Activated macrophages for treating skin ulceration: gene expression in human monocytes after hypo-osmotic shock.

Macrophages play a major role in almost all stages of the complex process of wound healing. It has been previously shown that the incorporation of a hypo-osmotic shock step, in the process of monocyte-concentrate preparation from a blood unit, induces monocyte/macrophage activation. As the macrophages are produced using a unique, closed and sterile system, they are suitable for local application on ulcers in elderly and paraplegic patients. Enhanced phagocytosis by the activated cells, as well as increased secretion of cytokines such as IL-1, IL-6, were detected in a recent study which are in accord with the very encouraging clinical results. In the present study, we used DNA microarrays to analyse the differential gene expressions of the hypo-osmotic shock-activated monocytes/macrophages and compare them to non-treated cells. Of the genes that exhibited differences of expression in the activated cell population, 94% (68/72) displayed increased activity. The mRNA levels of 43/68 of these genes (63%) were found to be 1.5-fold or higher (1.5-7.98) in the activated macrophages cell population as compared to the non-treated cells. Only four genes were found to have lower mRNA levels in the activated cells, with ratios of expression of 0.62-0.8, which may suggest that the changes are insignificant. A significant number of the genes that showed increased levels of expression is known to be directly involved in macrophage function and wound healing. This may correlate with the increased secretion of different cytokines by the activated macrophages depicted previously. Other groups of genes expressed are known to be involved in important pathways such as neuronal growth and function, developmental defects and cancer. The hypo-osmotic shock induces a gene expression profile of cytokines and receptors in the activated cells. These may evoke potential abilities to produce a variety of protein products needed in the wound healing process and may bring to light possibilities for other therapeutic applications of these cells.

Nonmuscle myosin heavy chain IIA mutations define a spectrum of autosomal dominant macrothrombocytopenias: May-Hegglin anomaly and Fechtner, Sebastian, Epstein, and Alport-like syndromes.

May-Hegglin anomaly (MHA) and Fechtner (FTNS) and Sebastian (SBS) syndromes are autosomal dominant platelet disorders that share macrothrombocytopenia and characteristic leukocyte inclusions. FTNS has the additional clinical features of nephritis, deafness, and cataracts. Previously, mutations in the nonmuscle myosin heavy chain 9 gene (MYH9), which encodes nonmuscle myosin heavy chain IIA (MYHIIA), were identified in all three disorders. The spectrum of mutations and the genotype-phenotype and structure-function relationships in a large cohort of affected individuals (n=27) has now been examined. Moreover, it is demonstrated that MYH9 mutations also result in two other FTNS-like macrothrombocytopenia syndromes: Epstein syndrome (EPS) and Alport syndrome with macrothrombocytopenia (APSM). In all five disorders, MYH9 mutations were identified in 20/27 (74%) affected individuals. Four mutations, R702C, D1424N, E1841K, and R1933X, were most frequent. R702C and R702H mutations were only associated with FTNS, EPS, or APSM, thus defining a region of MYHIIA critical in the combined pathogenesis of macrothrombocytopenia, nephritis, and deafness. The E1841K, D1424N, and R1933X coiled-coil domain mutations were common to both MHA and FTNS. Haplotype analysis using three novel microsatellite markers revealed that three E1841K carriers--one with MHA and two with FTNS--shared a common haplotype around the MYH9 gene, suggesting a common ancestor. The two new globular-head mutations, K371N and R702H, as well as the recently identified MYH9 mutation, R705H, which results in DFNA17, were modeled on the basis of X-ray crystallographic data. Altogether, our data suggest that MHA, SBS, FTNS, EPS, and APSM comprise a phenotypic spectrum of disorders, all caused by MYH9 mutations. On the basis of our genetic analyses, the name "MYHIIA syndrome" is proposed to encompass all of these disorders.

Subgroup of patients with Philadelphia-positive chronic myelogenous leukemia characterized by a deletion of 9q proximal to ABL gene: expression profiling, resistance to interferon therapy, and poor prognosis.

A major deletion of the region proximal to the rearranged ABL gene on 9q was found in 14/94 (15%) of chronic myelogenous leukemia Philadelphia-positive patients by interphase fluorescent in situ hybridization with the BCR/ABL extra signal dual-color probe. Preliminary results indicated that the prognosis of the deletion 9q patients is probably worse than that of the non-deletion 9q patients. Twelve of the 14 deletion 9q patients were treated with alpha-interferon and none had a major cytogenetic response. The median duration of the chronic phase in patients not undergoing BMT was significantly shorter for the deletion 9q patients as compared to the non-deletion 9q patients (p =.0144). DNA microarray technology was performed in order to compare the gene expression patterns between the two groups of patients. A number of genes exhibiting differential expression, especially involving cell adhesion and migration, were identified. This finding may identify a sub-group of CML patients with different cell properties and a relatively poor prognosis.

Mutation analysis of the FAS and TNFR apoptotic cascade genes in hematological malignancies.

