Variability in duration of follow up may bias the conclusions of cohort studies of patients with patent foramen ovale.

Closure of patent foramen ovale (PFO) is expected to prevent paradoxical emboli. In the absence of randomized trials, its efficacy has been assessed by comparing uncontrolled cohort studies of medically treated patients with those treated by PFO closure. The objective of this study was to highlight a confounder of such studies, namely, the variability in the duration of follow-up. We searched the literature for cohort studies of patients with ischaemic strokes, including those with PFO. During the first year of follow-up, recurrence hazards in patients younger than 55 years were 1-4% in those with any ischaemic stroke, 1-6% in medically treated patients with PFO and 0-5% in those after PFO closure. In most studies, the recurrence hazards were highest immediately after the index stroke and declined thereafter. Still, hazards were commonly reported in terms of annual averages over a wide range of follow-up periods for the various cohort studies, thereby ignoring the possibility that the duration of the follow-up may in and of itself affect the derived average recurrence hazards. A disregard of the time variance of stroke recurrence may confound the conclusions from comparisons between uncontrolled studies of patients with stroke and PFO.

The time horizons of formal decision analyses.

Clinical decision analyses use time horizons that vary from hours to the patient's entire life. Analyses of decisions with a lifetime horizon commonly use Markov models, which simulate the patient's lifespan by dividing it into equal periods (cycles). At each cycle, the model exposes a hypothetical cohort to the competing hazards of normal aging and of the disease in question (disease-specific hazards), and the results are presented as years of life expectancy. This paper highlights two limitations of lifetime Markov models that have been ignored in recent publications. First, since there are no readily available data on changes in disease-specific hazards over time, these hazards are often derived from short-term follow-up studies, and assumed to be constant over the patient's entire life. Second, results may be better presented in terms of health states (i.e. proportions of patients expected to recover completely, recover with a disability or die) rather than life expectancy. Although well-known, these two limitations require re-emphasis. They may be avoided by restricting the time horizon of decision analyses and presenting results as health states as well as life expectancies. When a lifetime horizon is necessary, the performance of Markov models may be improved by the using of time-variant disease-specific hazards derived from long-term follow-up studies, or from theoretical models that simulate more closely the disease progression over time, rather than assuming constant disease-specific hazards.

The 'dark nucleus' and disruptions of follicular architecture: possible new histological aids for the diagnosis of the follicular variant of papillary carcinoma of the thyroid.

AIMS: In order to facilitate the diagnosis of malignancy in solitary thyroid nodules which are non-invasive low-grade tumours, i.e. follicular variant of papillary carcinoma (FVPC) for which few histological discriminators exist, a search was made for additional diagnostically useful histological features. METHODS AND RESULTS: Haematoxylin and eosin (H & E)-stained sections of 70 resection specimens of solitary thyroid nodules were re-evaluated by a panel of three pathologists, and their consensus and original diagnoses compared. In addition, H & E- and periodic acid-Schiff-stained sections were evaluated for various histological features and sections were stained by various immunohistochemical markers to evaluate their discriminative powers. The above features were also assessed in a group of 24 papillary carcinomas of the thyroid (PTCs) associated with regional metastases. The finding of 'Orphan Annie eye' nuclei was the best indicator of malignancy, and was closely related to the presence of nuclear grooves and cells with dense, dark nuclei. In addition, distorted follicular architectural features, i.e. 'interconnecting cell masses' and 'fenestration', were also significant indicators of malignancy. Tumours diagnosed as FVPCs had a significantly lower incidence of associated lymph node metastases than the classical PTCs. CONCLUSIONS: The use of optically clear nuclei as a diagnostic criterion when found only focally may not be sufficiently stringent in distinguishing FVPCs from follicular adenomas. When classical histological indicators of malignancy are equivocal, the diagnosis of FVPC may be facilitated by the above-mentioned features.

Teaching doctor-patient interviewing skills using an integrated learner and teacher-centered approach.

