Potential influence of tacrolimus and steroid avoidance on early graft function in pediatric renal transplantation.

With the increasing adoption of steroid-sparing immunosuppression protocols in renal transplantation, it is important to evaluate any adverse effects of steroid avoidance on graft function. Early graft function, measured by CrCl was retrospectively studied in 158 consecutive pediatric renal transplant recipients from 1996 to 2005, receiving either steroid-free or steroid-based immunosuppression. Patients receiving steroid-free immunosuppression vs. steroid-based immunosuppression had no difference change in CrCl (DeltaCrCl) in the first week post-transplantation (p = 0.12). When stratified by corticosteroid usage, patients with higher tacrolimus trough levels (> or =14 ng/mL) had slower graft function recovery in the first week post-transplantation than those with lower tacrolimus trough levels (p = 0.008) in the steroid-free group only. Despite initial slower graft function recovery in this subgroup, there was no negative impact on graft function in the steroid-free group; in fact steroid-free patients trended towards better CrCl at six months (p = 0.047) and 12 months (p < 0.001) post-transplant than the steroid-based group. With the improved immunological outcomes with steroid avoidance, close surveillance should be performed of tacrolimus levels to avoid levels >14 ng/mL. In patients with slow recovery of early graft function, short-term perioperative steroids may be considered.

Risks and benefits of splenectomy in myelofibrosis with myeloid metaplasia: a retrospective analysis of 26 cases.

BACKGROUND AND OBJECTIVES: To evaluate the outcomes of splenectomy in myelofibrosis and myeloid metaplasia (MMM). METHODS: We retrospectively reviewed our records of 26 patients with MMM who underwent an open splenectomy at Boston University Medical Center between 1979 and 1995. Fourteen patients had agnogenic myeloid metaplasia (AMM) and 12 had myelofibrosis with antecedent myeloproliferative disorders (MF). The main indications for splenectomy were progressive transfusion-dependent anemia, painful splenomegaly, and hypercatabolic symptoms associated with cytopenia. RESULTS: Median time to splenectomy after the diagnosis of MMM was 29 months ranging from 1 to 96 months. Three patients (11%) died within 1 month after the surgery because of sepsis. The most common peri- and postoperative complications were pneumonia and other bacterial infections (42%), cardiac events (19%), acute bleeding (15%), ileus (15%), and venous thrombosis (12%). Of the eight surviving patients who underwent splenectomy for transfusion dependent anemia, six (75%) had improvement in their hematocrit levels with abolishment of blood transfusions. A durable symptomatic palliation was achieved in all patients. Liver enlargement was noted in seven patients at 1-year evaluation. None of these patients developed hepatic failure. Leukemic transformation occurred in 8 of 18 patients (44%) postsplenectomy. The median overall survival for the entire group was 58.5 and 28 months from the diagnosis of MMM and the time of splenectomy, respectively. There was no difference in survival rates between patients with AMM and MF. CONCLUSIONS: Splenectomy is an effective palliative procedure with an acceptable morbidity in selected patients with MMM. Progressive transfusion-dependent anemia should also be considered an indication for splenectomy in the absence of leukemic evolution.

Induction of cyclooxygenase-2 by overexpression of the human catalase gene in cerebral microvascular endothelial cells.

Prostaglandin (PG) formation by the inducible (type 2) cyclooxygenase (COX-2) and reactive oxygen species (ROS) have been proposed to play important roles in cerebrovascular pathological processes. To explore the relationship between ROS and COX-2 expression, adenovirus (Ad) vectors containing cDNA for human antioxidant enzymes including catalase (AdCAT:), copper/zinc superoxide dismutase (AdCu/ZnSOD), and manganese superoxide dismutase (AdMnSOD) were transferred into murine cerebral microvascular endothelial cells. AdCAT: (100 multiplicity of infection) infection increased the content and enzymatic activity of cellular Cat threefold and decreased the intracellular peroxide level. The expression of COX-2 mRNA and protein in cell lysates was up-regulated, and the amount of PGE(2) formed from exogenous arachidonic acid increased following AdCAT: infection in a dose-dependent manner, paralleling the expression of COX-2 protein. The AdCAT:-induced increase in PGE(2) formation was inhibited by NS-398, a selective inhibitor of COX-2 enzymatic activity. AdCAT: infection did not change the expression of the constitutive (type 1) COX protein. Although AdCu/ZnSOD and AdMnSOD infection increased the expression of superoxide dismutase proteins, COX-2 expression was not induced. An in vitro nuclear transcription assay indicated that overexpression of the Cat gene increases the transcription of the COX-2 gene. Furthermore, the stability of COX-2 mRNA induced by lipopolysaccharide was increased after AdCAT: gene transfer. These results indicate that AdCAT: gene transfer induces the transcriptional activation of the COX-2 gene and increases COX-2 mRNA stability. Therefore, peroxide may have regulatory effect on COX-2 function in the cerebral microcirculation.