OBJECTIVE: The existence of properly functioning apoptotic pathways is of utmost importance in the maintenance of a normal cell count. Several groups have searched for mutations in the FAS receptor, a well-characterized apoptotic protein carrying a death domain, and reported the existence of rare mutations in multiple myeloma, T-acute lymphoblastic leukemia (T-ALL), and adult T-cell leukemia. Our aim was to expand these searches by looking for mutations in the death domains of FAS, FADD, TNFR, TRADD, and RIP, in the promoter region of FAS, and in the protease domain of caspase 10, in a larger variety of hematological malignancies, some of which express an apoptosis-resistant phenotype. METHODS: We extracted RNA and DNA samples from 92 hematological malignancies: chronic lymphocytic leukemia (CLL; 31 cases), chronic myelogenous leukemia (CML; 28 cases), essential thrombocythemia (ET; 8 cases), acute lymphocytic leukemia (ALL; 6 cases), acute myeloblastic leukemia (AML; 6 cases), hairy-cell leukemia (HCL; 3 cases), Burkitt's lymphoma (3 cases), polycythemia vera (PV; 3 cases), myelofibrosis (2 cases), and chronic myelomonocytic leukemia (CMML; 2 cases) and performed PCR-SSCP and sequence analysis on these samples. RESULTS: Five polymorphic patterns were found: three in the death domain of the FAS gene in CML patients, one in the promoter of this gene in a CLL patient, and the fifth in the death domain of the TRADD gene in a CML patient. No mutations, altering amino acids, were found in these genes in any of the aforementioned malignancies. CONCLUSIONS: These observations imply that mutations in the death domains of FAS, FADD, TNFR, TRADD, and RIP and in the protease domain of caspase 10 are not a major cause for failure of apoptosis in hematological malignancies, mainly CML and CLL. Regulatory and epigenetic abnormalities in these apoptotic cascade members and aberrations in other components of all death machinery should be looked for.

Autosomal-dominant giant platelet syndromes: a hint of the same genetic defect as in Fechtner syndrome owing to a similar genetic linkage to chromosome 22q11-13.

Families with 3 different syndromes characterized by autosomal dominant inheritance of low platelet count and giant platelets were studied. Fechtner syndrome is an autosomal-dominant variant of Alport syndrome manifested by nephritis, sensorineural hearing loss, and cataract formation in addition to macrothrombocytopenia and polymorphonuclear inclusion bodies. Sebastian platelet syndrome is an autosomal-dominant macrothrombocytopenia combined with neutrophil inclusions that differ from those found in May-Hegglin syndrome or Chediak-Higashi syndrome or the Dohle bodies described in patients with sepsis. These inclusions are, however, similar to those described in Fechtner syndrome. Other features of Alport syndrome, though, including deafness, cataracts, and nephritis, are absent in Sebastian platelet syndrome. Epstein syndrome is characterized by macrothrombocytopenia without neutrophil inclusions, in addition to the classical Alport manifestations-deafness, cataracts, and nephritis-and it is also inherited in an autosomal-dominant mode. We mapped the disease-causing gene to the long arm of chromosome 22 in an Italian family with Fechtner syndrome, 2 German families with the Sebastian platelet syndrome, and an American family with the Epstein syndrome. Four markers on chromosome 22q yielded an LOD score greater than 2.76. A maximal 2-point LOD score of 3.41 was obtained with the marker D22S683 at a recombination fraction of 0.00. Recombination analysis placed the disease-causing gene in a 3.37-Mb interval between the markers D22S284 and D22S693. The disease-causing gene interval in these 3 syndromes is similar to the interval described recently in an Israeli family with a slightly different Fechtner syndrome than the one described here. Recombination analysis of these 3 syndromes refines the interval containing the disease-causing gene from 5.5 Mb to 3.37 Mb. The clinical likeness and the similar interval containing the disease-causing gene suggest that the 3 different syndromes may arise from a similar genetic defect.

Amplification of immunological functions by subcutaneous injection of intermediate-high dose interleukin-2 for 2 years after autologous stem cell transplantation in children with stage IV neuroblastoma.

BACKGROUND: Immunotherapy given post-autologous stem cell transplantation may eliminate residual tumor cells escaping the conditioning protocol. METHODS: Five children suffering from stage IV neuroblastoma were treated by recombinant interleukin-2 (IL-2) post-autologous peripheral blood stem cell transplantation. The patients' peripheral mononuclear cells were monitored for CD3+ and CD56+ levels, their proliferative response and killing of various cell lines targets. RESULTS: An increase in the level of total lymphocytes, mainly due to expansion of T cells, and enhanced proliferative response to phytohemaglutinin were observed. Elevated cytotoxicity against K562 and neuroblastoma target cells was detected in four patients and against K562 targets in one patient. Toxicity included mild thrombocytopenia, and fever in four patients and mild to moderate encephalopathy which necessitated withdrawing one patient from the protocol. Three of five patients studied are alive today, one of them whose IL-2 was stopped, is in relapse. Two patients have died. CONCLUSIONS: Immunotherapy with s.c. intermediate-high dose IL-2 is feasible and results in expansion of T cells and in stimulation of killing activity against several targets including in some cases, neuroblastoma tumor cells.

Genetic linkage of autosomal-dominant Alport syndrome with leukocyte inclusions and macrothrombocytopenia (Fechtner syndrome) to chromosome 22q11-13.

Fechtner syndrome is an autosomal-dominant variant of Alport syndrome, manifested by nephritis, sensorineural hearing loss, cataract formation, macrothrombocytopenia, and polymorphonuclear inclusion bodies. As opposed to autosomal-recessive and X-linked Alport syndromes, which have been genetically well studied, the genetic basis of Fechtner syndrome remains elusive. We have mapped the disease-causing gene to the long arm of chromosome 22 in an extended Israeli family with Fechtner syndrome plus impaired liver functions and hypercholesterolemia in some individuals. Six markers from chromosome 22q yielded a LOD score >3.00. A maximum two-point LOD score of 7.02 was obtained with the marker D22S283 at a recombination fraction of 0. Recombination analysis placed the disease-causing gene in a 5.5-Mb interval between the markers D22S284 and D22S1167. No collagen genes or genes comprising the basement membrane have been mapped to this region.