BACKGROUND AND OBJECTIVE: We describe an approach for the resolution of difficulties that some preclinical medical students appeared to have when acquiring patient interviewing skills. SETTING: Two medical schools in Israel. TYPE OF STUDY: Descriptive. OBSERVATIONS: Students' difficulties were related to the inconsistency between the patient-centered approach that was emphasized in the preclinical teaching programs and the disease-centered (biomedical) approach that was practiced on the wards. Others were confused by ambiguous vocabulary and by the multiplicity of rules that they had to remember. Still others appeared to resent attempts to teach them what they thought was elementary courtesy, to reject counterintuitive interviewing rules, and to be bored by the repetitive nature of the practice sessions. TEACHING INTERVENTION: We used an integrated learner- and teacher-centered approach, which is based on the premise that students learn more effectively when autonomous and self-motivated than when responding to instructions from others. Rather than the students being lectured, they were asked to identify the problems in doctor-patient communication and to propose solutions. We conducted live demonstrations of patient- and disease-centered interviews and encouraged students to discuss the advantages and disadvantages of each of them. Lastly, we supervised students as they interviewed patients with increasingly difficult communication problems. CONCLUSIONS: The described approach is consistent with current theories of adult learning. It permits the instructor's input and also supports students' autonomy in identifying and resolving problems in patient interviewing and in choosing the balance between patient- and disease-centered interviewing styles according to the patient's needs. The feasibility of our approach is conditional on the availability of instructors who feel comfortable conducting group discussions, are familiar with the literature on doctor-patient relations, and are experienced enough to demonstrate different interviewing techniques using live patients.

Characterization and distribution of an oncofetal antigen (M2A antigen) expressed on testicular germ cell tumours.

M2A antigen is an oncofetal antigen associated with germ cell neoplasia, present in testis on fetal gonocytes and re-expressed on carcinoma in situ (CIS) and germ cell tumours. We developed a panel of monoclonal antibodies (mAb), M2A (IgG2a), D1-26 (IgG2b) and D2-40 (IgG1), to this antigen in order to characterize its structure and study its distribution among germ cell tumours. M2A antigen was purified by sequential lectin and antibody affinity chromatography and characterized as a monomeric M, 40 000 surface sialoglycoprotein, extensively glycosylated with O-linked carbohydrate structures, but devoid of N-linked sugars. Terminal sialic acid residues were required for reactivity with mAb M2A and D1-26, but not D2-40. Sections of 69 testicular germ cell tumours, fixed in formalin and embedded in paraffin, were stained with mAb D2-40 to examine the distribution of M2A antigen. Uniform membrane staining was observed in seminomas, and focal staining in 69% of embryonal carcinomas, 29% of teratomas and 25% of yolk sac tumours. CIS in the vicinity of all germ cell tumours also displayed uniform membrane staining. The characterization of M2A antigen, and the development of mAb which react with it in conventionally preserved archival specimens, provide important initiatives to study the origin and progression of germ cell neoplasia.

Treatment of X-linked hereditary nephritis in Samoyed dogs with angiotensin converting enzyme (ACE) inhibitor.

X-linked hereditary nephritis (HN) in Samoyed dogs is a model for human HN (Alport's syndrome). Angiotensin converting enzyme (ACE) inhibitors have been shown to slow the progression of renal disease in animal models and human patients. To determine the effect of ACE inhibitor treatment on X-linked HN in Samoyed dogs, a group of affected and a group of normal males were each randomly divided into two subgroups, which were either treated with an ACE inhibitor or left untreated. ACE inhibitor treatment caused significant increases (P < 0.05) in plasma renin activity in normal and affected dogs, confirming its effectiveness, but did not lower systemic blood pressure. Three of four affected treated dogs had improved weight gains and, overall, treated dogs survived 1.36 times longer than affected untreated dogs (P < 0.05). ACE inhibitor treatment of affected dogs significantly delayed (P < 0.05) the onset of an increase in serum creatinine concentration, tended to delay the decline of glomerular filtration rate and effective renal plasma flow (ERPF), significantly improved (P < 0.05) the ERPF at 110-154 days of age, and significantly slowed (P < 0.01) the rate of increase of proteinuria. Affected treated dogs showed a significant (P < 0.05) transient reduction in glomerular basement membrane splitting. Thus, ACE inhibitor treatment of Samoyed dogs with X-linked HN produced beneficial effects with respect to renal function, renal structure, and survival.

A novel anti-seminoma monoclonal antibody (M2A) labelled with technetium-99m: potential application for radioimmunoscintigraphy.

