Cathepsin D (CD) up-regulation has been associated with human malignancy and poor prognosis. Thrombin upregulated CD mRNA and protein in eight tumor cell lines as well as in human umbilical vascular endothelial cells (HUVEC). Thrombin increased the secretion of CD by 3-to 8-fold and enhanced chemotaxis (f2-fold) in 4T1 murine mammary CA cells, which was completely inhibited with the knockdown of CD. Secreted 4T1 CD induced neoangiogenesis by 2.4-fold on a chick chorioallantoic membrane, which was blocked in CD-KD cells. The addition of pure CD (2 ng) to the chick chorioallantoic membrane increased angiogenesis by 2.1-fold, which was completely inhibited by Pepstatin A (Pep A). CD enhanced human HUVEC chemotaxis and Matrigel tube formation by 2-fold, which was then blocked by Pep A. CD enhanced HUVEC matrix metalloproteinase 9 (MMP-9) activity by f2-fold, which was completely inhibited by Pep A as well as a generic MMP inhibitor, GM6001. The injection of CD-KD 4T1 cells into syngeneic mice inhibited tumor growth by 3-to 4-fold compared with empty vector (EV) cells. Hirudin, a specific thrombin inhibitor, inhibited the growth of wild-type and EV cells by 2-to 3-fold, compatible with thrombin up-regulation of CD. CD and thrombin also contributed to spontaneous pulmonary metastasis; 4-fold nodule inhibition with CD versus EV and 4.6-fold inhibition with hirudin versus EV (P < 0.02). Thus, thrombin-induced CD contributes to the malignant phenotype by inducing tumor cell migration, nodule growth, metastasis, and angiogenesis. CD-induced angiogenesis requires the proteolytic activation of MMP-9. [Cancer Res 2008;68(12):4666-73]

Heterozygous mutations of ELA2, encoding the protease neutrophil elastase (NE), cause either autosomal dominant cyclic neutropenia or severe congenital neutropenia (SCN). Three hypotheses have been proposed for how allelic mutations produce these different disorders: 1) disruption of proteolytic activity; 2) mislocalization of the protein; or 3) destabilization of the protein resulting in induction of the unfolded protein response. As with other dominant diseases with reduced reproductive fitness, sporadic cases can result from new mutations not inherited from either parent. Here we report an exceptional genetic phenomenon in which both a cyclic neutropenia patient and an SCN patient each possess two new ELA2 mutations. Because of the rarity of the phenomenon, we investigated the origins of the mutations and found that both arise nonmosaically and in cis from the paternally-inherited allele. Moreover, these cases offer a unique opportunity to investigate molecular pathways distinguishing these two forms of hereditary neutropenia. We have characterized the mutants separately and in combination, with respect to their effects on proteolysis, subcellular trafficking, and induction of the unfolded protein response. Each pair of mutations acts more or less additively to produce equivalent net effects on reducing proteolytic activity and induction of the unfolded protein response, yet each has different and somewhat opposing effects on disturbing subcellular localization, thus offering support for a role for protein mistrafficking as a disease mechanism.

Ozonated autohemotherapy (O3-AHT) is used in the treatment of atherosclerotic ischemia of lower limbs (AILL). The impact of ozone on serum lipids and endothelium injury is of particular interest since these factors are important in the development of atherosclerotic lesions. To evaluate this issue, a prospective, placebo-controlled study was designed. Twelve hemodialyzed subjects with AILL received autohemotherapy with oxygen as a control followed by O3-AHT with ozone concentration of 50 micro g/ml. Serum lipids and plasma activity of von Willebrand factor (vWF) were measured. After O3-AHT, total cholesterol significantly decreased compared to the baseline (-8.34%) [P < 0.01]. LDL cholesterol was also significantly lower than the initial value (-17.71%) [P < 0.001]. No significant changes in the activity of vWF were found after the first session of O3-AHT and after all nine sessions of O3-AHT. The study demonstrated that O3-AHT did not affect deleteriously the endothelium in patients with chronic renal failure on maintenance hemodialysis. It may stimulate beneficial changes in serum lipid profile manifesting as a decrease in the total- and LDL-cholesterol levels.

The incidence of pediatric venous thromboembolism (VTE) has been increasing in the past few decades and can be associated with significant mortality and morbidity. There are known risk factors associated with VTE, including estrogen therapy. However, the relationship between testosterone and VTE remains unclear. Here, we present a 17-year-old female-to-male transgender patient without a history of inherited thrombophilia, who developed pulmonary embolism while receiving testosterone injections for gender dysphoria. Despite the limited data on testosterone and the risk of VTE, health care providers should counsel patients and family about the possible increased risk of VTE when starting testosterone.

Heterozygous mutations in the gene, ELA2, encoding neutrophil elastase, cause cyclic neutropenia and are the commonest cause of severe congenital neutropenia, a genetically heterogeneous leukemia-predisposing condition also known as Kostmann syndrome. The cataloged mutations have divergent effects on the enzyme, defying singular explanations for how mutations cause disease. Until now, all mutations have appeared to be constitutional and heritable. We describe two exceptional patients with Kostmann syndrome and cyclic neutropenia who each respectively demonstrate somatic mosaicism in the bone marrow of multiple different ELA2 sequences representing acquired mutations of constitutionally wild type alleles or, conversely, acquired reversion of constitutionally mutant alleles. The first patient has Kostmann syndrome, lacks a family history of neutropenia, and comes from a consanguineous population where recessive inheritance is anticipated. Sequencing of PCR-amplified genomic DNA from peripheral blood revealed two novel, apparently heterozygous ELA2 mutations, V69L and V72L, absent in both parents. The mother did possess an adjacent intronic polymorphism inherited by the patient, and we employed this polymorphism to determine the parent of origin of the mutation by sequencing genomic PCR fragments subcloned in bacteria. Remarkably, either or both mutation, as well as a third (V72M), appeared on either maternal or paternal alleles. The mutations have thus arisen independently in multiple different cells of the bone marrow. We propose that Kostmann syndrome here results from inheritance of an unidentified second gene and that the resulting neutropenia paradoxically leads to the selection in the bone marrow of counteracting, “neutrophilic” ELA2 mutations. The second patient has cyclic neutropenia lessening in severity since birth. Sequencing PCR-amplified genomic DNA from peripheral blood revealed two different, apparently heterozygous ELA2 mutations, IVS4+1 G>A (ΔV161-F170), the commonest ELA2 mutation observed in cyclic neutropenia, and T99I, not previously reported. Both mutations were absent in the unaffected parents but present in the patient’s buccal cells, demonstrating their de novo constitutional origins. Although the parents lacked distinguishing polymorphisms, the presence of two mutations allowed us to assess for somatic mosaicism. We subcloned genomic PCR fragments in bacteria and found that, in addition to wild type and both mutations in cis, some alleles contained just one or the other mutation, indicating that the mutations have undergone stepwise reversion in multiple, independent cells in the bone marrow. In colony formation assays of circulating stem cells, reversion to wild type occurred exclusively in CFU-GM, but not more primitive CFU-GEMM, suggesting selection in myeloid progenitors. Selection and counter-selection in these two complementary patients imply that there are two opposing categories of ELA2 mutations: some cause neutropenia and others suppress it. This may be the first description of an unexpected genetic phenomenon, where different mutations in a single gene can either cause or ameliorate the same disease, and may explain the divergent effect of various mutations.