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Two deacetyl-thymosin alpha(1) analogues containing Phe (4Br) or D-Phe (4Br) residue-[D-Phe(4Br)(21)]deacetyl-thymosin alpha(1) and [Phe(4Br)(21)]deacetyl-thymosin alpha(1), respectively-were synthesized by the manual solid-phase method and their immunological effects on the low E-rosette-forming lymphocytes of uraemic patients were examined. One of the synthetic analogues, [Phe(4Br)(21)deacetyl-thymosin alpha(1), demonstrated a restorative effect on the low E-rosette-forming lymphocytes of uraemic patients, which was stronger than that of deacetyl-thymosin alpha(1), but the other analogue, [D-Phe(4Br)(21)]deacetyl-thymosin alpha(1), showed no restorative effect under the same conditions.

The wide range of epithelial and mesenchymal lines of differentiation seen in hepatoblastoma suggests that this tumor derives from a pluripotent stem cell. To test this hypothesis, seven hepatoblastomas of various subtypes were investigated for the presence of cells with the features of the oval cells found during hepatocarcinogenesis in rodents that are thought to be closely related to hepatic stem cells. Because similar cells, referred to as "small cells," have been described in human liver disease with chronic ductular reaction, five liver biopsies from infants with biliary atresia were also investigated. The specimens were investigated by electron microscopy, immunoelectron microscopy, and immunostaining for cytokeratins 7, 8, 18, and 19. Small epithelial cells (SEC) corresponding to the oval cells of the rat and the "small cells" in humans were found in both biliary atresia and hepatoblastoma. These cells were oval and exhibited intercellular junctions, tonofilament bundles, and a biliary epithelium-type cytokeratin profile. SEC were found in small numbers in fetal hepatoblastoma and in moderate numbers in embryonal hepatoblastoma. In small cell hepatoblastoma, nearly all the tumor cells exhibited SEC-like ultrastructural features and a corresponding cytokeratin profile. Thus, cells exhibiting morphological and immunophenotypic features of hepatic stem cells are detectable in hepatoblastoma. Their numbers vary according to the subtype, reflecting the differing degrees of differentiation of the various subtypes, consistent with the theory propounded in the literature that embryonal and, with further differentiation, fetal tumor cells derive from precursor small cells. The findings support the hypothesis that hepatoblastoma derives from a pluripotent, probably entodermal or even less committed, stem cell.

The control of neuritic extension and guidance is critical for the development, maturation, and regeneration of functional neuronal circuits. We identified a neuronal 64-85 kDa phosphoprotein, the expression of which in mouse brain is regulated during development, reaching a peak at approximately 5 d postnatal, when maturation of neurons and synaptic connections is highly active. The amino acid sequence of the mouse protein deduced from its cloned cDNA reveals similarities with that of the neuritic outgrowth- and guidance-related product of the unc-33 gene in Caenorhabditis elegans. The regulation of its phosphorylation in response to nerve growth factor, as well as its localization in neurites and growth cones and at the neuromuscular junction, further indicates that Ulip (for Unc-33-like phosphoprotein) is not only a structural but likely is also a functional mammalian homolog of Unc-33, potentially involved in the control of neuritic outgrowth and axonal guidance.

Ovarian cancers have a high ability to invade the peritoneal cavity and some are stimulated by estrogens. In an attempt to understand the mode of action of estrogens on these cancer cells and to develop new markers, we have characterized estrogen-regulated proteins. This study was aimed at identifying a protein secreted by ovarian cancer cells whose level was increased by estradiol [Galtier-Dereure, F., Capony, F., Maudelonde, T. & Rochefort, H. (1992) J. Clin. Endocrinol. Metab. 75, 1497-1502]. By using microprotein sequencing, the 110-kDa protein was identified as fibulin-1, a protein of the extracellular matrix that binds to fibronectin, laminin, and nidogen. The amount of immunoprecipitated fibulin-1 secreted into the medium and present in the cell extract was increased up to 10-fold by estradiol in three estrogen-responsive ovarian cancer cell lines. By immunohistochemistry fibulin-1 was located in the stroma of several ovarian cancers and cysts. The findings highlight a potential role for fibulin-1 in the spread of ovarian cancer in the peritoneal cavity and/or in distal metastases.

About 90 percent of human pancreatic carcinomas show allelic loss at chromosome 18q. To identify candidate tumor suppressor genes on 18q, a panel of pancreatic carcinomas were analyzed for convergent sites of homozygous deletion. Twenty-five of 84 tumors had homozygous deletions at 18q21.1, a site that excludes DCC (a candidate suppressor gene for colorectal cancer) and includes DPC4, a gene similar in sequence to a Drosophila melanogaster gene (Mad) implicated in a transforming growth factor-beta (TGF-beta)-like signaling pathway. Potentially inactivating mutations in DPC4 were identified in six of 27 pancreatic carcinomas that did not have homozygous deletions at 18q21.1. These results identify DPC4 as a candidate tumor suppressor gene whose inactivation may play a role in pancreatic and possibly other human cancers.

Molecular genetics is a tool that can be learned as a language to assist clinicians in the management of colorectal cancer patients. Following a brief review of the genetic controls of colorectal cancer, the author focuses on the models of the Registry for Familial Adenomatous Polyposis and the Registry for Hereditary Nonpolyposis Colon Cancer to demonstrate most vividly the impact molecular genetics is currently having on the practical management of colon cancer. Recent discoveries of K-ras oncogene mutations in stool cultures and the prognostic implications of mutations of the TP53 and DCC genes are discussed in the context of future applications to the management of patients.

We investigated the effect of activated protein C (APC) on lipopolysaccharide (LPS)-induced pulmonary vascular injury in rats to investigate the possible usefulness of APC as a treatment for adult respiratory distress syndrome. Intravenously administered LPS (5 mg/kg) significantly increased pulmonary vascular permeability. APC prevented the LPS-induced increase in pulmonary vascular permeability observed at 6 hours. Heparin plus antithrombin III (ATIII) and active site-blocked factor Xa (DEGR-Xa), a selective inhibitor of thrombin generation, inhibited LPS-induced coagulopathy but did not prevent LPS-induced pulmonary vascular injury. LPS-induced pulmonary vascular injury was significantly attenuated in rats with nitrogen mustard-induced leukocytopenia and in rats treated with ONO-5046, a potent granulocyte elastase inhibitor. Administration of LPS also increased pulmonary accumulation of leukocytes, as evaluated by measurement of myeloperoxidase activity in the lungs. APC significantly reduced LPS-induced increases in pulmonary accumulation of leukocytes at 1 hour. Neither ATIII plus heparin nor DEGR-Xa inhibited leukocyte accumulation. Active site-blocked APC (DIP-APC) prevented neither the LPS-induced pulmonary accumulation of leukocytes nor the LPS-induced increase in pulmonary vascular permeability. These results suggest that the mechanism of APC inhibition of LPS-induced pulmonary vascular injury was independent of its anticoagulant activity and was related to its ability to inhibit accumulation of leukocytes. In addition, these findings suggest that the serine protease activity of APC may be essential to its inhibitory effect on LPS-induced pulmonary accumulation of leukocytes and subsequent pulmonary vascular injury.