OBJECTIVE: To study the potential usefulness of monoclonal antibody (mAb) M2A specific for seminoma to image tumour nodules in a preclinical nude mouse model. MATERIALS AND METHODS: MAb M2A was labelled with technetium-99m (99mTc) following reduction and was administered intraperitoneally to nude mice bearing subcutaneous HEY cell xenografts against which the antibody was originally raised. Biodistribution and gamma scintigraphy studies were performed 24 h after administration of 99mTc-M2A. RESULTS: Biodistribution studies showed specific targeting of 99mTc-M2A to HEY tumours in comparison with control mAb 99mTc-6E8 and 99mTc-2G3 which do not bind to HEY cells. Subcutaneous HEY cell tumours (0.5-1.0 g) were successfully imaged using gamma-scintigraphy following administration of 99mTc-M2A. CONCLUSION: The results of this study indicate the potential usefulness of 99mTc-M2A as a clinical reagent for imaging seminoma metastases.

Canine X chromosome-linked hereditary nephritis: a genetic model for human X-linked hereditary nephritis resulting from a single base mutation in the gene encoding the alpha 5 chain of collagen type IV.

Many families with X-chromosome linked hereditary nephritis (HN) have mutations in the gene on the X chromosome that codes for the alpha 5 chain of collagen type IV. Canine X-linked HN is an animal model for human X-linked HN. To study the alpha 5(IV) gene in this model, we used the nucleotide sequence published for the human alpha 5(IV) cDNA to construct sets of primers covering approximately 95% of the complete cDNA. cDNA from both affected and normal dog kidneys was amplified by PCR in nine overlapping regions. The nucleotide sequence encoding the noncollagenous domain NC1 hybridized to the human X chromosome and was 93% identical at the DNA level and 97% identical at the protein level to the human alpha 5(IV) NC1 domain, confirming that the canine alpha 5(IV) cDNA had been amplified. Sequence analysis of the alpha 5(IV) cDNA detected a single nucleotide substitution, G-->T, in affected dogs, changing a codon for a conserved glycine residue (GGA) to a stop codon (TGA). When genomic DNA was amplified, the same abnormality was found in exon 35. Using the canine NC1 domain cDNA as a probe for Northern analysis, two transcripts of approximately 8.6 kb and approximately 6.7 kb were identified in both normal and affected male dog kidney RNA. However, the abundance of both transcripts was decreased by a factor of approximately 10 in the affected dog. These results establish at the molecular level that canine X-linked HN is a model for human X-linked HN. This model provides an opportunity to determine the efficacy of new therapies and to investigate the role of the alpha 5(IV) chain in type IV collagen assembly.

Targeting of [111In]biocytin to cultured ovarian adenocarcinoma cells using covalent monoclonal antibody-streptavidin conjugates.

Three monoclonal antibodies (mAb) directed against the human ovarian adenocarcinoma cell line HEY, were substituted with maleimide and covalently bonded to thiolated streptavidin. The conjugates were separated from unreacted reagents by successive affinity chromatography on protein A-Sepharose and iminobiotin columns. Purified conjugates consisted of an immunoglobulin (Ig) monomer bound to a streptavidin tetramer through a covalent bond between the Ig molecule and one of the streptavidin subunits. The conjugates were able to specifically target [111In]biocytin to HEY cells in vitro in the presence of human serum and ascitic fluid from ovarian cancer patients.

Senior-Loken syndrome. Case reports of two siblings and association with sensorineural deafness.

Two siblings with Senior-Loken syndrome are described. The need for a full evaluation of renal function and hearing in children with a retinal dystrophy is emphasised.

Production of anti-NC1 antibody by affected male dogs with X-linked hereditary nephritis: a probe for assessing the NC1 domain of collagen type IV in dogs and humans with hereditary nephritis.