Comparative genomic sequence analysis of the human and mouse cystic fibrosis transmembrane conductance regulator genes.

The identification of the cystic fibrosis transmembrane conductance regulator gene (CFTR) in 1989 represents a landmark accomplishment in human genetics. Since that time, there have been numerous advances in elucidating the function of the encoded protein and the physiological basis of cystic fibrosis. However, numerous areas of cystic fibrosis biology require additional investigation, some of which would be facilitated by information about the long-range sequence context of the CFTR gene. For example, the latter might provide clues about the sequence elements responsible for the temporal and spatial regulation of CFTR expression. We thus sought to establish the sequence of the chromosomal segments encompassing the human CFTR and mouse Cftr genes, with the hope of identifying conserved regions of biologic interest by sequence comparison. Bacterial clone-based physical maps of the relevant human and mouse genomic regions were constructed, and minimally overlapping sets of clones were selected and sequenced, eventually yielding approximately 1.6 Mb and approximately 358 kb of contiguous human and mouse sequence, respectively. These efforts have produced the complete sequence of the approximately 189-kb and approximately 152-kb segments containing the human CFTR and mouse Cftr genes, respectively, as well as significant amounts of flanking DNA. Analyses of the resulting data provide insights about the organization of the CFTR/Cftr genes and potential sequence elements regulating their expression. Furthermore, the generated sequence reveals the precise architecture of genes residing near CFTR/Cftr, including one known gene (WNT2/Wnt2) and two previously unknown genes that immediately flank CFTR/Cftr.

Comparative response to splenectomy in Coombs-positive autoimmune hemolytic anemia with or without associated disease.

We reviewed our experience in 30 patients with direct Coombs-positive (DAT+) autoimmune hemolytic anemia (AHA) who underwent splenectomy. Twelve patients had idiopathic "warm" AHA (group I) and 18 had AHA associated with systemic diseases (group II). Complete response to splenectomy was defined as having normal hemoglobin and reticulocyte count lasting for at least 6 months without subsequent medical therapy. Subnormal but greater than 50% improvement in these parameters with or without medical therapy was considered to be a partial response. Median age was 64 (23-81) in group I and 68 (23-76) in group II. Median follow-up duration was 18 and 10.9 months, respectively. Nine of 11 (82%) evaluable patients with idiopathic AHA and 3 of 16 (19%) patients with associated disease achieved a complete response. Partial response was obtained in 2 (18%) and 6 (37%) patients in groups I and II, respectively. Both complete-response and overall-response rates were statistically different between two groups (P = 0.001 and 0.02). Postoperative courses of group I patients were uneventful except for one who developed a subphrenic abscess. Five patients in group II developed bacterial infections, which were mostly pneumonias. Our findings indicate that splenectomy is an effective treatment approach with low morbidity and mortality in patients with refractory idiopathic AHA. It should, however, be considered cautiously in AHA patients with underlying systemic diseases because of its decreased efficacy and increased surgical morbidity in this subgroup.

Construction of a high-resolution physical map of the approximate 1-Mb region of human chromosome 7q31.1-q31.2 harboring a putative tumor suppressor gene.

