Observations on tissue grafts established in rabbit ear chambers. A combined light and electron microscopic study.

An in vivo microscopic and ultrastructural study of tissues transferred to the transparent rabbit ear chamber is presented. Fragments of liver, kidney, thyroid, and myometrium were successfully auto- or allografted into the chamber from donors of all ages and allowed continuous in vivo observation of parenchymal structure and function, as well as of the graft vasculature which plays such an active role during rejection. Circulation was quickly reestablished by anastomosis of graft vessels to those of the ear chamber membrane and only minor reversible parenchymal changes occurred. Both vessels and parenchyma retained the characteristics of the organs of origin on both light and electron microscopy and were observed functioning in vivo for periods up to 1 yr in the case of autografts, and until rejection occurred in allografts. In the latter case, rejection did not occur in tissues obtained from adult and neonatal donors for nearly 3 months, while tissues of fetal origin were generally rejected much earlier. The kidney grafts provide a unique opportunity for a close comparative study of mammalian fetal and adult glomerular blood flow under varying rates of perfusion, and the tubular epithelium could be observed regenerating after initial acute tubular necrosis. Renal tubules from fetal, neonatal, and adult donors were all capable of metaplastic change to form a highly ciliated epithelium. Grafts of these tissues will allow the fine detail of the processes of rejection to be studied continuously in vivo.

A study of allograft kidney rejection occurring simultaneously in whole organ and ear chamber grafts in the rabbit.

When portions of adult renal tissue are allografted into the rabbit ear chamber, they usually survive for periods of up to several months (6). When a kidney from the same donor is grafted as a whole organ, the ear chamber grafts then reject with the whole organ in 7 days. During that time serial needle biopsies of the whole organ are compared with the in vivo appearance of the ear chamber grafts. This establishes that the changes occurring in the ear chamber grafts are monitoring the rejection process proceeding in the whole organ grafts. Dramatic vascular changes herald the earliest stages of unmodified rejection. A highly characteristic form of individual discrete platelet adhesion to both endothelium and adherent leukocytes is observed which is associated with the release reaction. At times as many as 20 such discrete platelets are clearly visible in profile in one high-power field. This demonstrates in vivo a mechanism whereby vascular and parenchymal damage may be produced by platelet contents, without previous aggregation or thrombus formation being necessary.

Glomerular lesions after renal transplantation.

Significant changes in glomeruli on light microscopy has been observed in 27 of 109 cadaveric renal allografts which functioned beyond 6 months. Tissue was available for study from all but two allografts. The histologic lesions were classified as follows: recurrent glomeruloneophritis, 9 cases (3 focal scierosis, 2 mesangial immunoglobulin A[IgA] disease, 2 mesangiocapillary glomerulonephritis, 1 dense deposit disease, 1 familial nephritis); de novo glomerulonephritis, 1 case (diffuse proliferative glomerulonephritis with crescents); and glomerular change of uncertain etiology, 17 cases (10 mesangiocapillary, 5 focal scierosis, 1 focal proliferative and 1 mesangial proliferative). These lesions were not distinguishable on light, fluorescent and electron microscopy from those in patients with spontaneous renal disease. All patients with glomerular lesions had proteinuria, and all but 3 had microscopic hematuria. Glomerular lesions were not significantly associated with early clinical rejection episodes or HLA compatibility. Presensitization of HLA antigens was significantly related to the occurence of a nonrecurrent glomerular lesion. Vescoureteral reflux was significantly more frequent in those with glomerular change (14 of 24) than in those without (13 of 48). Glomerular lesions were associated with a higher rate of graft loss due to renal transplant failure; renal function in survivors was significantly worse than in those without glomerular lesions.

Extending the lifetime of anticoagulant oligodeoxynucleotide aptamers in blood.

