, teratoma was the most common type of neoplasm in late recurrences. Excluding teratoma coexisting with other types of neoplasms, yolk sac tumor was the most frequent type of tumor in patients with late recurrence. It occurred in 47% of patients, either alone or with teratoma, another nonteratomatous germ cell tumor type, or a "nongerm cell malignant tumor." Unusual types of yolk sac tumor, including glandular, parietal, clear cell, and pleomorphic patterns, were seen frequently in late recurrences and often raised differential diagnostic problems with "nongerm cell" carcinomas. A smaller number of late recurrences consisted of other types of neoplasms. Twenty percent of patients with late recurrence had a nonteratomatous germ cell tumor other than yolk sac tumor, either alone, with yolk sac tumor, or with a "nongerm cell malignant tumor." Most of these nonteratomatous germ cell tumors other than yolk sac tumor were embryonal carcinoma, although rarely seminoma and choriocarcinoma were encountered. "Nongerm cell malignant tumors," including both sarcomas and carcinomas of various types, occurred in 23% of late-recurrence patients, either alone or with a nonteratomatous germ cell tumor. Late recurrences were seen in many different sites in these patients, including the retroperitoneum, abdomen, pelvis, liver, mediastinum, lung, bone (femur, vertebra, and rib), lymph nodes outside the retroperitoneum and mediastinum (supraclavicular, neck, and axillary regions), scrotum and inguinal regions, adrenal gland, chest wall, and buttocks. Follow-up data were available for 79 of the 91 patients studied. Duration of follow-up ranged from 2 months to 13 years after the patient's first late recurrences; the mean length of follow-up was 4.8 years. Patients whose late recurrences consisted of teratoma only had the most favorable outcomes, with 79% having no evidence of disease at last follow-up. Patients whose late recurrences consisted of pure "nongerm cell malignant tumor" or pure germ cell tumor (yolk sac tumor or other types) had a much worse prognosis: Only 36% to 37% were alive with no evidence of disease. Patients with two different types of nonteratomatous malignancies in their late recurrences had a dismal clinical course: Only 17% with both yolk sac tumor and other nonteratomatous germ cell tumor had no evidence of disease, whereas no patient with both nonteratomatous germ cell tumor and "nongerm cell malignant tumor" was disease free. Late recurrences consisting of teratoma alone often have a favorable outcome, but the prognosis in all other patients is poor. Furthermore, late recurrence is not likely to respond to chemotherapy and is best treated by surgical excision when possible.     

Alpha1 acid glycoprotein binds to imatinib (STI571) and substantially alters its pharmacokinetics in chronic myeloid leukemia patients.

PURPOSE: Imatinib (Glivec) is a potent inhibitor of bcr/abl, an oncogenic fusion protein that causes chronic myelogenous leukemia (CML). alpha1 acid glycoprotein (AGP) binds to imatinib with high affinity and inhibits imatinib activity in vitro and in vivo in an animal model. A pharmacokinetics analysis of imatinib was undertaken in CML patients. EXPERIMENTAL DESIGN: Imatinib plasma concentrations were measured in 19 CML patients treated with imatinib (400 or 600 mg/day). Five patients received a concomitant short-term course of clindamycin (CLI). RESULTS: A positive correlation between AGP and imatinib plasma levels was observed. CLI administration decreased imatinib plasma concentrations, evaluated as area under the curve (AUC) and peak concentrations (C(max)). The effects of a bolus of CLI was studied in three patients on imatinib 23 h after the last imatinib dose. Within 5-10 min in three of three cases, CLI caused a decrease in imatinib plasma concentrations of 2.6-, 2.7-, and 4.7-fold, respectively. In vitro experiments using fresh blasts from CML patients showed that AGP, at concentrations observed in the patients, decreased imatinib intracellular concentrations up to 10 times and blocked imatinib activity. The incubation with CLI restored imatinib intracellular concentrations and biological activity. CONCLUSION: AGP exerts significant effects of the pharmacokinetics, plasma concentrations, and intracellular distribution of imatinib in CML patients; these data indicate that plasma imatinib levels represent unreliable indicators of the cellular concentrations of this molecule.