Biomedical subjects
Publications and source records attributed to M Kremer.
Colicky lower abdominal pain can have several causes. Besides the typical urological reasons, differential diagnosis should include surgical, internal, and gynecological causes. In particular, in cases of ambiguous calcifications on plain abdominal films - as shown in the presented case - a mature teratoma of the ovary should be taken into consideration.
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Fast-track surgery is an interdisciplinary multimodal concept of minimally invasive surgery or new incision lines and "cutting old plaits" (e.g., the use of drains or tubes). It uses modern intraoperative anesthesia (e.g., fluid restriction) and analgesia, including new drugs and novel ways of administration (e.g., thoracic epidural analgesia) for postoperative pain relief, in combination with the immediate mobilization of the patient and early oral nutrition after the operation. This approach requires a cooperating team of motivated nurses, physiotherapists, anesthesiologists, and surgeons, in addition to continuous improvement of the processes involved. Moreover, extended patient education and information about the procedures and the expected time course are of the highest importance, as the active role of the patient is to be emphasized. This chapter describes the development and implementation of fast-track surgery in colorectal diseases at the Department of Surgery of the University Hospital of Heidelberg, Germany. Preliminary results of fast-track surgery suggest a significant and clear overall benefit for the patient. A shorter hospital stay and reduced systemic morbidity in addition to no increase in postoperative complications on an out-patient basis were found. However, to exclude a "bloody discharge" of the patients, thorough follow-up and quality control are mandatory. Although in the initial phase increased personnel care is necessary, in the new German reimbursement system with G-DRGs (German diagnosis-related groups) fast-track surgery seems to save resources in the long term.
Pulmonary involvement in ulcerative colitis may manifest as a variety of disorders. Ulcerative colitis-related interstitial lung disease is exceedingly rare and has been reported to be steroid-responsive. We describe the first case of a patient with acute exacerbation of ulcerative colitis-induced usual interstitial pneumonia, who did not respond to corticosteroid therapy and died 12 weeks after the onset of pulmonary symptoms. Early recognition of pulmonary disease in patients with ulcerative colitis is necessary to initiate further diagnostic work-up and may aid treatment decisions.
The expression and functional properties of connexin36 (Cx36) have been investigated in two neuroblastoma cell lines (Neuro2A, RT4-AC) and primary hippocampal neurons transfected with a Cx36-enhanced green fluorescent protein (EGFP) expression vector. Transfected cells express Cx36-EGFP mRNA, and Cx36-EGFP protein is localized in the perinuclear area and cell membrane. Upon differentiation of cell lines, Cx36-EGFP protein was detectable in processes with both axonal and dendritic characteristics. Small gap junction plaques were found between adjacent cells, and electrophysiological recordings demonstrated that the electrical properties of these gap junctions were virtually indistinguishable from those reported for native Cx36. Mutagenesis of Cx36 led to the identification of a structural element that interferes with normal protein localization. In contrast, site directed mutagenesis of putative protein phosphorylation motifs did not alter subcellular localization. This excludes phosphorylation/dephosphorylation as a major regulatory step in Cx36 protein transport.
This paper summarizes recent thinking on stimulating industrial research and development (R&D) for neglected infectious diseases and argues that it is critical to enlarge the value of the market for medicines and vaccines through, for example, global purchase funds. The most important economic barriers to R&D are that the commercial markets are small and that individual purchasing power is severely limited, even though the number of patients may be very large. Since R&D costs for all diseases are high, this means that returns will not cover investments. Various mechanisms have been proposed to address this economic imbalance (accepting that other barriers will also need to be considered). Economic devices which reduce the costs of R&D--push factors--are useful, but our review suggests that high costs do not explain the shortfall in R&D. Economic devices which address the lack of viable markets have been termed pull factors and are designed to create or secure a market, thereby improving the likelihood of a return on investments. One pull mechanism is the commitment in advance to purchase a product that meets specified criteria, if invented. The purchase-precommitment approach has a number of attractive features. For example, it only rewards successful outputs rather than supporting research that may not succeed. Pull programmes effectively mimic the market and lead companies to favour lines of attack that they believe will lead to marketable products. Overall, a combination of push and pull mechanisms is likely to represent an attractive approach. This could combine, for example, increased funding for public laboratories, public-private partnerships in R&D, purchases of underutilized existing products, and a precommitment to purchase new drugs and vaccines when developed.