The hemochromatosis gene (HFE) maps to 6p21.3, in close linkage with the HLA Class I genes. Linkage disequilibrium (LD) studies were designed to narrow down the most likely candidate region for HFE, as an alternative to traditional linkage analysis. However, both the HLA-A and D6S105 subregions, which are situated 2-3 cM and approximately 3 Mb apart, have been suggested to contain HFE. The present report extends our previous study based upon the analysis of a large number of HFE and normal chromosomes from 66 families of Breton ancestry. In addition to the previously used RFLP markers spanning the 400-kb surrounding HLA-A, we examined three microsatellites: D6S510, HLA-F, and D6S105. Our combined data not only confirm a peak of LD at D6S105, but also reveal a complex pattern of LD over the i82 to D6S105 interval. Within our ethnically well-defined population of Brittany, the association of HFE with D6S105 is as great as that with HLA-A, while the internal markers display a lower LD. Fine haplotype analysis enabled us to identify two categories of haplotypes segregating with HFE. In contrast to the vast majority of normal haplotypes, 50% of HFE haplotypes are completely conserved over the HLA-A to D6S105 interval. These haplotypes could have been conserved through recombination suppression, selective forces and/or other evolutionary factors. This particular haplotypic configuration might account for the apparent inconsistencies between genetic linkage and LD data, and additionally greatly complicates positional cloning of HFE through disequilibrium mapping.

This report concerns the synthesis and chemical characterization of novel series of N-substituted 2 beta-carbomethoxy-3 beta-(4'-iodophenyl)tropane (beta-CIT, 2) analogs and their neuropharmacological evaluation for affinity at dopamine (DAT), serotonin (5-HTT), and norepinephrine membrane transporters in rat brain tissue. N-Substituted analogs of beta-CIT with a 2 beta-carbomethoxy ester moiety showed lower DAT affinity than beta-CIT for the DAT, and some were more selective for the 5-HTT over the DAT. 2 beta-Carbomethoxy(iodophenyl)nortropane analogs of beta-CIT with the N-substituents difluoroethyl, mesoxypropyl, iodopropyl, and methylpropionyl all yielded > 10-fold lower DAT affinity than beta-CIT itself, whereas the N-(fluoropropyl)-2 beta- isopropyl ester analog (1) of beta-CIT exceeded beta-CIT (2, an N-methyl-2 beta-carbomethoxy ester) in DAT affinity. Several N-haloalkyl-substituted beta-CIT analogs yielded high 5-HTT affinity (Ki < 0.6 nM), ranking: N-fluoropropyl (5) > N-chloropropyl (4) > or = N-bromopropyl (3) > beta-CIT (2) > N-3'-phtalimidopropyl (11), with particularly high (ca. 30-fold) 5-HTT-over-DAT selectivity found in the N-fluoropropyl (5) and N-fluoroethyl (6) compounds, compared to only 3.o-fold 5-HTT selectivity in beta-CIT itself. Highly 5-HTT selective agents such as 5 and 6 may be useful as brain-imaging ligands for serotonin neurons or as mood-elevating drugs, while the high affinity and selectivity for the DA transporter found in N-(fluoropropyl)-2 beta-(carboxyisopropyl)-3 beta-(4'-iodophenyl)-nortropane (1) and N-(fluoropropyl)-2 beta-carboxymethoxy-3 bet-(4'-iodophenyl)nortropane (FP-beta-CIT, 5) support their use as improved markers for DA neurons.

The congenital dyserythropoietic anemias (CDAs) are a group of relatively rare inherited anemias that share in common ineffective erythropoiesis and morphologic abnormalities of mature red blood cells and their precursors. Three major types of CDA and a number of variants have been described. The diagnosis and categorization of these disorders are facilitated by microscopic examination of the blood and bone marrow and by serologic testing. Management of patients currently consists of observation and supportive care. Because patients with CDAs may be at significant risk for secondary hemochromatosis, they require monitoring for this condition. Splenectomy may be of benefit in certain cases in which the anemia is particularly severe. Over the past few years advances have been made in understanding the pathogenesis of these disorders, and it now appears that CDA II results from enzymatic defects in the cellular glycosylation pathway.

As part of an effort to characterize the hemochromatosis gene, we selected three non-chimeric yeast artificial chromosomes (YACs) overlapping with the YAC B30 previously described and forming an 800 kilobase contig covering the HLA-A/HLA-F region. The precise physical map of these YACs and of the corresponding genomic region were established. Nine concentrated sites of CpG cutter elements, potentially HTF islands, were mapped. In addition, several probes have been generated as tools for mapping and examining transcripts produced in the region. This allowed for the characterization and localization of two new coding sequences, provisionally named HCG (for hemochromatosis candidate gene) and numbered VIII and IX.

A human colon carcinoma cell line designated OUMS-23 has been established from metastatic pericardial fluid of a male familial adenomatous polyposis patient with colon cancer. Since 1984, the epithelial cells have been maintained in culture. Ultrastructural studies revealed the presence of numerous microvilli on the cell surface and desmosomes between the adjacent cells. The cells secreted carcinoembryonic antigen into the culture medium (15 ng/10(6) cells-1 24 h-1). The cells expressed heat-stable placental-type-like alkaline phosphatase, whereas the normal counterparts expressed tissue-unspecific alkaline phosphatase. Karyotypic analysis showed that the cell line was of human origin and that the chromosome number was broadly distributed between 53 and 118. Southern blot analysis of the APC gene revealed no abnormalities in OUMS-24 cells, while Northern blot analysis demonstrated that the expression of the gene was about one-half that of the normal human fibroblasts. No mutations at the "hot spots" of codons 12 and 61 of H-, K- and N-ras proto-oncogenes were detected in the cells. The cells could grow in soft agar at a cloning efficiency of 6.5%, and upon transplantation into nude mice the cells formed tumors, which were diagnosed as differentiated adenocarcinoma.

