Protein truncation test (PTT) to rapidly screen the DMD gene for translation terminating mutations.

We have developed a rapid and sensitive method to screen the Duchenne muscular dystrophy (DMD) mRNA for translation terminating mutations by a combination of RT-PCR (Reverse Transcription and Polymerase Chain Reaction) and in vitro transcription/translation applied to white blood cell mRNA. This technique was termed the protein truncation test (PTT). Here we demonstrate the detection of a point mutation in a DMD patient and his mother, a carrier. The PTT can also be used for carrier detection when no patient material is available, or in the case of spontaneous mutations. We developed a protocol to screen the total coding region of the DMD gene in 5-10 PTT reactions. Furthermore, PTT could be of diagnostic value in any disease where premature terminations form a substantial part of the total mutation spectrum.

Application of in vitro Myo-differentiation of non-muscle cells to enhance gene expression and facilitate analysis of muscle proteins.

Introduction of the myogenic-determination gene MyoD forces non-muscle cell cultures into myogenesis, thereby inducing expression of muscle-specific proteins and facilitating their analysis. In several MyoD-transfected fibroblasts, immunohistochemical detection showed expression of desmin after three days, of titin after five days and of dystrophin after seven days. Cell fusion (myotube formation) could be observed after five days. After nine days a fraction of the cells showed a striated titin pattern, indicating an advanced state of muscle differentiation. Dystrophin (the protein absent in Duchenne Muscular Dystrophy patients) can be detected in MyoD-transfected and differentiated fibroblasts from healthy individuals, and is absent in those of patients. MyoD-transfection increases transcription of the dystrophin gene, facilitating RNA-based mutation detection. Using RNA from MyoD-transfected, differentiated fibroblasts of a deceased patient with an unknown, non-deletion mutation, we were able to identify a CGA-->TGA nonsense mutation in the rod domain at basepair 6492 and to establish a rapid mutation specific test for future diagnosis of the mutation in his relatives.

Effector cells of low-dose IL-2 immunotherapy in tumor bearing mice: tumor cell killing by CD8+ cytotoxic T lymphocytes and macrophages.

The presence and cytotoxicity of tumor infiltrating cells is described in mice during effective immunotherapy with interleukin 2 (IL-2). DBA/2 mice were inoculated i.p. with 2 x 10(4) tumor cells on day 0 and treated with daily i.p. injections with 20,000 units IL-2 on days 10-14. Mice bearing a large syngeneic i.p. tumor burden (SL2 lymphoma, P815 mastocytoma, L5178Y lymphoma, or L1210 lymphoma) could be cured by i.p. immunotherapy with these low doses of IL-2. In the peritoneal cavity of these mice an infiltrate of mononuclear cells was present. Similar numbers of lymphocytes (10(6)-10(7)) and macrophages (+/- 10(7)) were present in control tumor bearing mice and IL-2 treated tumor bearing mice. The ratio of CD4+/CD8+ T lymphocytes in the peritoneal cavity of mice rejecting the SL2 tumor was smaller than 0.5, whereas this ratio is about 2 in naive mice. In the spleens of IL-2 treated tumor bearing mice only a minor decrease of CD4+/CD8+ ratio was observed from 2.1-2.4 to 0.9-1.9. T cells isolated from the peritoneal cavity of mice inoculated with SL2 tumor cells and treated with IL-2, were highly cytotoxic to SL2 cells: at E:T ratio 2:1 the cytotoxicity index was 37 +/- 3. This cytotoxicity was specific and mediated by CD8+ T lymphocytes. Macrophages that were present in the peritoneal cavity of mice treated with IL-2 were also highly cytotoxic. The C.I. of these cells was 63-76% at E:T ratio 1:1. Cytotoxic macrophages were also present in untreated tumor bearing mice. The i.p. injections of IL-2 (20,000 units/day) caused a four-fold increase in the local NK-activity in the peritoneal cavity in naive mice. These IL-2 injections did not generate LAK-activity in vivo. Specificity of the in vivo tumor rejection was tested by injection SL 2 i.p. on day 0 and P815 i.p. on day 10, or vice versa, followed by IL-2 treatment. Only the tumor cells that were injected on day 0 were rejected. These in vivo experiments point to specific tumor rejection. In conclusion, both cytotoxic macrophages and CTL's are present in a sufficient number and with sufficient cytotoxicity to explain the killing of tumor cells in the peritoneal cavity. The CTL-activity seems of decisive importance for tumor rejection as this is induced by IL-2.

Converting accounts receivable into cash.

In recent years, increasing numbers of healthcare providers have converted their accounts receivable into cash through a process called securitization. This practice has gained popularity because it provides a means to raise capital necessary to healthcare organizations. Although securitization transactions can be complex, they may provide increased financial flexibility to providers as they prepare for continuing change in the healthcare industry.

