Gene conversion is a likely cause of mutation in PKD1

Approximately 70% of the gene responsible for the most common form of autosomal dominant polycystic kidney disease ( PKD1 ) is replicated in several highly homologous copies located more proximally on chromosome 16. We recently have described a novel technique for mutation detection in the duplicated region of PKD1 that circumvents the difficulties posed by these homologs. We have used this method to identify two patients with a nearly identical cluster of base pair substitutions in exon 23. Since pseudogenes are known to be reservoirs for mutation via gene conversion events for a number of other diseases, we decided to test whether these sequence differences in PKD1 could have arisen as a result of this mechanism. Using changes in restriction digest patterns, we were able to show that these sequence substitutions are also present in N23HA, a rodent-human somatic cell hybrid that contains only the PKD1 homologs. Moreover, these changes were also detected in total DNA from several affected and unaffected individuals that did not harbor this mutation in their PKD1 gene copy. This is the first example of gene conversion in PKD1 , and our findings highlight the importance of using gene-specific reagents in defining PKD1 mutations.      

Shwachman syndrome: Exocrine pancreatic dysfunction and variable phenotypic expression

BACKGROUND & AIMS: Shwachman syndrome is an inherited condition with multisystemic abnormalities, including exocrine pancreatic dysfunction. The aim of this study was to evaluate the occurrence and progression of features in a large cohort of patients. METHODS: Clinical records of 25 patients with Shwachman syndrome were reviewed. RESULTS: Mean birth weight (2.92 +/- 0.51 kg) was at the 25th percentile. However, by 6 months of age, mean heights and weights were less than the 5th percentile. After 6 months of age, growth velocity was normal. Severe fat maldigestion due to pancreatic insufficiency was present in early life (fecal fat, 26% +/- 17% of fat intake; age, < 2 years). Serial assessment of exocrine pancreatic function showed persistent deficits of enzyme secretion, but 45% of patients showed moderate age-related improvements leading to pancreatic sufficiency. Neutropenia was the most common hematologic abnormality (88%), but leukopenia, thrombocytopenia, and anemia were also frequently encountered. Patients with hypoplasia of all three bone marrow cellular lines (n = 11) had the worst prognosis; 5 patients died, 2 of sepsis and 3 of acute myelogenous leukemia. Other findings included hepatomegaly and/or abnormal liver function test results and skeletal abnormalities. CONCLUSIONS: A wide and varied spectrum of phenotypic abnormalities among patients with Shwachman syndrome is described. Pancreatic acinar dysfunction is an invariable abnormality. Patients with severe bone marrow involvement may have a guarded prognosis. (Gastroenterology 1996 Dec;111(6):1593-602)     

Prostaglandin E2 potentiates platelet aggregation by priming protein kinase C

Prostaglandin E2 (PGE2) is produced by activated platelets and by several other cells, including capillary endothelial cells. PGE2 exerts a dual effect on platelet aggregation: inhibitory, at high, supraphysiologic concentrations, and potentiating, at low concentrations. No information exists on the biochemical mechanisms through which PGE2 exerts its proaggregatory effect on human platelets. We have evaluated the activity of PGE2 on human platelets and have analyzed the second messenger pathways involved. PGE2 (5 to 500 nmol/L) significantly enhanced aggregation induced by subthreshold concentrations of U46619, thrombin, adenosine diphosphate (ADP), and phorbol 12-myristate 13-acetate (PMA) without simultaneously increasing calcium transients. At a high concentration (50 mumol/L), PGE2 inhibited both aggregation and calcium movements. PGE2 (5 to 500 nmol/L) significantly enhanced secretion of beta-thromboglobulin (beta TG) and adenosine triphosphate from U46619- and ADP-stimulated platelets, but it did not affect platelet shape change. PGE2 also increased the binding of radiolabeled fibrinogen to the platelet surface and increased the phosphorylation of the 47-kD protein in 32P- labeled platelets stimulated with subthreshold doses of U46619. Finally, the amplification of U46619-induced aggregation by PGE2 (500 nmol/L) was abolished by four different protein kinase C (PKC) inhibitors (calphostin C, staurosporine, H7, and TMB8). Our results suggest that PGE2 exerts its facilitating activity on agonist-induced platelet activation by priming PKC to activation by other agonists. PGE2 potentiates platelet activation at concentrations produced by activated platelets and may thus be of pathophysiologic relevance.     