Some patients with hereditary nephritis (HN) who have received a renal transplant have been shown to form antibody with specificity for the NC1 domain of collagen type IV, a major constituent of glomerular basement membranes (GBM). We attempted to duplicate this phenomenon in a family of dogs with X-linked HN, a model for human X-linked HN, by immunizing affected male dogs with normal dog NC1 domain. A collagenase digest was prepared from normal dog GBM, the NC1 domain was separated into dimer (approximately 50 kDa) and monomer (24 kDa and 26 kDa) components by SDS-PAGE, and injected into two affected male dogs. Antisera obtained from both dogs contained antibody which reacted with the NC1 domain of dog and human GBM by a plate-binding radioimmunoassay, bound to the dimer and 26 kDa monomer bands by Western blotting, and staining dog and human GBM by immunofluorescence (IF). The affected male dog antiserum reacted equally by radioimmunoassay with the NC1 domain isolated from GBM of unaffected, affected male, and carrier female dogs in the family with X-linked HN, and bound by Western blotting to dimers and the 26 kDa monomer band of the NC1 domain of GBM in each group of dogs. However, the affected male dog antiserum differentiated these dogs by IF; it produced global staining of GBM of unaffected dogs, failed to stain GBM of affected male dogs, and produced segmental staining of GBM of carrier female dogs. Absorption of the affected male dog antiserum with normal dog NC1 domain eliminated the staining of dog GBM by IF, whereas staining persisted after absorption with affected male dog NC1 domain. The abnormal staining patterns of GBM seen by IF in the affected male and carrier female dogs and the results of the absorption studies imply an abnormality of one or more determinants in the 26 kDa monomer band of the NC1 domain of their GBM. Amino acid sequencing of this band identified the alpha 1(IV) chain of collagen type IV, a finding that has implications for the pathogenesis of canine X-linked HN. Absent and segmental staining respectively were also seen by IF in GBM of a male and female patient with HN, using the affected male dog antiserum. Thus, the results obtained in affected male and carrier female dogs with X-linked HN may also be relevant to patients with this disease.

Immunoreactivity of S100 beta in heart, skeletal muscle, and kidney in chronic lung disease: possible induction by cAMP.

S100 protein is a calcium-binding protein composed of two subunits S100 alpha and S100 beta, which are expressed selectively by specific cell types. The distribution of S100 beta was examined among various tissues obtained at autopsy from 18 subjects with chronic lung disease and 10 control subjects. The presence of S100 beta in individual cell types was demonstrated by immunoperoxidase staining using polyclonal and monoclonal antibodies specific for S100 beta. In the 10 control subjects, positive staining was seen in a number of cell types that normally produce S100 alpha and S100 beta, (e.g., glial cells, melanocytes, chondrocytes) or only S100 beta, (e.g., Schwann cells). There was no staining of myocardial cells, skeletal muscle fibers, or kidney tubules, which normally produce S100 alpha but not S100 beta. In contrast, in the 18 subjects with chronic lung disease, all of the above cell types stained positively for S100 beta, showing that in these subjects cell types that ordinarily expressed only S100 alpha also expressed S100 beta. We suggest that the observed induction of S100 beta in these cell types seen in subjects with chronic lung disease was mediated by an elevation of cAMP levels secondary to bronchodilator therapy with beta-adrenergic agonists and phosphodiesterase inhibitors.

Heterogeneity in expression of S100 beta mRNA in human melanoma and pleomorphic adenoma demonstrated by in situ hybridization.

S100 protein is a widely used immunohistochemical marker for identification of a number of tumors including malignant melanoma and pleomorphic adenoma of the salivary gland. To extend the detection techniques for S100 protein to the level of its mRNA, sections of malignant melanoma and pleomorphic adenoma were hybridized in situ with a 35S-labeled anti-sense RNA probe complementary to the mRNA for the beta subunit of human S100 protein. Both tumors were labeled with the anti-sense RNA probe but not with a sense RNA probe. In addition, sections of normal and tumor tissues which were known not to express S100 protein on the basis of immunohistochemical studies were not labeled with the anti-sense RNA probe. These results established the specificity of the in situ hybridization technique for the detection of S100 protein mRNA. Although most of the tumor cells in both malignant melanoma and pleomorphic adenoma were labeled with the anti-sense RNA probe, unlabeled tumor cells were also present in their vicinity, suggesting there was a heterogeneity among the cells in both tumor types with respect to S100 protein mRNA expression.

Renal disease in carrier female dogs with X-linked hereditary nephritis. Implications for female patients with this disease.