We have investigated (123)I and (125)I DNA aptamer analogs of anticoagulant DNA aptamers to thrombin exosite 1 and exosite 2 for thrombus imaging potential. Two severe problems are rapid clearance from circulating blood and blood nuclease. With aptamers (unlike antisense) the nucleotide analogs used in polymerase chain reaction-selection cycles also must be used in the radiotracer. We investigated 3'-biotin-streptavidin (SA) bioconjugates of the aptamers to alleviate these problems. Blood nuclease assays and biodistribution analysis were used in the mouse and rabbit. We found that 3'-biotin protected the aptamers significantly from blood nuclease in vitro, but it did not slow in vivo clearance. In contrast, the 3'-biotin-SA bioconjugates were resistant to blood nuclease in vitro and were also longer-lived (10-20 times) in vivo. Bioconjugate aptamers retained affinity for thrombin. Two solutions emerge: 1) In noncirculating blood (within a thrombus) 3'-biotin extends aptamer lifetime, whereas 2) in circulating blood (the transport medium), where more aggressive clearance is encountered, 3'-SA extends aptamer lifetime.

Loin pain as a presenting symptom in idiopathic glomerulonephritis.

Loin pain may be a major presenting symptom in patients with glomerulonephritis. Most of these patients show an underlying focal and segmental proliferative glomeruloneyphritis and there may be associated deposits of IgA and Igg in the mesangium. In this group of patients, vascular lesions are often prominent in the absence of hypertension. Episodes of recurrent macroscopic hematuria also occur, but the pain cannot be attributed to colic due to blood clots in the ureter.

Intraepithelial and invasive neoplasia of the vulva in association with human papillomavirus infection.

Colposcopic examination of 335 women with cytologically detected human papillomavirus (HPV) revealed involvement of the cervix in 316 patients (94%), vagina in 276 (82%) and vulva in 148 (44%). A symptom complex of pruritus and superficial dyspareunia was found in 98 of the 148 patients with vulvar infection (66%). Histologic examination revealed HPV-associated vulvar intraepithelial neoplasia (VIN) in 11 of 148 biopsies (7.4%). Follow-up of the patients with HPV infection with or without VIN showed a spontaneous regression rate of 56% but also demonstrated progression to VIN 3 in two patients and to invasive carcinoma of the vulva in one.

Neonatal whole blood hyperviscosity: the important factor influencing later neurologic function is the viscosity and not the polycythemia.

The neurologic outcome of 23 seven-year old children who had cord blood hyperviscosity was compared with that of children with normal cord blood viscosity in a randomised, controlled and blinded study. Viscosity was measured using a coaxial narrow-gap couette viscometer. Sixteen (69.6%) of the children with hyperviscous cord blood had a disability; this incidence being three times greater (22.7%) than in children whose cord blood was not hyperviscous (P < 0.01). In three children with cord-blood hyperviscosity, the disability was severe. No child had a severe disability with normal cord blood viscosity. Of the eight children whose cord blood was hyperviscous, but not polycythemic, six (75.0%) had a disability and in one child the disability was severe. These results demonstrate an association between cord blood hyperviscosity and later neurologic development. Cord studies are non-invasive and result in the rapid diagnosis of the neonatal hyperviscosity syndrome, so allowing earlier treatment. This may be crucial in altering the effects of hyperviscosity on the developing brain in the early neonatal period. Because the neurologic outcome of children was similar whether polycythemia was present or not, the prime factor was the viscosity and not the hematocrit level. We suggest it may be necessary to perform cord blood viscosity studies routinely.

Human papilloma virus infection and associated intraepithelial neoplasia of the cervix, vagina and vulva.

A prospective colposcopic and histological study was performed on 333 patients with cytologically detected human papilloma virus infection (HPV) without cytological evidence of associated intraepithelial neoplasia. The colposcopic changes seen in the cervix, vagina and vulva were documented and target biopsies were taken from abnormal areas in these 3 sites. Colposcopic and histological evidence of HPV infection was found in the cervix of 314 patients (94%), vagina in 274 patients (82%) and vulva in 146 patients (44%). Histologically proven cervical intraepithelial neoplasia was found in 94 of 333 patients biopsied (28%), vaginal intraepithelial neoplasia in 3 of 333 patients biopsied (0.9%) and vulval intraepithelial neoplasia in 9 of 146 patients biopsied (6.2%). This study identified the colposcopic changes of HPV infection to be widespread throughout the lower female genital tract. The diagnosis of intraepithelial neoplasia undetected by cytology in 28% of patients with HPV infection indicates that such patients require colposcopy and target biopsy according to the colposcopic findings.