We have described herein various skin diseases which are caused by essential trace element deposition, deficiency, allergy, etc. Pigmentation of hemochromatosis and hemosiderosis are recognized by hemosiderin deposition in the dermis. Acrodermatitis enteropathica is caused by a deficiency of Zn and is classified as either a hereditary type or as an acquired type. The former is autosomal recessive and the latter is caused by a low intake of Zn. Wilson's disease and Menkes' kinky hair syndrome, which are caused by abnormal Cu metabolism, elicit hyperpigmentation and morphological changes of the hair, respectively. It appears that kinky hair formation results from low activity of sulfhydryl oxidase which is a Cu enzyme. Bowen disease, which is carcinoma in situ, is caused by As toxicosis. Some cases, such as palmo-plantar-pustulosis, lichen planus and oral lichen planus are caused by allergies to metals used in dental surgery, especially Ni, Co, Cr and Sn.

Immunohistochemical detection of estrogen and progesterone receptors (ER and PgR, respectively) was performed in 67 cases of Japanese female invasive breast carcinoma on methacarn-fixed paraffin-embedded sections using monoclonal antibodies against ER (1D5) and PgR (10A9) with an antigen-retrieval technique based on microwave exposure in citrate buffer solution. Staining localized in nuclei and specimens containing > or = 20% specifically stained tumor cell nuclei were considered ER- or PgR-positive; the positive rate was 37% (25/67) for ER and 45% (30/67) for PgR. The immunohistochemical (IHC) results were compared with cytosolic receptors obtained from tissue homogenates as measured by dextran-coated charcoal (DCC) assay, in which tumors were considered ER- and/or PgR-positive when receptor concentration was greater than 3 or 5 fmol/mg cytosolic protein, respectively, and agreement between the two methods was 71.6% for ER and 80.6% for PgR. The sensitivity and specificity were 53.5 and 91.7% for ER-IHC, and 77.4 and 83.3% for PgR-IHC, respectively. In relation to age of the patient and stage of the cancer, ER immunoreactivity correlated with patient's age (> or = 55 vs 55, P = 0.032), and proportionally increased with aging (P = 0.0084) using the Kruskal-Wallis statistics. PgR-immunoreactivity correlated with nodal involvement (P = 0.031) by the TNM system. However, no correlations were found between the results of the DCC assay and any of the clinical parameters examined. Thus, immunohistochemical assay may provide valuable information in predicting prognosis and response to endocrine therapy.

According to a "two-hit" model, dominantly inherited predisposition to cancer entails a germline mutation, while tumorigenesis requires a second, somatic, mutation. Non-hereditary cancer of the same type requires the same two hits, but both are somatic. The original tumor used in this model, retinoblastoma, involves mutation or loss of both are somatic. The original tumor used in this model, retinoblastoma, involves mutation or loss of both copies of the RB1 tumor-suppressor gene in both hereditary and non-hereditary forms. In fact, most dominantly inherited cancers show this relationship. New dominantly inherited cancers show this relationship. New questions have arisen, however. When a tumor-suppressor gene is ubiquitously expressed, why is there any specificity of tumor predilection? In some instances, it is clear that two hits produce only a benign precursor lesion and that other genetic events are necessary. As the number of necessary events increase, the impact of the germline mutation diminishes. The number of events is least for embryonal tumors, and relatively small for certain sarcomas. Stem-cell proliferation evidently plays a key role early in carcinogenesis. In some tissues it is physiological, as in embryonic development and in certain tissues in adolescence. In adult renewal tissues, the sites of the common carcinomas, mutation may be necessary to impair the control of switching between renewal and replicative cell divisions; the APC gene may be the target of such a mutation.

Predictive genetic testing offers the opportunity for persons at risk to obtain information about their carrier status concerning Huntington's disease (HD). HD is associated with the expansion of a (CAG) repeat in the gene IT15. HD chromosomes contain about 40-75 repeat units, whereas normal chromosomes show a range between 11 and 33 repeats. The HD repeat is highly unstable during transmission, involving both increases and decreases in size with the largest expansions occurring in male meioses. The number of (CAG) copies is inversely correlated with the onset age of the disorder. Investigation of sporadic cases revealed that new mutations for HD are more frequent than estimated. Analysis of the repeat length allows direct DNA diagnosis of affected individuals and asymptomatic persons at risk. From 1989 to March 1993 in our institute predictive testing by indirect DNA methods was requested by 108 persons at risk. After cloning of the HD gene in 1993, direct testing was requested by 113 persons at risk between April 1993 and April 1994 in Göttingen. In about 400 patients differential diagnosis was performed with the direct method. In approximately 82% of the patients the expanded (CAG) repeat was found, and the HD diagnosis could be confirmed.

Markers of endothelial cell activation were measured in 28 patients presenting with various forms of limited or focal type cutaneous vasculitis. Plasma levels of tissue plasminogen activator antigen (t-PA:Ag), plasminogen activator inhibitor type 1 antigen (PAI-1:Ag) and PAI-1 activity, fibrin plate, von Willebrand factor antigen (vWF:Ag), tissue factor (TF) and soluble thrombomodulin (sTM) were measured. In comparison with the control group (n = 20) there was a significant increase in t-PA:Ag, vWF:Ag and TF (P < 0.05, Mann-Whitney U-test) in the cutaneous vasculitis group. This study confirms that measurable degrees of endothelial activation occur in cutaneous vasculitis. Cutaneous vasculitis includes a diverse group of clinical conditions, which are associated with inflammatory changes in cutaneous blood vessels with local fibrin deposition. The aetiology and pathogenesis of the majority of these entities remain unknown. Causative mediators are thought to include immune complexes, anti-endothelial cell antibodies, cytotoxic lymphocytes and viruses. Histologically, immune complexes and complement are frequently detected on the vessel wall, and serologically anti-endothelial antibodies are often detected in patients with vasculitis and in systemic lupus erythematosus (SLE) which correlate with the severity of cutaneous vasculitis, arthritis and nephritis. Lymphocyte-mediated toxicity to endothelial cells has been reported in a small number of patients with giant cell arteritis and Takayasu's arteritis. The vascular endothelium plays a central part in the control of haemostasis. Under physiological conditions endothelial cells present an anticoagulant surface to blood constituents, partially due to surface expression of heparan sulphate and thrombomodulin (TM). Heparan sulphate binds antithrombin III (ATIII), thereby accelerating inactivation of intrinsic coagulation enzymes. Thrombomodulin is an endothelial cell surface glycoprotein which promotes anticoagulation by forming a complex with thrombin which then activates protein C. Activated protein C together with a cofactor, protein S, inactivates FVa and FVIIIa. von Willebrand factor (vWF) is synthesized by endothelial cells, stored in Weibel-Palade bodies and released into the circulation upon endothelial stimulation. vWF mediates the binding of platelets to the subendothelium and is the carrier molecule for FVIIIC. The endothelium controls fibrinolysis by producing t-PA and its inhibitor PAI-1. Inflammatory cytokines such as interleukin-1 (IL-1) and tumour necrosis factor (TNF) activate endothelial cells, causing a shift from an antithrombotic to prothrombotic state, including expression of tissue factor, increased synthesis of PAI-1 and decreased expression of TM. Fibrin deposition and intravascular thrombosis are seen in cutaneous vasculitis syndromes, suggesting local endothelial cell activation. The aim of this pilot study was to assess whether perturbation of the endothelium in cutaneous vasculitis could be detected in the patients' plasma samples. If so, further studies to assess any correlation in levels of these markers with disease activity might prove useful in the future.

