Cisplatin-based chemotherapy in advanced basal and squamous cell carcinomas of the skin: results in 28 patients including 13 patients receiving multimodality therapy.

This study reports the results of cisplatin (CDDP)-based chemotherapy (CT) as sole therapy and as neoadjuvant (NA) therapy in 28 consecutive patients (pts) with advanced basal cell (BC) and squamous cell (SC) cancers of the skin. CT in 24 pts consisted of CDDP 75 mgm/m2 and doxorubicin (Dox) 50 mg/m2 intravenously (IV) every 3 weeks with Dox being omitted in four pts due to severe preexisting cardiac disease. Thirteen of the 28 pts received CT in the NA setting, five before surgery and eight before radiation therapy (RT). Response rates to CT were complete remission (CR) in eight of 28 (28%) pts, partial remission (PR) in 11 of 28 (40%) for an overall response rate of 68%. Thirteen pts received a second treatment modality with five of 13 pts having a CR to CT alone before the second modality and seven converting to CR postsecond modality for a total CR rate of 12 of 13 (92%) in the multimodality group. Duration of responses in the CT-only group ranged from 4 to 82 months; however, only two patients remain in remission in this group. Of the twelve CRs from the multimodality therapy group, 11 of 12 (91%) pts remain in CR with duration of response ranging from 3 to 81 months. Toxicities were manageable, with no toxic deaths and only five pts stopped CT secondary to side effects. This study suggests the combination of CDDP and Dox is highly effective in BC and SC cancers of the skin and by itself can produce long unmaintained remissions, but when combined with a second modality of therapy, it is capable of producing not only long unmaintained CRs but probable cures in the majority of pts.

Mineral contents of brans passed through the human GI tract.

Concentrations of Cu, Fe, Zn, Ca, and phytate were measured in dry-milled corn bran, wheat brans, and soybean hulls prior to being baked in bread and after passage through the human GI tract. Significant changes in mineral concentrations in the retrieved remnants compared to the starting materials were as follows: Cu, Fe, and Zn increased by factors of from two to four and Ca increased at least ten-fold in dry milled corn bran; Ca increased and phytate decreased in wheat brans; Zn increased and Fe decreased in soy hulls. These studies show that indigestible remnants of wheat brans in the human colon associate preferentially with calcium and that dry milled corn bran remnants can be loaded with increased concentrations of all four minerals, and especially with calcium. Concentrations of minerals and phytate were significantly greater in whole fecal samples from wheat bran diets than in the corresponding retrieved bran remnants.

A prospective study of the management of the young Helicobacter pylori negative dyspeptic patient--can gastroscopies be saved in clinical practice?

BACKGROUND: Helicobacter pylori status has been suggested as a means of selecting young dyspeptic patients for gastroscopy as patients who are H. pylori negative and do not exhibit alarm symptoms or ingest non-steroidal anti-inflammatory medication have a low risk of serious organic disease. AIM: To determine if young patients with ulcer-like dyspepsia and found to be H. pylori negative on non-invasive testing could be reassured by this knowledge and not proceed to gastroscopy. PATIENTS: One hundred and sixty-one consecutive attendees aged 45 years or less with a presenting complaint of epigastric pain or discomfort were prospectively recruited from open access gastroscopy referrals and gastroenterology clinics. METHODS: Patients who were H. pylori negative on 13-carbon urea breath test were reassured of the likelihood of a normal gastroscopy, given lifestyle advice and also advised to take symptomatic therapy as required. Patients were reviewed at 6 weeks, 3 months and 6 months when symptoms and quality of life were reassessed. Patients proceeded to gastroscopy if at any review their dyspepsia score stayed the same or worsened. RESULTS: Fifty-five H. pylori negative patients were recruited (30 male, mean age 31 years), two patients did not attend subsequent review. Thirty-two (58%) came to gastroscopy. Endoscopic diagnoses included 25 which were normal, three with gastro-oesophageal reflux disease, three with peptic ulcer disease and one with gastric erosions. Dyspepsia and quality of life scores showed significant improvement over 6 months. CONCLUSIONS: This management strategy resulted in a 42% reduction in gastroscopies in H. pylori negative patients. Whilst the majority of patients endoscoped had normal findings, seven patients (22%) had pathology. Overall there were significant improvements in dyspepsia and quality of life at 6 month follow-up.

General practitioners' habits and knowledge in relation to the management of H. pylori-associated dyspepsia and their views about a locally available 13-carbon urea breath test.

We report the results of general practitioners' views on Helicobacter pylori-associated dyspepsia and use of screening tests in the community. The use of office serology tests in screening is of concern as independent validation in specialist units has been disappointing.

An extracellular fungal polysaccharide composed of 2-acetamido-2-deoxy-D-glucuronic acid residues.