Male dogs with X-linked hereditary nephritis (HN) serve as a model for studying male patients with this disease. In the present study, carrier female dogs were found to resemble female patients in showing a broad range of renal dysfunction. Of 37 carrier female dogs studied, all were healthy up to 5 years of age; however, all had proteinuria develop at 2 to 3 months, and focal segmental glomerulosclerosis (FSGS) was detected after 7 months. After 5 years, 4 of 13 dogs remained healthy and showed mild FSGS on renal biopsy; 4 had mild renal dysfunction develop and their kidneys showed extensive FSGS; 5 died prematurely of renal failure with end-stage kidneys. By immunofluorescence, using antibody to the NC1 domain of collagen type IV, segmental staining of glomerular basement membranes (GBM) was seen in all dogs before 3 to 4 years, and lesions of FSGS were negative. Thereafter, a transition to global staining of GBM was noted and lesions of FSGS became positive. Lens capsule and basement membranes in lung and choroid plexus showed discontinuous staining in two young carrier female dogs and continuous staining in one older carrier female dog. By electron microscopy, multilaminar splitting of some GBM was seen up to 4 years, and thereafter, splitting took on a compressed appearance, with the layers becoming apposed though still detectable. The authors conclude that: 1) carrier female dogs with X-linked HN are mosaics for an abnormality in the NC1 domain of GBM and other basement membranes; 2) FSGS develops in all carrier female dogs in glomerular capillary loops that possess an abnormal NC1 domain, and progresses to a variable extent in different dogs; and 3) the abnormality of NC1 in GBM of carrier female dogs appears to diminish with age, but this does not prevent progression of renal disease. Similar conclusions may apply to females with X-linked HN.

Pulmonary hemorrhage and necrotizing glomerulonephritis without glomerular immune deposits: report of two cases.

Two children with a syndrome of pulmonary hemorrhage and necrotizing, nonimmune glomerulonephritis are reported. A boy and girl, both of East Indian descent, developed recurrent lung infiltrates from the age of 3 months and 2 years, respectively. Both subsequently presented with pulmonary hemorrhage, fever, arthritis, hematuria, and nephrotic range proteinuria at 1.5 and 6 years of age, respectively. Renal biopsy in each case showed acute, severe, focal and segmental, necrotizing and crescentic glomerulonephritis without immune deposits. Subsequent renal biopsies revealed severe glomerular and tubulointerstitial scarring. No vasculitis or granulomas were seen on renal, skin, or lung biopsies. Antineutrophil cytoplasmic antibodies (ANCA) were not detected in sera taken 1.5 years in the boy and 3.5 years in the girl after the onset of renal disease. The boy was treated with prednisone, azathioprine, and plasmapheresis, but developed progressive renal impairment and commenced dialysis within 4 years. Subsequent to renal transplantation, he developed an immunoblastic lymphoma and died. The girl was treated initially with prednisone and later with cyclophosphamide. After 4 years, she had a normal glomerular filtration rate (GFR). While necrotizing nonimmune glomerulonephritis associated with pulmonary hemorrhage is rare, and cases are characteristically difficult to classify because of many overlapping features, it is possible that these children had a unique illness.

Dietary modification reduces splitting of glomerular basement membranes and delays death due to renal failure in canine X-linked hereditary nephritis.

Affected male (AM) Samoyed dogs with X-linked hereditary nephritis (HN) demonstrate splitting of all of their glomerular basement membranes (GBM) and rapidly develop renal failure within the first year of life, features reminiscent of those seen in male patients with X-linked HN. In contrast, carrier female (CF) dogs with X-linked HN show only isolated foci of splitting of GBM, and renal failure is never seen at such an early age. In the present study, we assessed whether a diet designed for dogs in renal failure could modify the changes seen in GBM of AM and CF dogs and improve the clinical outcome in the AM dogs. Beginning at 35 days of age, one group of dogs (unaffected, AM, and CF) was fed a regular diet, while a second group was fed a modified diet (i.e., restricted in protein, lipid, calcium, and phosphorus). AM dogs fed the modified diet showed less of a reduction in glomerular filtration rate than AM dogs fed the regular diet, indicative of a delay in the onset and a decrease in the severity of renal damage. Nevertheless, all of the AM dogs eventually died of renal failure regardless of diet. However, the onset and progression of renal failure were delayed and the severity of splitting of GBM was reduced in the AM dogs fed the modified diet; these dogs lived 53% longer than AM dogs fed the regular diet. CF dogs fed the modified diet also showed a reduced severity of splitting of GBM. In addition, when two CF dogs on the modified diet were switched to the regular diet, splitting of their GBM increased, indicating that continual administration of the modified diet was required to maintain the reduced rate of splitting. These studies indicate that dietary modification is beneficial in canine X-linked HN, and suggest that similar benefits (i.e., reduction in severity of splitting of GBM and delay in development of renal failure) might be observed in patients with HN who are treated with an appropriately modified diet.