In order to clarify the association between bcl-2 protein (Bcl-2) expression and genetic alteration, we investigated p53 and DCC (deleted in the colon carcinoma gene locus) gene abnormalities in Bcl-2-positive and -negative gastric carcinomas using a polymerase chain reaction/loss of heterozygosity (LOH) assay. Bcl-2 immunoreactivity was found in 25 of 178 (14%) gastric carcinoma cases. With these 25 positive cases, the proportion 18/87 (20.6%) of the total in early stages demonstrating invasion of the mucosa and/or submucosa was significantly greater (P = 0.013) than the 7/91 (7.7%) found for advanced tumors exhibiting invasion into or through the muscularis propria. However, there was no statistically significant variation between the proportions for differentiated (17/98 cases, 17.3%) and undifferentiated (8/80 cases, 10%) lesions. Sixteen Bcl-2-positive cases (9 cases were not studied because of insufficient specimen material to allow extraction of DNA) and 31 cases randomly selected from a Bcl-2-negative group were analyzed for the presence of p53-LOH or DCC-LOH and for p53 by immuno-histochemistry. The minority of the Bcl-2-positive group had p53-LOH and were immunopositive for p53 (P = 0.033, P = 0.028 respectively), while no association was found in the Bcl-2-negative category. In contrast, there was no correlation at all between Bcl-2 expression and DCC-LOH although the number of informative cases analyzed was too small to allow definite conclusions. These results indicate that Bcl-2 may be predominantly expressed at an early stage in gastric carcinomas, possibly in negative association with p53 gene abnormalities.

Accumulating evidences that carcinogenesis requires multiple gene alterations of oncogenes and tumor suppressor genes have recently emerged. In addition, genes related to invasion and metastasis are also important in understanding development of colorectal cancer. In this study, clinical significance and application of tumor suppressor genes and invasion related genes such as APC (adenomatous polyposis coli), DCC (deleted in colorectal carcinoma) tumor suppressor genes and invasion related gene, matrilysin were studied. In the mouse tumor induced by mutagen contained in cooked food, PhIP (2-amino-1-methyl-6- phenylimidazo [4,5-b] pyridine), nonsense mutations of APC gene that is similar to human colorectal cancer have been observed. These results suggested the quite interesting issue of mutagen contained in daily food having etiological role of colorectal cancer. DCC gene alteration, decreased expression of DCC mRNA was detected in 60% of advanced colorectal cancer. In all cases with liver metastasis, DCC expression was absent or markedly decreased, a finding that detection of DCC expression have an clinical importance that predicts metastatic potential of colorectal cancer. Matrilysin, the member of MMPs (matrix metalloproteinases) which degrade matrix components such as type IV collagen, laminin or fibronectin. In most of colorectal cancer, matrilysin was overexpressed in tumor cells. Matrilysin-transfected colorectal cancer cells showed more invasive ability in vitro and gained metastatic potential in SCID mice. Suppression of matrilysin expression by treated with all-trans retinoic acid (ATRA) or introduction of anti-sense matrilysin decreased the invasive ability in vitro. This result suggests that matrilysin plays an important role in invasion and metastasis and have a possibility of new anti-invasion therapy.

A venous mesenteric infarction in a 27-year-old patient is reported. This patient presented a genetic quantitative AT-III deficiency without anticoagulation therapy. Ultrasonography revealed portal vein thrombosis and laparoscopy showed mesenteric vein infarction. Laparotomy was performed mmediately and revealed segmental infarction of 60 cm of the jejunum which was resected; the portal vein was considered to be partially occluded on palpation. No strangulation or mechanical factors were identified. Immediately postoperatively the patient received therapeutic doses of heparin with AT-III concentrates to increase AT-III levels; no recurrent thrombotic episode was observed. A systematic second-look operation 24 hours postoperatively showed good bowel viability. Five days later, long-term anticoagulation with acenocoumarol was decided. Twelve days later, ultrasonography showed complete portal revascularization which was confirmed by a third surgical operation on D60.

Using chain-transfer polymerization, we have synthesized oligomers of poly(N-isopropylacrylamide) [poly(NIPAAm)] with one carboxyl group at the end of each oligomer chain. The lower critical solution temperature (LCST) of the oligomers is very close to that of homo-poly(NIPAAm) lacking the end carboxyl group. The carboxyl groups were activated in methylene chloride using N,N'-dicyclohexyl-carbodiimide (DCC) and N-hydroxysuccinimide (NHS). A conjugate of trypsin with the preactivated oligomer has been prepared. We studied the effect of oligomer to enzyme (O/E) ratio in the feed on the O/E ratio of the conjugate (the average number of oligomer chains conjugated to one trypsin molecule), assuming that only the primary amino groups of lysine residues and the amino terminal of trypsin would react. The O/E ratio of the conjugate was estimated by determination of the remaining primary amine groups on the trypsin molecule. More than 95% of the conjugate can be recovered by thermally induced precipitation.

Data obtained in experiments in 80 rats and in clinical observations of 24 patients with burns of the II and IIIA degrees showed the activation of vasculo-thrombocytic and procoagulant links of the hemostasis system with a simultaneous depression of the anticoagulative blood system. The thermal trauma is followed by a dramatic decrease of the concentration of heparin, fibrinogen, decreased activity of antithrombin-III, fibrinolysis and plasminogen activators. There was a simultaneous growth of concentration of antiplasmins (alpha 2-macroglobulin and alpha 1-antitrypsin).