Recovery of Medicare and Medicaid overpayments in bankruptcy.

Mr. Roest argues in favor of the Third Circuit's decision in University Medical Center v. Sullivan to advance the proposition that the Doctrine of Recoupment should be applied narrowly in health care bankruptcy cases. The article begins by introducing key provisions of the Medicare Act and Bankruptcy Code, and by distinguishing between recoupment and setoff. The article then focuses on the Third Circuit's decision, giving both a sketch of the court's decision and a commentary on the holding. The article concludes with a discussion of recoupment-related issues left open by the University Medical Center decision.

Cadmium-induced inhibition of proliferation and differentiation of embryonal carcinoma cells and mechanistic aspects of protection by zinc.

Murine embryonal carcinoma cells have been used in in vitro models to study the effects of cadmium chloride on proliferation and differentiation of early embryonic cells. This approach allows the various cell types within the early embryo as well as several developmental mechanisms to be dissected and studied in isolation using larger numbers of cells than would be readily available from the embryo itself. The present study shows that both embryonal carcinoma cell proliferation and differentiation into parietal endoderm are inhibited by cadmium chloride. The effects are counteracted by the additional presence of zinc chloride. The uptake of cadmium into the cells is inhibited in the presence of zinc chloride, suggesting that competition between these metals for passage into the cells contributes to the mechanism underlying the protective effect of zinc. In addition, metallothionein gene expression is enhanced more rapidly after simultaneous incubation with zinc chloride, indicating that the attenuating effect of zinc on cadmium toxicity is also partly attributable to detoxification by metallothioneins.

Protein truncation test (PTT) for rapid detection of translation-terminating mutations.

Currently available techniques used to recognize point mutations in genetic disease are time consuming and are capable of screening only small pieces of DNA. Moreover, they detect all sequence differences including phenotypically silent changes. Consequently, they are not convenient to analyse mutations in large, multi-exonic genes, where a large fraction of pathological point mutations arises from early termination, as is the case for the one third non-deletion/duplication cases of Duchenne Muscular Dystrophy. We have developed a rapid and sensitive method, the Protein Truncation Test (PTT). PTT is based on a combination of RT-PCR, transcription and translation and selectively detects translation-terminating mutations. We demonstrate its effectiveness to detect point mutations in DMD-patients and carrier females. PTT should be widely applicable diagnostically in any disease where early terminations contribute substantially to the disease cause.

Rapid detection of translation-terminating mutations at the adenomatous polyposis coli (APC) gene by direct protein truncation test.

Familial adenomatous polyposis (FAP) is usually associated with protein truncating mutations in the adenomatous polyposis coli (APC) gene. The APC mutations are known to play a major role in colorectal carcinogenesis. For the identification of protein truncating mutations of the APC gene, we developed a rapid, sensitive, and direct screening procedure. The technique is based on the in vitro transcription and translation of the genomic PCR products and is called the protein truncation test. Samples of DNA from individual FAP patients, members of a FAP family, colorectal tumors, and colorectal tumor-derived cell lines were used to show the effectiveness of this method.

Splicing mutations in DMD/BMD detected by RT-PCR/PTT: detection of a 19AA insertion in the cysteine rich domain of dystrophin compatible with BMD.

We have used an RNA based mutation detection method to screen the total coding region of the dystrophin gene of a Duchenne and a Becker muscular dystrophy patient in whom DNA based mutation detection methods have so far failed to detect mutations. By RT-PCR and the protein truncation test (PTT) we could identify point mutations in both cases. DMD patient DL184.3 has a T-->A mutation in intron 59 at position -9, creating a novel splice acceptor site for exon 60. As a result seven intronic bases are spliced into the mRNA, causing a frameshift and premature translation termination 20 codons downstream. Since this patient had died and only fibroblasts were available, we applied MyoD induced myodifferentiation of stored fibroblasts to enhance muscle specific gene expression. With the results of this mutation analysis, prenatal diagnosis could subsequently be performed in this family. BMD patient BL207.1 carries a G-->C mutation at position +5 of intron 64, disrupting the splice donor consensus sequence and activating a cryptic splice donor site 57bp downstream. The inclusion of these 57 intronic bases in the mRNA leaves the reading frame open and results in the insertion of 19 amino acids into the cysteine rich domain of dystrophin. Interestingly, this insertion in a part of the dystrophin considered to interact with the dystrophin binding complex of the sarcolemma is apparently compatible with mild BMD-like clinical features. Both mutations reported are missed by analysis of multiplex PCR products designed for deletion screening of the coding region. Extrapolation from existing point mutation detection efficiencies by DNA and RNA based methods emphasises that RNA based methods are more sensitive and that most of the remaining undetected mutations may affect splice or branch sites or create cryptic splice sites.