BACKGROUND: The production of autologous composite skin equivalents for the treatment of full-thickness skin defects in burns is time consuming and costly because of laboratory procedures which have to be performed manually. In the present study keratinocytes were seeded into INTEGRA Artificial Skin and placed in a perfusion culture system in order to evaluate the possibility of producing composite grafts in an automated system with the aim of establishing a cost-effective method of industrial production. METHODS: Composite grafts of INTEGRA and human keratinocytes were raised in perfusion culture and grafted onto athymic mice to evaluate their potential to reconstitute a full-thickness skin substitute in vivo compared to grafts from standard stagnant cultures. RESULTS: Cultured composites from perfusion cultures showed no significant histological differences compared to those from stagnant cultures; however, a tendency of improved cell growth and a more surface-oriented localization was observed. Cell proliferation and surface-bound differentiation were not impaired by the use of carbonate-independent buffering (HEPES), which is necessary for perfusion culture. The composite grafts from perfusion culture exhibited identical wound adherence and complete healing and histologically represented a multi-layered, keratinizing human epidermis. CONCLUSION: Engineering of differentiated composite skin equivalents is possible in a perfusion culture system, which offers technical and procedural and possibly even biological advantages compared to standard stagnant culture methods. The development of automated perfusion culture systems for the production of composite grafts in sizes required clinically (scale-up) will be the next step in the cost-effective engineering of large-scale composite skin equivalents.
Nasal NK/T-cell lymphoma is a unique form of lymphoma highly associated with Epstein-Barr virus, and with a characteristic geographic distribution. Recently, we showed that p53 is overexpressed in a high percentage of nasal NK/T-cell lymphomas. The aim of this study was to analyze the status of the p53 gene, and correlate it with the expression of p53 protein and its downstream target, the cyclin-dependent kinase inhibitor p21, in a series of 25 cases of well-characterized nasal NK/T-cell lymphoma from Mexico. The highly conserved exons 5 to 8 of the p53 gene were amplified by polymerase chain reaction and screened for mutations by denaturing high-pressure liquid chromatography. Abnormal polymerase chain reaction products detected by denaturing high-pressure liquid chromatography and additional selected cases were sequenced. In addition, the incidence of loss of heterozygosity at the p53 locus was analyzed in 12 cases. Of the 25 patients, 17 were male and 8 female (M:F ratio, 2.1:1), with a median age of 43 years (range, 21 to 93 years). Morphologically, most of the cases were composed of a mixture of medium-sized cells and large transformed cells (21 cases), and four cases were composed exclusively of large transformed cells. Three different groups determined by p53 gene status and expression of p53 protein were identified: group 1 was p53 +/p53 mutated (five cases, all with p53 missense mutations). Morphologically, three of the five cases were composed of large cells. All five cases revealed overexpression of p53 in the majority of the tumor cells with a mean of 86%. Unexpectedly, three of these cases also showed overexpression of p21. Four of the five patients presented with clinical stage IVB and died with disease. Group 2 was p53+/p53 wild-type (10 cases). Histologically, nine cases were of the mixed type, and one of the large cell type. The percentage of p53 overexpressing cells was lower than in the previous group with a mean of 23%. p21 was positive in 7 of the 10 cases. Six patients in this group presented with clinical stages I to II and four patients with advanced disease (stage III and IV). Five patients are alive 12 to 120 months later (mean, 24 months), three with no evidence of disease. Group 3 was p53-/p53 wild-type (10 cases). All cases showed mixed cell morphology. p21 was positive in 5 of 10 cases. Four patients presented with clinical stage I to II and six patients with advanced disease. Four patients are alive with no evidence of disease 9 to 60 months later (mean, 10 months). Overall, p53 mutations were present in 24% (5 of 21) of the evaluable cases, all of them overexpressing p53 in the majority of tumor cells. Cases with p53 mutations were associated with large cell morphology (P = 0.0162) and presented more often with advanced stage disease. Loss of heterozygosity at chromosome 17p was found only in 2 of the 12 (17%) cases investigated, both cases showed p53 mutations of the remaining allele. P21 overexpression (60% of cases) is frequent in nasal NK/T-cell lymphoma and seems to be independent of p53 gene status. The overexpression of p53 and p21, independent of p53 mutations, although as yet not clear, might be the result of Epstein-Barr virus infection, and warrants further investigation.