We study the models for calcium (Ca) dynamics developed in earlier studies, in each of which the key component is the kinetics of intracellular inositol-1,4,5-trisphosphate-sensitive Ca channels. After rapidly equilibrating steps are eliminated, the channel kinetics in these models are represented by a single differential equation that is linear in the state of the channel. In the reduced kinetic model, the graph of the steady-state fraction of conducting channels as a function of log10(Ca) is a bell-shaped curve. Dynamically, a step increase in inositol-1,4,5-trisphosphate induces an incremental increase in the fraction of conducting channels, whereas a step increase in Ca can either potentiate or inhibit channel activation, depending on the Ca level before and after the increase. The relationships among these models are discussed, and experimental tests to distinguish between them are given. Under certain conditions the models for intracellular calcium dynamics are reduced to the singular perturbed form epsilon dx/d tau = f(x, y, p), dy/d tau = g(x, y, p). Phase-plane analysis is applied to a generic form of these simplified models to show how different types of Ca response, such as excitability, oscillations, and a sustained elevation of Ca, can arise. The generic model can also be used to study frequency encoding of hormonal stimuli, to determine the conditions for stable traveling Ca waves, and to understand the effect of channel properties on the wave speed.

Using a positional cloning strategy to identify the hemochromatosis gene (HFE), we isolated seven cDNAs by cDNA selection from a region of 400 kilobases (kb) located near the HLA-A and HLA-F loci. In this paper, we report the study of one of the corresponding genes, referred to as HCG V (hemochromatosis candidate gene), localized 150 kb centromeric to HLA-A. This gene was found to be expressed ubiquitously in the form of a 1.8 kb transcript, and to be apparently well conserved during evolution. The gene spanned 3.1 kb and is organized in three exons and two introns. The cDNA of 1620 base pairs (bp) showed an open reading frame of 378 bp, encoding for a 126 amino acid polypeptide which displayed a strong identity with the predicted product of a mouse Tctex-5 gene (t complex, testis expressed) localized in the t complex on chromosome 17. The HCG V gene was assessed as a potential candidate for hemochromatosis in regard to its localization in the linkage disequilibrium area between HFE and polymorphic markers. The study of deletions and point mutations in hemochromatosis patients revealed a single bp polymorphism within the coding region; however, no associated disease changes were found. Therefore we conclude that HCG V is unlikely to be involved in the pathogenesis of hemochromatosis.

An objective of this research is to verify the mechanism of anticoagulant activity of surface-immobilized heparin in the presence of plasma proteins. The competition and binding interaction between immobilized heparin and antithrombin III (ATIII)/thrombin have been described in vitro. However, the strong ionic character of heparin leads to its specific and nonspecific binding with many other plasma proteins. Most notably, fibronectin contains six active binding sites for heparin which may interfere with the subsequent binding of heparin with ATIII or thrombin. Heparin was covalently immobilized through polyethylene oxide (PEO) hydrophilic spacer groups onto a model surface synthesized by random copolymerization of styrene and p-aminostyrene. The binding interaction of immobilized heparin with ATIII was then determined in the presence of different fibronectin concentrations. The binding interaction was studied by first binding immobilized heparin with ATIII, followed by the introduction of fibronectin; heparin binding with fibronectin, followed by incubation with ATIII, and simultaneous incubation of surface immobilized heparin with ATIII and fibronectin. The extent of ATIII binding to heparin in each experiment was assayed using a chromogenic substrate for ATIII, S-2238. The results of this study demonstrate that the displacement of ATIII from immobilized heparin was proportional to the fibronectin concentration, and was reversible. Furthermore, the binding sequence did not play a role in the final concentration of ATIII bound to immobilized heparin.

We studied the urinary concentrating capacity in experimental hemochromatosis. Sprague-Dawley rats were randomized into iron (Fe)-loaded (injected sc with 1.2 g elemental iron/kg body weight as iron dextran) and pair-fed control groups. The urinary concentrating ability was studied after 10 months of iron loading. At basal condition, urine osmolality (Uosm) was significantly lower (P < 0.05) in the Fe-loaded rats compared with the control animals despite comparable urinary arginine-vasopressin (AVP) excretion in the two groups. Although 48-h water deprivation resulted in comparable rises in plasma concentration and urinary excretion of AVP in the two groups, maximal Uosm in the Fe-loaded animals was significantly lower than that seen in the control group (P < 0.01). Moreover, the observed urinary concentrating defect could not be corrected by pharmacological doses of exogenous AVP. There was no significant difference in renal chloride, sodium, calcium, or magnesium handling at either basal or sodium depleted states. Histologic studies showed marked iron deposition in the cortex and outer medulla accompanied by mild tubular atrophy particularly in the distal convoluted tubules. Thus, chronic experimental iron overload leads to nephrogenic diabetes insipidus marked by AVP-resistant urinary concentrating defect.

Low molecular weight heparins (LMWHs) are obtained by partial enzymatic or chemical depolymerisation and further chromatographic separation of standard unfractionated heparin. Most pharmacokinetic studies of LMWHs in humans have utilised the anticoagulant action against activated clotting factors. LMWH contains both high and low affinity fragments to antithrombin-III. Antithrombotic efficacy of LMWH is dependent on both fragments. LMWH levels peak 10 minutes after intravenous administration and are detectable for 5-8 hours. Following subcutaneous administration, maximum anti-factor Xa levels are demonstrable up to 12 hours or longer. Anti-factor IIa activity is eliminated faster than anti-factor Xa activity when measured with chromogenic substrates, but not with thrombin generation. The half-lives of the various LMWHs and their bioavailability, as measured by their anti-factor Xa or anti-factor IIa activities differ to a certain extent and clinical efficacy cannot be predicted from such data. On the basis of clinical studies, the antithrombotic potential of s.c. LMWHs for general surgery lies approximately around 2000 anti-factor Xa units and for high-risk patients around 4000 anti-factor Xa units, with no increased bleeding tendency. This is derived from investigations with enoxaparin, reviparin and dalteparin and is not valid for other LMWH derivatives.

Molecular alterations play a key role in the pathogenesis of gastrointestinal cancers. In the present paper we describe relevant molecular alterations in human pancreatic adenocarcinomas. Overexpression of growth factor receptors (EGF receptor, c-erbB2, c-erbB3, TGF beta receptor I-III), growth factors (EGF, TGF alpha, TGF beta-1-3, aFGF, bFGF), adhesion molecules (ICAM-1, ELAM-1) and gene mutations (p53, K-ras, DCC, APC) are present in a significant number of these tumors. These changes stimulate tumor growth and enhance the metastatic behavior of pancreatic cancer cells and thereby may contribute to shorter postoperative survival following tumor resection.