Reports of frequent loss of heterozygosity (LOH) of markers on human chromosome 7q in malignant myeloid disorders as well as breast, prostate, ovarian, colon, head and neck, gastric, pancreatic, and renal cell carcinomas suggest the presence of a tumor suppressor gene (TSG). Functional assays have demonstrated that the introduction of an intact copy of human chromosome 7 (hchr7) can restore senescence to immortalized human fibroblast cell lines having LOH of markers within 7q31-q32 and can inhibit the tumorigenic phenotype of a murine squamous cell carcinoma cell line. To facilitate the cloning of the putative TSG, we have constructed a high-resolution physical map of this region of hchr7, specifically that encompassing the markers D7S522 and D7S677 within 7q31.1-q31.2. By using a lower resolution yeast artificial chromosome-based map as a starting framework, we established complete clone coverage of the implicated critical region in bacterial-artificial chromosomes (BACs) and P1-derived artificial chromosomes (PACs). The resulting BAC/PAC-based contig map has provided suitable clones for the systematic sequencing of the entire interval. In addition, we have already identified 29 clusters of overlapping expressed-sequence tags (ESTs) and 4 known genes contained within these clones. Together, the physical map reported here coupled with the evolving sequence and gene maps should hasten the identification of the putative TSG residing within this region of hchr7.

Is splenectomy more dangerous for massive spleens?

BACKGROUND: Reports vary about whether risks are greater for removal of massive (> or = 1500 g) spleens than for smaller (< 1500 g) spleens. We sought to determine the hazards of splenectomy. METHODS: We reviewed 223 consecutive adults with elective splenectomies for hematologic diseases. Morbidity and mortality rates were combined with published data to create a meta-analysis. RESULTS: Patients with massive spleens are more likely to have postoperative complications (relative risk [RR] 2.1, 95% confidence interval [CI] 1.3 to 3.4; P = 0.003) and death (RR 4.7, 95% CI, 1.5 to 15.1; P = 0.01). However, when the investigation is restricted to comparable diagnoses, patients with massive spleens do not differ from those with smaller spleens regarding complications (RR 1.4, 95% CI, 0.8 to 2.7; P = 0.3) or mortality (RR 2.1, 95% CI, 0.5 to 9.7; P = 0.4). These observations are confirmed by metaanalysis. Furthermore, multivariate analysis indicts age as a critical risk of complications and death. CONCLUSIONS: Increased age and underlying illness are the predominant factors associated with morbidity and mortality following splenectomy for hematologic disease. Adjusting for age and diagnosis, spleen size is not a hazard.

A physical map of human chromosome 7: an integrated YAC contig map with average STS spacing of 79 kb.

The construction of highly integrated and annotated physical maps of human chromosomes represents a critical goal of the ongoing Human Genome Project. Our laboratory has focused on developing a physical map of human chromosome 7, a approximately 170-Mb segment of DNA that corresponds to an estimated 5% of the human genome. Using a yeast artificial chromosome (YAC)-based sequence-tagged site (STS)-content mapping strategy, 2150 chromosome 7-specific STSs have been established and mapped to a collection of YACs highly enriched for chromosome 7 DNA. The STSs correspond to sequences generated from a variety of DNA sources, with particular emphasis placed on YAC insert ends, genetic markers, and genes. The YACs include a set of relatively nonchimeric clones from a human-hamster hybrid cell line as well as clones isolated from total genomic libraries. For map integration, we have localized 260 STSs corresponding to Genethon genetic markers and 259 STSs corresponding to markers orders by radiation hybrid (RH) mapping on our YAC contigs. Analysis of the data with the program SEGMAP results in the assembly of 22 contigs that are "anchored" on the Genethon genetic map, the RH map, and/or the cytogenetic map. These 22 contigs are ordered relative to one another, are (in all but 3 cases) oriented relative to the centromere and telomeres, and contain > 98% of the mapped STSs. The largest anchored YAC contig, accounting for most of 7p, contains 634 STSs and 1260 YACs. An additional 14 contigs, accounting for approximately 1.5% of the mapped STSs, are assembled but remain unanchored on either the genetic or RH map. Therefore, these 14 "orphan" contigs are not ordered relative to other contigs. In our contig maps, adjacent STSs are connected by two or more YACs in > 95% of cases. With 2150 mapped STSs, our map provides an average STS spacing of approximately 79 kb. The physical map we report here exceeds the goal of 100-kb average STS spacing and should provide an excellent framework for systematic sequencing of the chromosome.