The pathogenesis and clonal evolution of gastric diffuse large B-cell lymphoma (DLBCL) and its relationship to extranodal marginal zone B-cell lymphoma (MZBL), mucosa-associated lymphoid tissue (MALT) type, are still controversial. The aim of this study was to establish the clonality of morphologically distinct areas of gastric lymphomas as well as their genetic relationship to each other. Six gastric lymphomas, consisting of two MZBL, MALT type, two DLBCL, and two "composite" lymphomas were subjected to laser capture microdissection and subsequent PCR-based amplification of the immunoglobulin heavy chain gene. One DLBCL showed a biclonal pattern of rearranged immunoglobulin heavy chain (IgH) genes of two different areas without evidence of a common origin. Two composite DLBCL with areas of extranodal MZBL, MALT type, were also biclonal and displayed different IgH gene rearrangements in the small-cell and in the large-cell components, respectively. Sequencing of the CDR3 region revealed unique VH-N-D and D-N-JH junctions, thus corroborating the presence of two genuinely distinct tumor clones in each of these three cases. In contrast, the remaining three gastric lymphomas (one DLBCL and two MZBL, MALT type) showed IgH gene rearrangements in which CDR3 regions were identical in the different tumor areas. Our results suggest that gastric DLBCL may be composed of more than one tumor cell clone. Further, DLBCL may not necessarily evolve by transformation of a low-grade lymphoma, but may also originate de novo. An ongoing emergence of new tumor clones may considerably hamper molecular diagnosis and follow-up of gastric DLBCL.
The association of mycosis fungoides (MF) and Hodgkin's lymphoma is a relatively frequent occurrence, but the potential clonal relationship of the two neoplasms is still controversial. We report a case of a patient with a history of MF in Clinical Stage 1A who developed retroperitoneal lymphadenopathy 9 years after the initial diagnosis of MF. A bone marrow biopsy obtained at this time showed nodular involvement by a mixed cellular infiltrate with large, atypical cells consistent with Hodgkin and Reed-Sternberg (RS) cells. These atypical cells were positive for CD30 and CD15 and did not express B- or T-cell markers. In addition, they lacked evidence of infection by Epstein-Barr virus, both by immunohistochemical staining for latent membrane protein 1 and by in situ hybridization for EBER1/2. The background population consisted mainly of small T cells without morphological or phenotypical signs of malignancy. Review of the skin biopsy obtained 9 years before showed the typical features of MF. Polymerase chain reaction analysis of the T-cell receptor T-gene confirmed the presence of a clonal T-cell rearrangement in the skin specimen. The bone marrow biopsy, however, showed a polyclonal pattern both for the T-cell receptor gamma-gene, as well as for immunoglobulin heavy chain genes. Isolation of RS cells stained for CD30 was performed by laser capture microdissection. Polymerase chain reaction analysis of several groups of RS cells showed a reproducible biallelic rearrangement of IgH genes, which was confirmed by cloning and sequencing of polymerase chain reaction products. To our knowledge, this is the first case in which a distinct clonal origin of MF and Hodgkin's lymphoma arising in the same patient is clearly demonstrated, based on molecular analysis of microdissected RS cells.