Autologous bone marrow transplantation in acute myelogenous leukemia: in vitro treatment with myeloid cell-specific monoclonal antibodies

Second or third chemotherapy-induced remissions in acute myelogenous leukemia (AML) are limited by early relapse of the leukemia. We developed monoclonal antibodies (MoAbs) that are cytotoxic to myeloid leukemia cells to treat bone marrow from these patients ex vivo for autologous transplantation. In this pilot study, bone marrow was harvested from ten patients with AML in remission, treated with one or two complement-fixing MoAbs, PM-81 and AML-2-23, which react with myeloid differentiation antigens, incubated with rabbit complement, and cryopreserved. These MoAbs were chosen because they have broad reactivity with AML cells but not with pluripotent progenitor cells. At the time of transplant, 6 patients were in second complete remission, 1 each was in third complete or partial remission, and 2 were in early first relapse. The patients were treated with cyclophosphamide (60 mg/kg a day for 2 days) and total body irradiation (200 cGy twice a day for 3 days) and given infusions of MoAb-treated bone marrow. Full bone marrow reconstitution was observed in eight patients; two patients did not recover platelets. Seven of the ten patients are surviving and disease-free at 21.0, 15.0, 13.0, 10.0, 6.0, 3.0, and 2.0 months posttransplant. Treating bone marrow with MoAbs to myeloid differentiation antigens does not interfere with pluripotential stem cell engraftment. Longer follow-up and a controlled study are necessary to prove that the apparent efficacy of this therapeutic approach in some patients is attributable to MoAb-mediated killing of leukemia cells.     

Interleukin-6 enhances growth factor-dependent proliferation of the blast cells of acute myeloblastic leukemia

The effects of recombinant interleukin-6 (IL-6) on the proliferation of blast precursors present in the peripheral blood of patients with acute myeloblastic leukemia (AML) was investigated. IL-6 had little effect by itself; however, it synergized with granulocyte macrophage colony- stimulating factor (GM-CSF) and interleukin-3 (IL-3) in the stimulation of AML blast colony formation. Responsiveness of blast progenitors to IL-6 was heterogeneous. On normal bone marrow cells the same synergy was observed on granulocyte and monocyte precursors (GM-CFC), while there was no significant effect on erythroid and multipotential precursors.     

Effect of propranolol on bile acid- and cholera enterotoxin-stimulated cAMP and secretion in rabbit intestine.

Stimulation of net secretion by deoxycholic acid (DCA) in the colon and by cholera enterotoxin (CE) in the jejunum is mediated by cAMP. Propranolol (Pr) inhibits adenylate cyclase (AC) activity and net secretion induced by bile acid in the colon. The aim of this study was to assess the organ specificity of DCA and CE as well as the selectivity of Pr inhibition. Three colonic and three jejunal loops were prepared in each of 8 rabbits treated intravenously with Pr, 4 mg per kg, 1/2 hr before loop construction and in each of 10 untreated control rabbits. One milliliter of DCA, 6 mM, CE, 10 mug per ml, or heat-inactivated CE or 0.9% NaCl, as basal controls were injected in random order into each of the loops. The volume of luminal fluid and mucosal AC were measured in each intestinal loop 5 hr later. DCA in the colon stimulated AC 2-fold (P less than 0.01) and luminal fluid 15-fold (P less than 0.01). CE in the jejunum stimulated AC 2.3-fold (P less than 0.01) and luminal fluid 9-fold (P less that 0.01). No significant effects on volume or AC occurred in response to CE in the colon or to DCA in the jejunum. Pr pretreatment completely prevented the stimulation of AC and luminal fluid by DCA in the colon but did not affect the action of CE in the jejunum of the same animals. Thus, DCA and CE are organ-specific stimulants of cAMP systems, and Pr is a selective inhibitor of certain inducers of cAMP and net secretion.     