2006 expressed-sequence tags derived from human chromosome 7-enriched cDNA libraries.

The establishment and mapping of gene-specific DNA sequences greatly complement the ongoing efforts to map and sequence all human chromosomes. To facilitate our studies of human chromosome 7, we have generated and analyzed 2006 expressed-sequence tags (ESTs) derived from a collection of direct selection cDNA libraries that are highly enriched for human chromosome 7 gene sequences. Similarity searches indicate that approximately two-thirds of the ESTs are not represented by sequences in the public databases, including those in dbEST. In addition, a large fraction (68%) of the ESTs do not have redundant or overlapping sequences within our collection. Human DNA-specific sequence-tagged sites (STSs) have been developed from 190 of the ESTs. Remarkably, 180 (96%) of these STSs map to chromosome 7, demonstrating the robustness of chromosome enrichment in constructing the direct selection cDNA libraries. Thus far, 140 of these EST-specific STSs have been assigned unequivocally to YAC contigs that are distributed across the chromosome. Together, these studies provide > 2000 ESTs highly enriched for chromosome 7 gene sequences, 180 new chromosome 7 STSs corresponding to ESTs, and a definitive demonstration of the ability to enrich for chromosome-specific cDNAs by direct selection. Furthermore, the libraries, sequence data, and mapping information will contribute to the construction of a chromosome 7 transcript map.

A collection of 1814 human chromosome 7-specific STSs.

An established goal of the ongoing Human Genome Project is the development and mapping of sequence-tagged sites (STSs) every 100 kb, on average, across all human chromosomes. En route to constructing such a physical map of human chromosome 7, we have generated 1814 chromosome 7-specific STSs. The corresponding PCR assays were designed by the use of DNA sequence determined in our laboratory (79%) or generated elsewhere (21%) and were demonstrated to be suitable for screening yeast artificial chromosome (YAC) libraries. This collection provides the requisite landmarks for constructing a physical map of chromosome 7 at < 100-kb average spacing of STSs.

Tiazofurin effects on IMP-dehydrogenase activity and expression in the leukemia cells of patients with CML blast crisis.

Tricot et al have reported that the nucleoside analog tiazofurin can induce hematologic remissions in patients with chronic myelogenous leukemia in blast crisis (CML-BC). These reports prompted us to begin a derivative, phase II trial of tiazofurin in CML-BC to determine if the promising findings reported by these investigators could be reproduced. In our ongoing trial patients receive tiazofurin by IV infusion (2200-4400 mg/m2 over 1 hr) once every 24-48 hrs for up to 10 days. Each of 3 patients, treated to date on this trial, experienced substantial hematologic responses with normalization of WBC counts and complete or partial clearance of blasts from the blood within 4-11 days of treatment. These responses were relatively brief, in that leukemic blasts reaccumulated in the marrow and blood of patients within 4 weeks following treatment, but were re-induced with subsequent courses of treatment. Of interest, the rates of blast cell reaccumulation appeared to increase progressively following sequential courses of treatment. Tiazofurin, which inhibits IMP-dehydrogenase (IMPDH) and blocks guanine ribonucleotide synthesis, has been shown to increase IMPDH mRNA expression in various cell lines in vitro, as an apparently compensatory response to guanylate deprivation. Studies of IMPDH mRNA expression in the leukemic blasts of CML-BC patients receiving tiazofurin treatment showed that this same phenomenon occurs in vivo. Since IMPDH activity is linked to the proliferative activity of neoplastic cells an amplification of IMPDH message expression induced by tiazofurin may lead to an increased sensitivity of the leukemic clone to cycle active agents.

Collection of mobilized blood progenitor cells for hematopoietic rescue by large-volume leukapheresis.