The black yeast-like fungus NRRL YB-4163, now tentatively identified as Rhinocladiella elatior Mangenot, has been found to produce an extracellular microbial polysaccharide composed mainly of 2-acetamido-2-deoxy-D-glucuronic acid residues. Polysaccharide (PS) YB-4163, when isolated in good yield as the neutral potassium salt, dissolves readily in water to produce extremely viscous solutions, which form stable foams and emulsions. By depolymerizing PS YB-4163 with [14C]methanol-HCl, the polysaccharide can be both identified and quantitated radiochemically by determining the individual [14C]methyl glycosides after their separation by paper chromatography. When the methyl glycosides of PS YB-4163 were reduced with NaB3H4, only the methyl glycosides of 2-acetamido-2-deoxy-D-[6-3H]glucose were found. Analysis of the monosaccharide released from carboxyl-reduced PS YB-4163 by acid hydrolysis or methanolysis also showed 2-acetamido-2-deoxy-D-glucuronic acid to be the main constituent. Previously, the only polysaccharides known to be composed entirely or hexosaminuronic acid have been cellular products from pathogens. Of these, the antigenic polysaccharide (SPSA) from Staphylococcus aureus is composed entirely of 2-amino-2-deoxy-D-glucuronic acid, but its amino groups are substituted equally with acetyl and N-acetylalanyl groups. The specific optical rotation of PS YB-4163, [alpha]20D -75 degrees (c 0.5, water), is similar to that of SPSA (-91 degrees), and suggests beta-D-linkages that must be either (1 leads to 3) or (1 leads to 4).

Cisplatin-associated hemolytic-uremic syndrome. Successful treatment with a staphylococcal protein A column.

Cisplatin-associated hemolytic-uremic syndrome (HUS), an under-reported form of HUS induced by chemotherapy, typically pursues a fulminant and lethal course. We report the cases of two patients with squamous cell carcinoma of the head and neck who developed massive hemolysis, profound thrombocytopenia, and dialysis-dependent renal failure after therapy with cisplatin. Plasma exchange was ineffective in both patients, but plasma perfusion with a staphylococcal protein A column produced a dramatic and permanent response in the second patient. These cases show the importance of considering HUS as a cause of renal failure in such patients who receive cisplatin-based chemotherapy, and support the role of staphylococcal protein A plasma perfusion as treatment for this condition.

Tuberculosis in a kiwi (Apteryx mantelli).

Generalised tuberculosis was found in a captive kiwi (Apteryx mantelli) at autopsy. Sequential faecal sampling of the other kiwis and falcons (Falco novaeseelandiae) held in the aviary revealed an excretor of Mycobacterium avium.

Severe central nervous system toxicity from high-dose cytarabine: expressive aphasia occurring after the second day of treatment.

A patient with acute erythroleukemia secondary to polycythemia vera was treated iv with high-dose cytarabine at a dose of 3 g/m2 every 12 hours. After the fourth dose, he developed an expressive (Broca's type) aphasia and somnolence, which progressed until after the treatment was stopped at the seventh dose. The somnolence cleared partially during the next 2 weeks but recurred after cytarabine was restarted at a dose of 100 mg/m2 daily for 7 days. The patient died 5 days later of refractory extreme thrombocytopenia and bleeding. The normal spinal fluid obtained during life and the postmortem findings were consistent with previously reported findings with central nervous system toxicity from high-dose cytarabine. Three unusual aspects of this case are early onset of symptoms, expressive aphasia as the dominant manifestation, and exacerbation of symptoms with standard doses (100 mg/m2/day).

The NIST Surface Structure Database -- SSD version 4.

SSD-4, the fourth version of the NIST Surface Structure Database, has appeared in early 2002 as a significant upgrade to both the database and graphics software; it now also includes nearly 1300 structures published through the end of 2000. SSD is an interactive PC-based database of critically selected surface structures determined with a variety of experimental techniques. The data include not only atomic coordinates, bond lengths and bond angles, but also information about experimental preparation, measurement and analysis methods. The software provides advanced search and display facilities, as well as interactive three-dimensional color visualization and analysis tools of great flexibility. In addition, the program can generate publication-quality color or grayscale prints of any structure, with a host of user-selectable options (such as view angle, perspective, colors, ball and bond styles, and labels).

Salmonella pathogenicity island 2 influences both systemic salmonellosis and Salmonella-induced enteritis in calves.

We have used signature-tagged mutagenesis to identify mutants of the host-specific Salmonella enterica serotype Dublin which were avirulent in calves and/or BALB/c mice. A mutant with a transposon insertion in the sseD gene of Salmonella pathogenicity island 2 (SPI-2), which encodes a putative secreted effector protein, was identified. This mutant was recovered from the bovine host but not from the murine host following infection with a pool of serotype Dublin mutants. However, a pure inoculum of the sseD mutant was subsequently shown to be attenuated in calves following infection either by the intravenous route or by the oral route. The sseD mutant was fully invasive for bovine intestinal mucosa but was subsequently unable to proliferate to the same numbers as the parental strain in vivo. Both the sseD mutant and a second SPI-2 mutant, with a transposon insertion in the ssaT gene, induced significantly weaker secretory and inflammatory responses in bovine ligated ileal loops than did the parental strain. These results demonstrate that SPI-2 is required by serotype Dublin for the induction of both systemic and enteric salmonellosis in calves.