Coagulation inhibitors and fibrinolytic parameters were studied in twelve patients on continuous ambulatory peritoneal dialysis (CAPD) and ten patients on haemodialysis (HD). Patients on CAPD exhibited higher levels of ATIII and proteins C and S than those on HD. No significant differences were noted in tPA and PAI levels. Both groups of patients showed higher levels of tPA than controls. Besides, patients on HD had significantly lower levels of ATIII and protein C than controls. PAI levels in both patient groups were similar to those of the controls, but tPA levels were higher in patients than in controls. These results indicate that HD is associated with marked diminution in the circulating levels of coagulation inhibitors. This is in contrast to CAPD patients who showed elevated levels of these inhibitors, despite their significant loss in the dialysate. The finding of enhanced fibrinolysis in both patient groups may be a natural protective mechanism against the development of a thrombotic tendency.

Genetic hemochromatosis is one of the most common inherited disorders in Caucasian populations. The disease frequency in Caucasian populations in Australia, Europe, and the United States is 1:300-400. The basic genetic defect remains unknown, although the hemochromatosis gene is closely linked to histocompatibility antigen (HLA) A, thus allowing early diagnosis in members of affected families. Many factors-environmental, genetic, and nongenetic in nature-influence the degree of iron loading in affected individuals. In particular, pathologic and physiologic blood loss and blood donation influence iron stores in hemochromatosis. The iron concentration in the liver is an important determinant of survival because a hepatic iron concentration in excess of 400 mumol/g dry weight is usually associated with cirrhosis. Patients with cirrhosis secondary to hemochromatosis are at risk of heptocellular carcinoma and complications of portal hypertension. The combination of improved awareness of the condition and the use of HLA typing to identify affected family members has led to earlier diagnosis and therapy, and to an improvement in overall survival.

Benign familial chorea (BFC) is a rare neurological disease with an autosomal dominant transmission. The disorder is characterized by its early onset in childhood, a non-progressive course of choreatic movements and the absence of intellectual impairment. There is one study describing an expanded (CAG)n repeat in the gene IT15 (Huntington) on chromosome 4p (causative for Huntington's chorea) in a family reported to have BFC that was diagnosed on the basis of onset and non-progressive course. We failed to find an expansion of the (CAG)n repeats in an Austrian family having BFC. The three affected individuals of the family had 18-25 CAG repeats. These results indicate that the diagnostic criteria for BFC should include a normal result in the analysis of the (CAG)n repeat region of the Huntington gene.

The interactions between antithrombin III (ATIII), thrombin, and surface immobilized heparin were investigated. Carboxylated polystyrene modified with covalently immobilized albumin-heparin conjugate contain sites which can bind ATIII from buffer and plasma solutions. Approximately 65% of the ATIII molecules present at the heparinized surface either adsorbed from a buffer or plasma solution, exchanged with ATIII in buffer solution. The exchange between surface bound ATIII and ATIII in solution was repeated several times on the same heparinized surface. The number of binding sites that could bind and release ATIII was much higher when the heparinized surface was exposed to an ATIII containing buffer solution than to a plasma solution. The reduction in binding sites available for ATIII using plasma solutions as compared to buffer solutions could be explained by the competition of other plasma proteins with ATIII for the heparinized surface. It was observed that heparin binding proteins were able to compete with ATIII for binding to the immobilized heparin. Furthermore adsorption of proteins on the heparinized surface significantly reduced the availability of binding sites for ATIII. Exposure of thrombin to the heparinized surface resulted in thrombin activity at the surface. The thrombin activity on the heparinized surfaces was lower on surfaces with a higher ATIII concentration. The activity of surface bound thrombin was not affected by the presence of other plasma proteins. Enzymatically active thrombin molecules present at the heparinized surface were completely inactivated when the surface was exposed to a solution containing ATIII. The inactivation rate of surface bound thrombin by ATIII was higher than the rate of the uncatalyzed inactivation of thrombin in solution. Part of the Thrombin-Antithrombin III (TAT) complexes (10-20%) that were formed upon inactivation of thrombin remained bound to the heparinized surface. In general it was concluded that only the surface immobilized heparin molecules that can bind ATIII in a reversible way determine the anticoagulant properties of the surface. The mechanism of inactivation of a protease on a heparinized surface depends either on the catalytic effect of heparin on the inactivation rate of proteases by ATIII or on an increased uncatalytic inactivation due to increased concentrations of ATIII near the surface as compared to the concentration of ATIII in the bulk phase.

A characteristic feature of colorectal cancer genesis is its stepwise progression, which offers unique possibilities for studying its development. There are two principal kinds of mutation leading to uncontrolled cell proliferation and cancer. The first renders a stimulatory gene hyperactive--generation of an oncogene--and the second is the inactivation of a tumour suppressor gene. Current knowledge suggest that the change from normal mucosa to a small adenoma may be mediated by mutations of the APC gene and MCC gene on chromosome 5, by chromosome 5 deletion, by c-myc activation, and by DNA hypomethylation. The development to a large adenoma may be caused by Ki-ras mutation and further change to a dysplastic adenoma by deletion of the DCC gene on chromosome 18. The ability to become an invasive carcinoma may then be mediated by p53 mutations and deletion of chromosome 17p. Identification of genetic markers for metastatic disease is under progress.

Redox cycling is a characteristic of transition metals such as iron. Iron is hypothesized to have been actively involved in the birth of primitive life on earth through the generation of reducing equivalents in the presence of UV light. Iron is an essential metal in mammals for oxygen transport by hemoglobin and for the function of many enzymes including catalase and cytochromes. However, the "free" or "catalytic" form of iron mediates the production of reactive oxygen species via the Fenton reaction and induces oxidative stress. Serum "free" iron is observed in rare situations such as in severe hemochromatosis in which serum transferrin is saturated. However, it is known that superoxide can release "free" iron from ferritin and hemosiderin in the cell. "Free" iron is quite cytotoxic as well as mutagenic and carcinogenic. Iron compounds were first reported to induce sarcomas in rats by Richmond in 1959. Thereafter, several iron-induced carcinogenesis models were established, including the ferric nitrilotriacetate model by Okada and colleagues. Iron may have a role in the carcinogenic process of other transition metals such as copper and nickel, or other kinds of carcinogens such as nitrosamine and even virus-induced carcinogenesis. In humans, genetic hemochromatosis and asbestosis are two major diseases associated with iron-induced carcinogenesis. There is an increasing number of reports of an association between increased body iron stores and increased risk of cancer. Iron-induced oxidative stress results in two possible consequences: (1) redox regulation failure that leads to lipid peroxidation and oxidative DNA and protein damage; (2) redox regulation that activates a variety of reducing and oxystress-protective mechanisms via signal transduction. Both consequences appear to play a role in iron-induced carcinogenesis.