BACKGROUND: Mobilized blood progenitor cells rapidly reconstitute hematopoiesis in patients after dose-intensive chemotherapy. However, optimal timing and methods of mobilized blood progenitor cell collection have yet to be fully defined. STUDY DESIGN AND METHODS: The utility of large-volume leukapheresis (LVL; > 15 L blood processed) in collecting target doses of mononuclear cells (7 x 10(8)/kg) for use in autologous hematopoietic rescue was investigated. LVL was begun at a standardized interval (14 days) after a course of limited chemotherapy and subsequent daily recombinant human granulocyte-macrophage-colony-stimulating factor administration to mobilize blood progenitor cells into the circulation. With each LVL procedure, mononuclear cells, colony-forming units-granulocyte-macrophage (CFU-GM), burst-forming units-erythroid, mixed colonies, total clonogenic progenitor cells, and CD34+ cells collected per kg of patient weight were counted. After high-dose chemotherapy and infusion of cryopreserved mobilized blood progenitor cells, the days needed for neutrophils to reach levels of > 0.5 x 10(9) per L and for platelets to reach levels of > 20 x 10(9) per L were recorded. RESULTS: In 14 previously treated cancer patients, an average of 28.9 +/- 4.9 L of blood was processed per LVL (n = 35) to collect medians of 2.5 x 10(8) mononuclear cells per kg (range, 1.0-7.4), 14 x 10(4) CFU-GM per kg (0-208), 27 x 10(4) clonogenic progenitor cells per kg (0-370), and 2.8 x 10(6) CD34+ cells per kg (0-112.5). Fifty-seven percent of patients (8/14) required one or two LVL procedures to collect adequate blood progenitor cells (range, 1-4). After dose-intensive chemotherapy, 13 patients received medians of 6.8 x 10(8) mononuclear cells per kg (range, 5.1-9.9), 53 x 10(4) CFU-GM per kg (9-208), and 12 x 10(6) CD34+ cells per kg (3.6-112.5). Rapid hematopoietic reconstitution occurred with 10 days (range, 8-12) and 9 days (6-15), respectively, for neutrophil and platelet recoveries. CONCLUSION: Scheduled LVL, beginning on Day 14 after the administration of granulocyte-macrophage-colony-stimulating factor following chemotherapy, is a convenient and efficient method of collecting blood progenitor cells. The mononuclear cells so obtained effected consistent and rapid hematopoietic reconstitution in a highly reproducible manner in a group of heavily treated patients.

Synchronization of plasma exchange and cyclophosphamide in severe and refractory autoimmune hemolytic anemia.

Two cases of severe autoimmune hemolytic anemia (AIHA) that failed multiple treatment modalities obtained complete and long-lasting remissions with a combination of three one volume plasma exchange (PE) on succeeding days followed 6 hours later on the 3rd day by cyclophosphamide (cyc) 750 mg/m2 IV, and cyc/prednisone (pred) qd tapering to either no therapy or minimal therapy over a 6 month period. Both cases remain without evidence of AIHA after 43 and 19 months follow-up. Possible non-exclusive mechanisms that explain this favorable outcome are enhanced cytotoxic effect of cyc on proliferating lymphocytes participating in the antibody rebound phenomena, suppression of B lymphocytes with daily cyc/pred, and/or formation of anti-idiotype antibodies.

Sialochemistry: a diagnostic tool?

Saliva has proven to be a discriminating element in forensic arenas, an effective indicator of acute diseases of salivary glands, and a promising probe for drug monitoring. With the advent of sensitive immunochemical assays, the compositional profile of human salivary secretions has been expanded considerably. Thus, the establishment of a range of "normal values" for a variety of "intrinsic" and "extrinsic" salivary components represented the initial step to use saliva as a diagnostic tool of oral health status. Unfortunately, numerous cross-sectional studies have shown a wide individual variation in the salivary composition of healthy populations, thus precluding its use as a diagnostic chair-side test for the screening of the most common chronic oral diseases (e.g. caries and periodontal disease). A possible explanation may arise from the wide functional versatility of salivary molecules. For instance, it has been recognized recently that in addition to its digestive properties, salivary amylase may modulate bacterial colonization, whereas histatins are not only antifungal but also bactericidal. Thus, low levels of already known antimicrobial salivary molecules (e.g., secretory IgA, lactoferrin, and lysozyme) could be compensated with higher concentrations of other molecules with antimicrobial activity, such as amylase and histatins. Consequently, for caries and periodontal diseases, longitudinal sialochemical studies may yield more insight than cross-sectional studies.