Morphologic evidence that analgesic-induced kidney pathology contributes to the progression of tumors of the renal pelvis.

BACKGROUND: Whether phenacetin-containing analgesics cause renal pelvic tumors by virtue of the weak mutagenicity of phenacetin, or indirectly through local effects of analgesic-induced renal papillary scarring, is debated. Because phenacetin consumption ceased in New South Wales, Australia in 1975, cases of renal pelvic carcinoma seen 14-15 years later (many of which were associated with long-standing analgesic-induced renal papillary pathology) provided an opportunity to examine the temporal relation between phenacetin exposure and those histologic characteristics of the tumors and adjacent renal tissue that may implicate analgesics in their etiology. METHODS: The authors conducted a "blinded" histopathologic review of tumors of the renal pelvis and adjacent noncancerous renal tissue from 100 cases for which epidemiologic data regarding risk factor exposure (specifically phenacetin-containing analgesics, tobacco, infection, and kidney stones) had been obtained in a population-based case-control study from New South Wales in 1989 and 1990. RESULTS: A history of consumption of phenacetin-containing analgesics was associated strongly with the presence and severity of diffuse renal papillary scarring, and less strongly with papillary calcification. The histologic grade of the renal pelvic tumors tended to rise significantly with consumption of phenacetin-containing analgesics in a dose-dependent fashion and with the degree of papillary scarring, but was not related to smoking. In multivariate analysis it was the degree of papillary scarring (to a greater extent than the amount of phenacetin consumption) that was associated significantly and strongly with a higher histologic grade. Only diffuse papillary calcification was associated significantly with squamous change in the renal pelvic tumors. CONCLUSIONS: Based on the results of the current study, the authors conclude that 1) in phenacetin-related tumors of the renal pelvis, the presence and severity of analgesic-induced renal papillary scarring correlates with tumor progression and 2) papillary calcification is a risk factor for squamous change in renal pelvic urothelioma.

Affinity labelling of tRNA nucleotidyltransferase from baker's yeast with tRNAPhe modified on the 3'-terminus.

2'-Deoxy-2'-amino-cytidylic acid can be incorporated into position 75 of tRNAPhe from yeast by tRNA nucleotidyltransferase yielding tRNAPhe-C-C(2'NH2). tRNAPhe-C-C(2'NH2) can be reacted with the N-hydroxysuccinimide esters of bromoacetic acid of mercuriacetic acid to yield the derivatives tRNAPhe-C-C(2'NHCOCH2Br) and tRNAPhe-C-C(2'NHCOCH2Hg+OH-). Each of these reactive tRNAs inactivates tRNA nucleotidyltransferase from yeast with similar kinetics. The enzyme can be protected against inhibition by its substrates tRNAPhe-C and tRNAPhe-C-C as well as ATP and CTP. A covalent, isolatable 1:1 complex between tRNAPhe-C-C(2'NHCOCH2Br) and the enzyme was formed, but could not be found when the enzyme had previously been inactivated with p-hydroxymercuribenzoate.

Diabetic vasculopathy in the rat: an in vivo microscopic study of the circulation.

A transparent tissue chamber adapted to the rat back was used for in vivo observation of vascular and circulatory changes in the streptozotocin diabetic rat. The chamber was implanted in a functional and richly vascularized bed of striated muscle. Extensive red cell rouleaux formation was observed within 2-7 days of induction of hyperglycaemia and was closely followed by development of constrictions and dilatations in both venules and arterioles. In 3 of 7 diabetic rats saccular microaneurysms, similar in form to those reported in diabetic animals and humans, developed in the vascular bed. The rat back chamber provides an opportunity for continuous in vivo study of vascular lesions developing in streptozotocin diabetes. Fixation under microscopic observation allows close correlation of functional changes in vivo with the ultrastructural appearance of specific tissue areas.