The tumor necrosis factor receptor superfamily at present consists of ten different transmembrane (type I) glycoproteins with characteristic limited sequence homology for the cysteine-rich repeats in the extracellular domain. In parallel the tumor necrosis factor ligand super-family has been recognized by discovery of ligands for all members of the receptor superfamily. These molecules are also transmembrane (type II) glycoproteins, with the exception of lymphotoxin-alpha which is the only entirely secreted protein of the tumor necrosis factor-like proteins. Several members of the ligand superfamily, including tumor necrosis factor and CD95L also exist in a biologically active soluble form. The tumor necrosis factor ligand superfamily contains at present ten different proteins. In addition, NGFR p75 binds to a second family of proteins (neurotrophins). These nerve growth factor-like dimeric soluble molecules are basic neurotrophic factors and the five members (NGF, BDNF, NT-3, NT-4, NT-5) are not related to the tumor necrosis factor superfamily ligands. The members of the tumor necrosis factor ligand superfamily (TNF, LT-alpha, LT-beta, CD27L, CD30L, CD40L, CD95L, 4-IBB, OX40L, TRAIL) share common biological activities, but some properties are shared by only some ligands, while others are unique. The diverse biological activities triggered through tumor necrosis factor receptors have been linked to the regulation of cellular activation, including immune responses and inflammatory reactions, but also with the pathology of a series of human diseases.

Inactivation of the "deleted in colon cancer" (DCC) gene on chromosome 18 is known to be associated with the tumorigenesis and metastasis of colorectal cancer. In the present study, we investigated the expression of DCC and the c-erbB-2 product in surgical specimens from 45 patients with breast cancer by immunohistochemical staining, and found the expression of DCC to be decreased in 23 (51%) tumors. In 8 years of follow-up, 11 of 22 (50%) patients with DCC-positive staining tumors, and 17 of 23 (74%) patients with DCC-negative tumors developed recurrence. The stratified analysis, according to the status of axillary lymph node metastasis, showed the same tendency. Overexpression of erbB-2 was detected in 13 (29%) of the 45 breast cancer specimens, but there were no differences in the relapse rate between patients with erbB-2 positive and those with erbB-2 negative tumors. Although the individual alteration of DCC or erbB-2 did not possess independent prognostic significance for the prediction of recurrence, patients with tumors having the double alteration of DCC-negative and erbB-2-positive showed adverse relapse-free survival (0.025 < P < 0.05). These findings suggest that a decrease in DCC expression and erbB-2 overexpression may influence the progression of breast carcinoma.

The DNA of 147 patients of European origin clinically diagnosed with idiopathic hemochromatosis and 193 controls was examined for mutations of the HLA-H gene at nt 845 and nt 187. One hundred twenty-one (82.3%) of the hemochromatosis patients were homozygous and 10 (6.8%) heterozygous for the 845A (C282Y) mutation. All of the homozygous patients were also homozygous for nt 187C, and all 845A heterozygotes had at least one copy of 187C. Thus, the nt 845 and nt 187 mutations were in complete linkage disequilibrium; nt 187 was a C on all chromosomes with the 845A mutation. Eight of the 10 heterozygotes for 845A were heterozygous for 187G(H63D). The excess of heterozygotes at both nt 187 and nt 845 suggested either the presence of as yet undiscovered mutations existing in trans with 845A and in linkage disequilibrium with 187G, or that the 187G itself is a deleterious mutation, which in concert with the 845A can give rise to hemochromatosis. None of the 193 normal controls were homozygous for 845A and 29/193 (15%) were heterozygous for 845A. Although 47/193 (24.3%) of normal controls were heterozygous for the 187G mutation only two of these carried the 845A mutation. If the 187G mutation complemented the 845A mutation with high penetrance in causing hemochromatosis, then the population frequency of the two genes would require that a high proportion of patients with hemochromatosis be heterozygous for 845A and 187G. Instead, the frequency of homozygotes for the 845A mutation was much higher than that of the 845A/187G genotype. Based on our data, the penetrance of the 845A/187G genotype is only 1.5% and based on the data of Feder et al. only 0.5%. In contrast, the penetrance of the homozygous 845A/845A genotype seems to be very high. Thus, screening for this genotype should be very useful.

Diamine silver fluoride [AgF: (NH3)2AgF] is a unique fluoride solution used clinically in Japan for primary teeth. AgF has been shown to reduce dental caries and dentine hypersensitivity. However, AgF also stains teeth black due to sulfide precipitation and thus is unacceptable for permanent teeth. In the present study, the potential value of ammonium hexafluorosilicate [SiF: (NH4)2SiF6], which has a formula similar to that of AgF but contains no Ag, was studied with respect to its ability to fluoridate apatite. Hydroxyapatite [HAP: Ca10(PO4)6(OH)2] powder was treated with several topical fluoride solutions, i.e. neutral sodium fluoride, acidulated phosphate fluoride (APF) along with AgF and SiF. Following treatment, the HAP powders were analyzed for their fluorine content, crystallinity changes, and solubility in acidic solution. The highest level of fluoridated HAP [FAP: Ca10(PO4)6(OH)xF2-x] was observed in HAP powder treated with SiF, even though more total fluorine in the form of FAP and CaF2 was observed in HAP powder samples treated with APF. Presumably as a consequence of this higher level of FAP formation after SiF treatment, the highest level of crystallinity along with the lowest solubility in acidic solution were observed in HAP powder treated with SiF solution. We concluded, therefore, that SiF may have potential value for use as a topical fluoride solution.

In a long-term experiment MCF-7 cells showed an exponential proliferation rate in fetal calf serum while they become quiescent in growth-factor- and steroid-free serum. The mitogenic activity of this cell line was increased by oestradiol and insulin, exposure to both hormones showing a synergic effect. These mitogen-mediated effects are inhibited by genistein a phytoestrogen which, by itself at the doses used, did not affect either the basal proliferation rate or the total protein content. Immunoblotting and Scatchard analysis reveal respectively that insulin increases the oestrogen receptor (ER) content and its binding capacity. The presence of genistein decreases the ER content and completely abolishes the binding capacity of this protein. Insulin is able to increase progesterone receptor (PR) levels while, in the presence of genistein, the above-reported effect was completely abolished. On the basis of the latter data the authors suggest that insulin is able to affect the phosphorylation status of ER and PR, inducing functional changes in both proteins, although this was in the absence of their natural ligands. Indeed, the presence in the medium of a tyrosine kinase inhibitor such as genistein could substantially decrease both ER and PR levels in these cells.