Could bacterial vaginosis be due to the competitive suppression of lactobacilli by aerobic microorganisms?

Bacterial vaginosis (BV) is characterized by lack of lactobacilli and predominance of anaerobic species. Ciprofloxacin is active against a great number of aerobic bacteria. The effect of ciprofloxacin on the vaginal microbial flora was studied both in healthy subjects and in patients with BV in order to test if BV may be due to competitive suppression of lactobacilli by aerobic species. About half of the patients were relieved of their symptoms. No adverse effects on the microbial profile or colonization by lactobacilli were produced by the drug. Lactobacilli known to be able to produce hydrogen peroxide were found to be significantly more common in healthy women than in women with BV. The species most frequently related to vaginal health were Lactobacillus jensenii and Lactobacillus rogosae.

Levels of salivary cystatins in periodontally healthy and diseased older adults.

Cystatins are cysteine protease inhibitors present in a variety of tissues and body fluids, including saliva. One possible function of these molecules may be to modulate tissue destruction in periodontal diseases. To investigate the potential role of salivary cystatins in these events, the levels of cystatins in saliva from periodontally healthy and diseased individuals were measured by enzyme-linked immunosorbent assay. Flow rates and total protein content were determined in all the samples collected, while cysteine protease inhibitory activity was assessed in submandibular-sublingual secretions. Statistical analysis showed no significant differences in the levels and activity of salivary cystatins in periodontally healthy and diseased individuals. These findings suggest that comparing the levels of cystatins in glandular salivas may not be a suitable indicator of periodontal disease status.

Reversal of bone marrow fibrosis and subsequent development of polycythemia in patients with myeloproliferative disorders.

Bone marrow fibrosis is a characteristic finding in agnogenic myeloid metaplasia and in the spent phase of polycythemia vera. It is commonly believed that the reticulin deposition is irreversible. However, we report four patients who demonstrated clinical and laboratory evidence of transition from myelofibrosis to polycythemia. The transition was documented by improvement in the hemoglobin concentration and by determination of the Cr51 red blood cell mass, accompanied by a resolution of the fibrosis on serial bone marrow biopsies. Two of the patients had been treated with alkylating agents and splenectomy, one with myelosuppressive therapy without splenectomy, and one with splenectomy alone. These findings indicate that bone marrow fibrosis in the chronic myeloproliferative disorders is not always an irreversible phenomenon. Pathogenetic implications will be discussed.

Bacterial vaginosis is not a simple ecological disorder.

Eighty-four patients with bacterial vaginosis were examined in an open randomized trial, the aim of which was to define clinical results and the microbiological panorama after topical treatment for 1 week with either an acetic acid jelly (A), an estrogen cream (B), a fermented milk product (C) or metronidazole (D). After exclusion because of chlamydia infection (15 cases) or for other reasons, 61 cases remained for complete evaluation 4 weeks after the start of treatment. Clinical cure was obtained in 3 cases out of 17 on regimen A, in 1 out of 16 on regimen B, in 1 of 14 on regimen C, and in 13 out of 14 on regimen D. The patients were conclusively either symptomless or symptomatic when examined on 113 occasions. Statistically significant reduction after treatment resulting in relief of symptoms was observed in the numbers of corynebacteria and anaerobic cocci, whereas lactobacilli increased in numbers. The instillation of high numbers of Lactobacillus acidophilus (C) into the vagina cured only 1 patient and did not influence the predominance of lactobacilli in the vagina at the follow-up examination. The difference in microbiological profile of women in symptomatic and asymptomatic conditions becomes still more apparent when the results of the present and previously published studies on the subject by the present group of investigators are combined. The symptomatic woman is significantly more often harboring corynebacteria, Gardnerella vaginalis, peptostreptococci, peptococci, eubacteria and Bacteroides species. Lactobacilli are significantly reduced in numbers. However, only 51% of our previously symptomatic, but now symptomless women show predominant growth of lactobacilli, which is less than expected for healthy women.(ABSTRACT TRUNCATED AT 250 WORDS)