The distinction between mantle cell lymphoma (MCL) and other small B-cell non-Hodgkin lymphomas (NHL) is important because MCL has a more aggressive clinical course. In bone marrow (BM) biopsy specimens, this distinction can be particularly difficult. Although cyclin D1 immunostaining and molecular detection of the t(11;14) translocation are highly specific markers for MCL, they fail to detect a proportion of cases. We have recently described that MCL typically lacks detectable expression of the cyclin-dependent kinase inhibitor p27(kip1) protein by immunostaining, which is expressed at high levels in most small B-cell NHL inversely correlated to the proliferation rate. We therefore examined whether p27(kip1) immunostaining could be a useful adjunct for the differential diagnosis of small B-cell NHL infiltrates in the BM. Trephine BM biopsy specimens of 96 patients, including well-characterized MCL (19 cases), B-cell chronic lymphocytic leukemia (27 cases), follicular lymphoma (18 cases), hairy cell leukemia (22 cases), and marginal zone lymphoma (10 cases) as well as 10 reactive BM, including five with benign lymphoid aggregates were investigated. In addition, the presence of a t(11;14) translocation involving the major translocation cluster was studied by PCR in all MCL. All cases of B-cell chronic lymphocytic leukemia, follicular lymphoma, and marginal zone lymphoma revealed a strong p27(kip1) nuclear staining in the majority of neoplastic cells. Fourteen (78%) cases of MCL were p27(kip1)-negative in the tumor cells, whereas four cases revealed a weak nuclear positivity. Seventeen (77%) cases of hairy cell leukemia were also either completely negative for p27(kip1) or showed a faint positive staining in a minority of the neoplastic cells. Nine of 19 cases (47%) of MCL showed a bcl1 rearrangement involving the major translocation cluster region. These findings demonstrate that p27(kip1) immunostaining is a valuable additional marker for the differential diagnosis of small B-cell NHL infiltrates in BM biopsies. The reduction or lack of p27(kip1) protein expression in MCL, as well as in hairy cell leukemia, might be an important event in the pathogenesis of these disorders.
A 23-year-old female presented with enlarged cervical lymph nodes, and a diagnosis of nonspecific lymphadenitis with formation of pyogranulomas was rendered. Despite an initial oral antibiosis and subsequent long-term intravenous and oral antibiosis under hospitalized conditions, the symptoms progressed. The lymph nodes became larger and then affected the cervical region bilaterally. Her general condition worsened, and an exanthema of the extremities accompanied by a reactive arthritis occurred. Serological assays of various viral and bacterial markers and blood cultures were negative. Application of a polymerase chain reaction (PCR) protocol allowing specific amplification of mycobacterial DNA revealed DNA of Mycobacterium chelonea in formalin-fixed, paraffin-embedded lymph node tissue. Sequencing of the PCR product showed a 97% homology with the known Mycobacterium chelonae sequence. Modification of the antibiotic therapy with clarithromycin, imipenem and amikacin resulted in a rapid regression of the symptoms. The clinical course, in combination with the difficulties in detecting the infectious agent, supports the usefulness of molecular pathological analyses specific for nontuberculous mycobacteria (NTM).