Huntingtin expression was examined by Western blot and immunoprecipitation studies of lymphoblastoid cell lines from Huntington's disease (HD) homozygotes, heterozygotes, and a phenotypically normal individual with a t(4p16.3;12p13.3) breakpoint in the HD gene. The latter produced a reduced level of normal huntingtin without evidence of an altered protein, indicating that simple loss of huntingtin activity does not cause HD. In juvenile onset HD heterozygotes, NH2- and COOH-terminal antisera revealed reduced relative expression from the mutant allele. Pulse-chase studies indicated that huntingtin is a stable protein whose differential allelic expression is not due to destabilization of the mutant isoform. No stable breakdown products specific to mutant huntingtin were detected in either HD homozygotes or heterozygotes. These data are consistent with HD involving either a gain of function or a dominant negative loss of function that operates within severe constraints and suggest that in either case the pathogenic process is usually saturated by the amount of abnormal huntingtin produced from a single mutant allele.

<h4>Purpose</h4>To present data to support the hypothesis that human retinoblastoma tumors form by a multistep process.<h4>Methods</h4>Survey of the karyotype of 90 human retinoblastomas, with emphasis on changes in chromosomal regions known to contain oncogenes and tumor suppressor genes.<h4>Conclusion</h4>The data support the hypothesis that retinoblastoma occurs after multiple and often varied steps, similar to other human malignancies.

Disseminated intravascular coagulation (DIC) syndromes can be defined as the formation of fibrin deposits within the microcirculation, occurring in definite clinical situations. Their biological counterpart is a consumption coagulopathy. The clinical profiles of DIC have been well known for decades, are multiform and range from latency to overwhelming haemorrhagic diatheses, including also characteristic but rare situations, such as purpura fulminans, acral cyanosis and pictures resembling thrombotic thrombocytopenic purpura or haemolytic-uraemic syndrome. Biological tests of DIC show a consumption coagulopathy, displayed on the standard haemostasis sheet; along with signs of paracoagulation and/or of secondary fibrinolysis (FDP). New tests have recently been introduced: D-dimers are specific and sensible; Antithrombin-III, protein C and alpha 2-antiplasmin also can sometimes be useful. The knowledge of the pathophysiology of DIC has made advances with passing years. Fibrin deposits may be non-occlusive, and indeed they are swiftly removed by a secondary fibrinolysis. Except in very rare situations, such as those leading to a cortical renal necrosis, and perhaps in some ARDS, there is little evidence relating DIC to organ failure syndromes. Moreover, there is no clear relationship between the severity of the consumption coagulopathy and the prognosis. For instance, the mortality is much lower in abruptio placentae, where the coagulopathy is very severe, than in septic shock, where it is usually moderate. In septic shock, the disorders of haemostasis were related initially to a platelet activation, then to an activation of the contact system (releasing kinins and triggering complement cascade), and nowadays to the activation of the extrinsic coagulation system. The treatment of DIC is mainly the treatment of its cause. Indications for heparin therapy should be strictly limited to a few exceptional circumstances. When haemorrhagic diathesis threatens, FPC and/or platelet transfusion may be indicated. Aprotinin can be useful in rare cases of overwhelming secondary fibrinolysis. Trials with antithrombin-III or C1-esterase inhibitors are in progress.

The study was carried out on 20 newborns with diagnosed premature ablation of placenta of 0,I,II clinical degree. In comparison to healthy newborns (n = 30) statistically significant differences were proved in: ATIII activity-48% (65%), plasminogen concentration-50% (63%), alpha 2antyplasmin activity 53% (67%); euglobulin clot fibrinolysis time 70 (95) min., and fibrinogen concentration 1.4 (1.8) g/l. The presented differences prove increased coagulation system activation in newborns from mothers with symptoms of premature ablation of placenta. Activity changes of hemostasis system in cases of premature ablation of placenta have the features of disorders accompanying intrauterine fetal anoxia.

The protein and DNA polymorphisms of human antithrombin III (ATIII) were studied by isoelectric focusing (IEF) and PCR techniques in a Chinese population sample (n = 51). Products of two alleles within the coding region of the ATIII locus were observed by IEF. The allele frequencies were: ATIII*1 = 0.9608, ATIII*2 = 0.0392. The PCR analysis of the fragment length polymorphism (FLP) for the ATIII 5' locus disclosed two alleles. Their frequencies were: ATIII5'*1 = 0.4118, ATIII 5'*2 = 0.5882. An association analysis between these two ATIII polymorphisms revealed the incompatibility between the ATIII 5'FLP and the ATIII IEF variants, indicating that the ATIII 5' FLP is not responsible for ATIII variants.

We postulated that the severity of iron overload in homozygous hemochromatosis probands is related to the expression of HLA-A3 or D6S105 allele 8. Therefore, we used these markers to characterize Alabama hemochromatosis probands and normal control subjects. We then quantified the blood removed by phlebotomy to exhaust body iron stores and maintain normal serum ferritin concentrations in our hemochromatosis probands. Induction and maintenance phlebotomy requirements were significantly greater in presumed HLA-A3 homozygotes or in D6S105 allele 8 homozygotes than in homozygous probands lacking these markers. Intermediate values were observed in probands who were HLA-A3 or allele 8 heterozygotes, respectively. We also analyzed data from males and females separately. Among subjects of the same sex, the induction and maintenance phlebotomy requirements in subjects presumed to be HLA-A3 homozygotes or in allele 8 homozygotes were greater than those of other groups. Our results support the hypothesis that the severity of iron overload in hemochromatosis is determined predominantly by genetic factors, and provide evidence that two or more mutations for hemochromatosis exist. However, the design of our study does not permit a distinction to be made between allelic and locus heterogeneity for the hemochromatosis gene(s).

The minimization of viral contamination in therapeutic proteins produced in transgenic goats' milk can be achieved by a combinatorial approach. It begins with reduction in the risk in the starting material followed by appropriate clearance/inactivation steps in the purification process. To minimize risk in the starting material, Genzyme Transgenics Corporation (GTC)'s closed goat herds are subjected to routine serological surveillance for known viral diseases, especially those transmitted through milk. Although scrapie is defined as a slow-acting virus of sheep and goats, its incidence in goats in the US is rare (only four cases) and all four were in goats co-mingled with scrapie-infected sheep. All GTC's domestic goats were selected for previous non-exposure to sheep, cows or scrapie. In addition, milk, which is the starting material for transgenic protein production, is categorized as non-infectious for prions. Standard operating procedures are in place at GTC Farm sites to minimize human, animal or vehicular vectoring of viral diseases and the transgenic production animals are milked according to high standard Good Agricultural Practices (GAP). The transgenic protein (ATIII) purification process contains steps that should provide a high level of viral reduction. Validation of viral and prion removal will also be undertaken.