Specificity of elongation factor Tu from Escherichia coli with respect to attachment to the amino acid to the 2' or 3'-hydroxyl group of the terminal adenosine of tRNA.

Modified Tyr-tRNATyr and Phe-tRNAPhe species from yeast having the aminoacyl residue bound specifically to the 2' and 3' position of the terminal adenosine, respectively, were investigated for their ability to form ternary complexes with Escherichia coli elongation factor Tu and GTP. Both Tyr-tRNATyr-CpCpA (2'd) and Tyr-tRNATyr-CpCpA(3' d) derivatives which are esterified with the amino acid on the 3' and 2' position respectively and which lack the vicinal hydroxyl were able to form ternary complexes. The stability of these ternary complexes was lower than in the case of native Tyr-tRNATyr-CpCpA. Tyr-tRNATyr-CpCpA(3' d) having the amino acid attached to the 2' position interacted considerably more strongly with EF-Tu - GTP than Tyr-tRNATyr-CpCpA(2' d). Ternary complex formation was observed with neither Phe-tRNAPhe-CpCpA(2'NH2) nor Phe-tRNAPhe-CpCpA(3'NH2). It is concluded that 2' as well as 3' isomers of native aminoacyl-tRNA can be utilized for ternary complex formation but in a following step a uniform 2'-aminoacyl-tRNA - EF-Tu - GTP complex is formed. Although the free vicinal hydroxyl group of the terminal adenosine is not absolutely required, replacement of the ester linkage through with the amino acid is attached to tRNA by an amide linkage leads to loss of ability to interact with elongation factor Tu.

Cytodiagnosis of pancreatic and biliary cancer by endoscopic duct aspiration.

Duct aspiration was performed immediately after endoscopic retrograde cholangiopancreatography (ERCP) in 33 patients with ductal changes which were suggestive of pancreatic or biliary cancer. Malignant cells were identified in 10 of 17 patients (60%) with cancer of the pancreas, and in two of 10 patients (20%) with cancer of the biliary tract. Atypical cells were identified in additional patients with cancer, but cytological characteristics did not permit a definitive diagnosis. No false positive diagnoses were made. Duct aspiration is a relatively simple extension of ERCP, and enhances diagnostic accuracy, particularly in patients with carcinoma of the pancreas.

An autoclavable glucose biosensor for microbial fermentation monitoring and control.

The design, construction, and characterization of a prototype-regenerable glucose biosensor based on the reversible immobilization of glucose oxidase (GOx) using cellulose binding domain (CBD) technology is described. GOx, chemically linked to CBD, is immobilized by binding to a cellulose matrix on the sensor-indicating electode. Enzyme immobilization can be reversed by perfusing the cellulose matrix with a suitable eluting solution. An autocavable sensor membrane system is employed which is shown to be practical for use in real microbial fermentations. The prototype glucose biosensor was used without failure or deterioration during fed-batch fermentations of Escherichia coli reaching a maximum cell density of 85 g (dry weight)/L. Medium glucose concentration based on sensor output correlated closely with off-line glucose analysis and was controlled manually at 0.44 +/- 0.2 g/L for 2 h based on glucose sensor output. The sensor enzyme component could be eluted and replaced without interrupting the fermentation. To our knowledge, no other in situ biosensor has been used for such an extended period of time in such a high-cell-density fermentation.

Synthesis and radioiodination of a stannyl oligodeoxyribonucleotide.