Parathyroid adenomas evaluated by Tl-201/Tc-99m pertechnetate subtraction scintigraphy and high-resolution ultrasonography

Thallium-201/technetium-99m pertechnetate subtraction scintigraphy of the parathyroid glands was performed in a prospective study of 33 patients who had undergone bilateral neck exploration for elevated serum calcium and serum parathyroid hormone levels. In 31 cases, the Tl-201/Tc-99m subtraction technique yielded an overall sensitivity of 81%, specificity of 99%, and accuracy of 94% for identifying solitary parathyroid adenomas. Tl-201/Tc-99m subtraction scintigraphy correctly identified 73% of parathyroid adenomas weighing less than 499 mg, 79% of those weighing 500-1,499 mg, and 100% of adenomas weighing more than 1,500 mg. In a subgroup of 24 patients with solitary parathyroid adenomas who underwent both scintigraphy and high-resolution sonography, the sensitivity, specificity, and accuracy of both procedures were similar.     

Child abuse study among Swedish physicians and medical students

Background: The purpose of the present paper was to examine the attitudes and experiences of reporting child abuse and neglect among primary care and hospital‐based physicians and to study the responses of physicians and medical students to case vignettes suggestive of possible physical abuse or neglect. Methods: Physicians at the child health centers in Göteborg primary care (n= 44) and the general pediatricians at the pediatric hospital (n= 21) in Göteborg answered a questionnaire regarding their attitude and experiences reporting child abuse and neglect. The physicians and medical students (n= 34) responded to three case vignettes in which child abuse and neglect could be suspected. Results: A majority of the physicians had reported child abuse and neglect to the social services (80%). No differences were found between primary care and hospital‐based physicians in terms of reporting or attitudes. Two‐thirds of the physicians had suspected child abuse and neglect and decided not to report, and the major reason for not reporting was a lack of confidence in social services organization. Twenty‐one percent had never reported a child for abuse or neglect during their working career. Medical students were more likely to report hypothetical cases than physicians. Conclusion: Many physicians have reported child abuse to social services but also have neglected to do so even when suspecting abuse. It is important that medical students’ willingness to report is continued when starting to work clinically and that all physicians should be continuously educated.     

A Southwest Oncology Group study on the use of a human tumor cloning assay for predicting response in patients with ovarian cancer

A total of 211 patients with epithelial ovarian cancer (168 with tumors refractory to prior chemotherapy and 43 with no prior chemotherapy) from 33 different Southwest Oncology Group institutions had their tumors sampled and specimens shipped to two central laboratories for drug-sensitivity testing in a human tumor cloning assay. The 168 patients with a prior history of chemotherapy failure (median of four prior chemotherapeutic agents) were treated with the most effective agent(s) found in the cloning assay (23 patients), and those patients whose tumors did not form colonies in vitro or did not manifest any sensitivity to agent(s) were treated with a clinician's choice of agent(s) (101 patients). The remaining 44 of the 168 patients were not treated with chemotherapy because of deteriorating performance status or early death. The complete and partial response rate in patients treated according to assay results was 28% versus 11% for the patients treated according to clinician's choice (P = 0.03). There was no statistically significant difference in survival between the two options (6.25 versus 7 months, respectively). The 43 patients with no history of prior chemotherapy were all treated with standard combination chemotherapy, and their clinical response was compared with their in vitro sensitivity to the same agents. Overall there was a 100% true-positive rate and 100% true-negative rate for the seven evaluable patients. From these data the authors conclude that use of the human tumor cloning assay may increase the response rate but not the survival for selected patients with advanced chemotherapy-refractory ovarian cancer. The study is weakened, however, by the many steps of patient selection necessitated by inadequate tumor colony formation in vitro and the inability to treat all patients (because of early death or a rapid decline in performance status). The assay does appear to be worthy of additional study for predicting response to combination chemotherapy in patients without a prior history of chemotherapy. Finally the use of central chemosensitivity testing laboratories is feasible for testing in vitro predictive assays in a cooperative group setting.