The identification of an increasing array of highly potent, endogenous peptide and protein factors termed cytokines, that can be efficiently synthesized using recombinant DNA technology, offers exciting new approaches for drug therapy. However, the physico-chemical and biological properties of these agents impose limitations in formulation and development of optimum drug delivery systems as well as on the routes of delivery. Oral mucosa, including the lining of the cheek (buccal mucosa), floor of mouth and underside of tongue (sublingual mucosa) and gingival mucosa, has received much attention in the last decade because it offers excellent accessibility, is not easily traumatized and avoids degradation of proteins and peptides that occurs as a result of oral administration, gastrointestinal absorption and first-pass hepatic metabolism. Peptide absorption occurs across oral mucosa by passive diffusion and it is unlikely that there is a carrier-mediated transport mechanism. The principal pathway is probably via the intercellular route where the major permeability barrier is represented by organized array of neutral lipids in the superficial layers of the epithelium. The relative role of aqueous as opposed to the lipid pathway in drug transport is still under investigation; penetration is not necessarily enhanced by simply increasing lipophilicity, for other effects, such as charge and molecular size, also play an important role in absorption of peptide and protein drugs. Depending on the pharmacodynamics of the peptides, various oral mucosal delivery systems can be designed. Delivery of peptide/protein drugs by conventional means such as solutions has some limitations. The possibility of excluding a major part of drug from absorption by involuntary swallowing and the continuous dilution due to salivary flow limits a controlled release. However these limitations can be overcome by adhesive dosage forms such as gels, films, tablets, and patches. They can localize the formulation and improve the contact with the mucosal surface to improve absorption of peptides and proteins. Addition of absorption promoters/permeabilizers in bioadhesive dosage forms will be essential for a successful peptide/protein delivery system.
Research in anesthesia risk management has focused primarily on adverse patient outcomes. Most risk management studies have evaluated the practices of the physician anesthesiologist, while minimal research has been conducted to examine anesthesia care provided by Certified Registered Nurse Anesthetists (CRNAs). For this reason, the American Association of Nurse Anesthetists Foundation supported an examination of closed malpractice claim files from St Paul Fire and Marine Insurance Company that involved insured CRNAs. A team of 8 CRNA researchers investigated 223 closed claim files that involved incidents from 1989 to 1997. The results indicated that preoperative physical status, patient age, surgical procedure, type of anesthetic, age of anesthesia provider, and the type of anesthesia providers, (e.g., CRNA alone vs CRNA and anesthesiologist working together) did not have a statistically significant relationship with adverse anesthetic outcomes. However, providing appropriate care, being vigilant, encountering a less severe adverse outcome, and not being able to prevent the outcome were associated with smaller monetary awards. The findings of this study support those of similar studies.
Electrical synapses (gap junctions) in neuronal circuits have become a major focus in the study of network properties such as synchronization and oscillation (Galarreta and Hestrin, 1999; Gibson et al., 1999). Despite the recent progress made in unraveling the contribution of gap junctions to network behavior, little is known about the molecular composition of the junctional constituents. By cloning gap junction proteins [connexins (Cxs)] from zebrafish retina and through functional expression, we demonstrate that the retina possesses a high degree of connexin diversity, which may account for differential functional properties of electrical synapses. Three new Cxs, designated as zebrafish Cx27.5 (zfCx27.5), zfCx44.1, and zfCx55.5, and the carp ortholog of mammalian Cx43 were cloned. By in situ hybridization and in situ RT-PCR, we demonstrate that the four fish connexin mRNAs show differential localization in the retina. Transient functional expression in paired Xenopus oocytes and in the neuroblastoma N2A cell line indicate an extreme range of electrophysiological properties of these connexins in terms of voltage dependence and unitary conductance. For instance, the new zfCx44.1 exhibited high sensitivity to voltage-induced closure with currents decaying rapidly for transjunctional potentials >10 mV, whereas zfCx55.5 channels showed an opposite voltage dependence in response to voltage steps of either polarity. Moreover, although zfCx44.1 channels showed unitary conductance as high as any previously reported for junctional channels (nearly 300 pS), zfCx55. 5 and zfCx27.5 exhibited much lower unitary conductances (<60 pS).