Galactose-conjugated antibodies in cancer therapy: properties and principles of action.

Galactose conjugation of antibodies causes them to be recognized by the hepatic asialoglycoprotein receptor and therefore cleared very rapidly from the blood. In these investigations, some effector functions of galactose-conjugated antibodies were assayed, and several applications to experimental tumors in vivo were demonstrated. Galactose conjugation did not interfere with two antibody functions in addition to antigen binding, namely complement-mediated cytotoxicity and antibody-dependent cell-mediated cytotoxicity. This conjugation procedure was originally developed for its potential use in localized immunotherapy, such as i.p. Injection of galactose-antibody conjugates i.p. demonstrated, more conclusively than other methods that have been used, that the presence of ascites causes prolonged retention of antibody in the peritoneal cavity and that this effect is correlated with the volume of ascites present. In mice bearing i.p. tumor xenografts, i.p. injection of galactose-antibody conjugates resulted in high tumor/nontumor ratios at 28 h after antibody injection, with values of 40:1, 43:1, 77:1, and 11:1 for the blood, kidney, lung, and spleen, respectively, although the ratio was only 4:1 for the liver. Control experiments demonstrated that i.p. injection of unconjugated antibody or a galactose-conjugated nonreactive antibody produced much lower tumor/nontumor ratios. In investigations of possible systemic application of galactose-antibody conjugates, we found that injection of large amounts of an inhibitor that binds competitively to the hepatic receptor, asialo-bovine submaxillary mucin, can block clearance of galactose-conjugated antibodies for 2-3 days. In this way, high blood levels of antibody can be maintained for 2-3 days, thus allowing penetration and binding to solid tumors, followed by very rapid blood clearance. With this approach, using a human carcinoma growing s.c. in nude mice, high tumor/nontumor ratios were obtained 4 days after injection, with mean values of 43:1, 18:1, 17:1, and 15:1 for the blood, kidney, lung, and spleen, respectively, although the ratio for the liver was only 1.7:1. The blood level at this time was 0.04 +/- 0.02% (SD) of the injected dose/g, while the tumor level was 1.69 +/- 1.29% of the injected dose/g. In conclusion, galactose-conjugated antibodies appear to have diverse applications in regional or systemic immunotherapy.

Immunohistochemical staining of germ cell tumors and intratubular malignant germ cells of the testis using antibody to placental alkaline phosphatase and a monoclonal anti-seminoma antibody.

Antibody to placental alkaline phosphatase (PLAP) has been previously used in immunoperoxidase (IMP) staining studies of germ cell tumors and intratubular malignant germ cells (ITMGC) of the testis, the latter believed to be the precursor of these tumors. In this study, we compared staining by IMP using monoclonal antibody (mAb) and polyclonal antibody to PLAP with that seen using a mAb, M2A, which was previously shown to react with testicular seminomas and ITMGC. Antibody to PLAP and M2A reacted with different cellular components, as assessed by IMP staining of placenta and prepubertal testis and by Western blotting of seminoma lysates. Antibody to PLAP stained pure seminomas (seven of seven), pure embryonal carcinomas (four of four), and the seminoma (three of three) and embryonal carcinoma (six of six) components of mixed testicular germ cell tumors. M2A stained pure seminomas (26 of 26) and the seminoma component (three of three) of the mixed tumors, but failed to stain pure embryonal carcinomas (zero of four) or the embryonal carcinoma component (zero of five) of the mixed tumors. Both antibody to PLAP and M2A stained ITMGC of the testis. Since M2A stained seminomas and ITMGC but not embryonal carcinomas, seminomas would appear to be more closely related to ITMGC than embryonal carcinomas. This result has led us to speculate on the histogenesis of testicular germ cell tumors.