Synthesis and radioiodination of a stannyl oligodeoxyribonucleotide were undertaken to evaluate a gamma ray emitting ODN ligand for thrombus imaging in vivo . Synthesis of the ODN was based on modified automatedbeta-cyanoethyl phosphoramidite chemistry with an organotin nucleoside (dU*) coupled to a thrombin binding aptamer sequence to give d(U*GGTTGGTGTGGTTGG). The synthesis accommodated dU*, which is destannylated by iodine or acids. Fourteen standard synthesis cycles were followed by one 'stannyl synthesis cycle', distinguished by Fmoc protection, omission of capping, oxidation by an organic peroxide and cleavage by ammonium hydroxide. The organotin nucleoside phosphoramidite {5'-[fluorenylmethoxycarbonyl]-5-(E)-[2-tri-n -butylstannylvinyl]-2'-deoxyuridine-3'-(2-cyanoethyl N,N-diisopropyl phosphoramidite)} was prepared from 5-iodo-2'-deoxyuridine. A customized mild rapid workup included deprotection with methylamine, and reverse phase HPLC with CH3CN/triethylammonium bicarbonate. Pure stannyl ODN was highly retained by reverse phase HPLC. Radioiodination of stannyl ODN (100 microg) provided 123I-labeling yields up to 97%. Five alternative oxidants were effective. High specific activity [123I]- ODN (15 000 Ci/mmol) was recovered, separated from unlabeled isomers. Excellent reverse phase HPLC resolution of ODN isomers (alternatively I, Cl, H or Br in vinyl deoxyuridine) was essential. The affinity of the iodovinyl aptamer analog (Kd = 36 nM) for human alpha-thrombin was similar to the native aptamer (Kd = 45 nM).

Identification of small cells in fetal and infant adipose tissue.

In developing adipose tissue, cells of size less than 25 micron may make a major contribution to adipose tissue cell populations. This study reports the separation, identification, and sizing of infant and foetal adipose tissue small cells. Subcutaneous adipose tissue was taken from the anterior abdominal wall of children aged 2 months and 22 months. Subcutaneous tissue was also obtained from a paraumbilical site in 4 fetuses of gestational age 16 weeks. Tissue samples were fixed in buffered osmium tetroxide solution containing collidine. Aliquots of cell preparations suspended in saline on microscope slides were viewed and separated using a dissecting microscope. A diameter distribution of particle size was obtained using at least 200 cells for each preparation. There was a substantial number of cells that were smaller than 20 micron. Their modal cell size was remarkably constant in fetuses and in 2-month-old and 22-month-old infants. Ultrastructural studies show that the tissue is composed of a relatively uniform population of cells within sparsely collagenous connective tissue. Although some of the cells show no intracytoplasmic aggregates of lipid material, others show variable amounts of lipid material which can occupy a very large part of the cell volume.

Cancer of the pancreas in young adults.

Two young women with cancer of the pancreas are described. The rising incidence and epidemiology of this cancer is discussed. The need for tissue diagnosis and some new aspects of treatment of pancreatic cancer relevant to these cases are presented.

Cord whole blood hyperviscosity: measurement, definition, incidence and clinical features.

Cord whole blood viscosity and haematocrit values (PCV) were determined in 2461 live birth infants. Viscosity measurements were performed on an Australian-designed coaxial narrow-gap viscometer. Normal viscosity values were determined for each week of gestation above 34 weeks. Hyperviscosity was defined as a viscosity value above 2 s.d. from the mean for each week of gestation and it occurred in 164 (6.7%) newborn infants. Although a close relationship existed between cord whole blood viscosity and PCV (r = 0.6597, P less than 0.0001), only 47.4% of polycythaemic infants (PCV greater than 65) were also hyperviscous and only 23.9% of hyperviscous infants were also polycythaemic. Hence, using the haematocrit to select which infants require viscosity studies fails to detect many hyperviscous newborn infants. Hyperviscosity was less common (3.6%, P less than 0.001) in infants who were born by Caesarean section and more common (16.5%, P less than 0.001) in those who were growth retarded. Of the hyperviscous infants, 84.5% were not growth retarded, most (87.8%) were term and most (86.6%) were delivered vaginally. Most hyperviscous newborn infants may thus remain undetected unless routine whole blood viscosity studies are performed.

The pathogenesis of hypertensive vascular changes in the rat: microscopic and ultrastructural correlation in vivo.

1. This study describes the changes observed in the rat microcirculation in vivo in the transparent tissue chamber of the rat back, during the florid hypertension produced by ligation of the rat aorta between both renal arteries. 2. Besides providing a new model for analysis of the action of therapeutic agents in hypertension, direct evidence of the variation in blood flow in hypertension is discussed. Fixation of the chamber tissues allows direct ultrastructural correlation with the altered function of particular vessels observed and recorded cinemicroscopically in vivo.