Glomerulopathy with fibronectin deposits (GFND, MIM 601894) is an autosomal dominant kidney disease that leads to terminal renal failure at a median age of 47 years. It represents a distinct entity of membranoproliferative glomerulonephritis (MPGN) type III and is characterized by the unique feature of massive glomerular deposits of fibronectin. We have recently localized a gene locus for GFND to human chromosome 1q32 by total genome linkage analysis in a large kindred, within a 4.1-cM critical interval between markers D1S2872 and D1S2891. This interval contains a cluster of genes for "regulators of complement activation" (RCA), which represent strong candidates for GFND. To identify positional candidate genes for GFND within the critical genetic interval, we here report the cloning of the entire critical GFND region in a complete YAC and partial PAC contig. We constructed a high-resolution transcriptional map, thereby defining positional and functional candidate genes for the disease. To evaluate their role in GFND, we performed functional studies on RCA proteins in GFND patients from the large kindred, as well as mutational analysis of the genes for complement receptor-2 (CR2), membrane cofactor protein (MCP), and decay accelerating factor (DAF). Although no loss-of-function mutation has been identified as yet, these data provide a basis for the examination of candidate genes for GFND and other genes for MPGN, which localize to the vicinity of the GFND region.
Although ductal carcinoma in situ (DCIS) of the breast is accepted as a potential precursor lesion for invasive ductal cancer (IDC), the critical genetic events associated with the tumor progression remain unknown. Since some extensive DCIS may show a small focus of IDC, these cases seem to be particularly suitable to investigate the primary abnormalities that determine the progression from in situ to early invasive cancer. We combined laser-microdissection with degenerative oligonucleotide-primed PCR (DOP-PCR) and comparative genomic hybridization (CGH) to detect copy number changes in 7 cases of extensive (>4 cm) DCIS with 1 small adjacent invasive focus. In 3 of the cases, single lymph node metastases (LN) were already present and were also investigated. Analysis of DCIS, IDC and LN components in the same patients revealed several consistent chromosomal changes present at all 3 sites: 1q, 7q, 8q, 16, 17, 19, 20q, 21q and 22q, the most frequent losses on 4q, 11q and 13q. DNA gain on 3p and 12q were more frequently found in IDC than in DCIS, suggesting the presence of proto-oncogenes activated during the progression to invasive cancer on these regions. Using paired analysis, resemblence of alterations found in DCIS and IDC could be quantified (odds ratio 7.0, p< or = 0.01). Gains on 6p, 10q, 14q and 15q and losses on 9p were identified in DCIS and IDC but not in LN, which may, therefore, represent early events in the carcinogenic process. Additional losses were found in the LNs on 2q, 3q, 5q, 6q, 12q and 16q. CGH results on chromosome 1 and 20 were confirmed by FISH and on chromosomal region 9p by microsatellite analyses. Our findings strongly underline the precursor status of high-grade DCIS, in which most of the chromosomal changes identified in IDC are already present. However, although the early stages of breast cancer, i.e., DCIS and the small foci of IDC were mainly characterized by DNA gains, the progression to metastatic tumor (LN) must have involved additional DNA losses on several regions.
In order to assess the current technical standard of diagnostic molecular pathology, we have conducted a multicenter trial with 34 participating pathology laboratories in Germany, Austria and Switzerland. Formalin-fixed, paraffin-embedded tissue blocks were selected from 15 cases, comprising 4 B-cell non-Hodgkin's lymphomas, 4 T-cell non-Hodgkin lymphomas, 4 cases with lymphadenitis, 2 cases with confirmed tuberculosis and 1 case of sarcoidosis. All participating laboratories received one 10-microm section from each of the 15 cases to detect clonality using immunoglobulin heavy chain (IgH) gene or T-cell receptor (TCR)-gamma gene rearrangement analysis in 12 and mycobacterial DNA in 3 cases. In addition, participants had to answer technical questions about the application of internal quality controls and performance of fragment length or sequence analysis. Correct results were reported in 80% and 90% for IgH and TCR-gammagene rearrangement analysis, respectively, and in 83% for mycobacterial DNA analysis. No significant differences in the quality of results were obvious when the individual techniques used for molecular analysis were compared. However, when two independent techniques were used by the same laboratory, a higher rate of correct results was obtained for IgH and TCR rearrangement analysis. In conclusion, this study demonstrates a high technical standard of molecular diagnostic adjuncts among the participating laboratories. Regular multicenter trials with a greater number of participating laboratories working in this field will be indispensable to ensure a continuing or increasing standard in diagnostic molecular pathology.