An improved method for preparing thick sections for immuno/histochemistry and confocal microscopy and its use to identify rare events.

Detection of rare events within solid tissues by immunocytochemistry is aided by imaging thick sections. Sections of 40--100 microm thickness of paraformaldehyde-fixed solid tissue can be prepared by use of a vibrating microtome and when immunolabelled these sections can be imaged in a confocal microscope. This approach provides excellent preservation of the structure of the sample and imposes minimal antigenic damage. In studies of the invasion of the bovine intestinal epithelium by Salmonella, this method has allowed detection of individual invading bacteria within large samples. The thick vibrating microtome sections were also used for the detection of rare apoptotic cell nuclei identified by TUNEL staining.

Extracellular polysaccharide from the black yeast NRRL Y-6272: improved methods for preparing a high-viscosity, pigment-free product.

When the extracellular polysaccharide from the black yeast NRRL Y-6272, composed of two parts N-acetyl-D-glucosamine and one part N-acetyl-D-glucosaminuronic acid, is isolated at maximum culture viscosity, adhering black pigment gives the polysaccharide preparations a gray-to-black appearance. Precipitation of the polysaccharide from cell-free culture supernatants with either ethanol of hexadecyltrimethylammonium bromide failed to remove the pigment. Various other methods were therefore tried for obtaining a high-viscosity polysaccharide product free of pigment. By systematically varying ingredients of defined and semidefined media, an improved medium was found that not only gave polysaccharide preparations of increased viscosity, but also increased yield. A key ingredient in this medium is L-asparagine. Also, adding autoclaved bovine serum albumin or egg albumin to this medium at the time of inoculation allowed a pigment-free polysaccharide to be isolated by standard procedures. None of several other proteins of synthetic polyamides tested were as effective as bovine serum albumin or egg albumin. In an alternate approach, pink mutants obtained by irradiation of the parent black strain with ultraviolet light, apparently produce the same extracellular polysaccharide free of any pigment but in lower yields or inferior in quality.

Salmonella SirA is a global regulator of genes mediating enteropathogenesis.

SirA of Salmonella typhimurium is known to regulate the hilA and prgH genes within Salmonella pathogenicity island 1 (SPI1). To identify more members of the SirA regulon, we screened 10,000 random lacZY fusions (chromosomal MudJ insertions) for regulation by SirA and identified 10 positively regulated fusions. Three fusions were within the SPI1 genes hilA (an SPI1 transcriptional regulator), spaS (a component of the SPI1 type III export apparatus) and sipB (a substrate of the SPI1 export apparatus). Two fusions were within the sopB gene (also known as sigD). sopB is located within SPI5, but encodes a protein that is exported via the SPI1 export apparatus. In addition, five fusions were within genes of unknown function that are located in SPI4. As spaS and sipB were likely to be hilA dependent, we tested all of the fusions (except hilA) for hilA dependence. Surprisingly, we found that all of the fusions require hilA for expression and that plasmid-encoded SirA cannot bypass this requirement. Therefore, SirA regulates hilA, the product of which regulates genes within SPI1, SPI4 and SPI5. Both sirA and hilA mutants are dramatically attenuated in a bovine model of gastroenteritis, but have little or no effect in the mouse model of typhoid fever. This study establishes the SirA/HilA regulatory cascade as the primary regulon controlling enteropathogenic virulence functions in S. typhimurium. Because S. typhimurium causes gastroenteritis in both cattle and humans, we believe that this information may be directly applicable to the human disease.

Mutation of invH, but not stn, reduces Salmonella-induced enteritis in cattle.

The induction of secretory and inflammatory responses in calves by Salmonella typhimurium and Salmonella dublin strains was compared, and the effects of mutations in the invH and stn genes were assessed. S. typhimurium induced greater secretory and inflammatory responses than S. dublin in bovine ileal loops, despite the fact that these serotypes were recovered from bovine ileal mucosa in comparable numbers (P. R. Watson, S. M. Paulin, A. P. Bland, P. W. Jones, and T. S. Wallis, Infect. Immun. 63:2743-2754, 1995). These results implicate serotype-specific factors other than, or in addition to, intestinal invasion in the induction of enteritis. The secretory and inflammatory responses induced by S. typhimurium and S. dublin in bovine ligated ileal loops were not significantly altered by mutation of stn, which suggests that stn does not have a major role in Salmonella-induced enteritis. The invH mutation significantly reduced the secretory and inflammatory responses induced in bovine ileal loops, and this correlated with a reduction in the severity of enteritis following oral inoculation of calves. The attenuation associated with the invH mutation did not appear to be due to an increased susceptibility to the innate host defense mechanisms, because the resistance of S. typhimurium to the bactericidal action of either bovine polymorphonuclear leukocytes or bovine serum was not significantly altered. However, lysis of macrophages following infection with S. typhimurium was significantly reduced by the invH mutation. The invH mutation prevented the normal secretion of several proteins, including SipC, by S. typhimurium, indicating that the function of the inv-spa-encoded type III protein secretion system was disrupted. Taken together, these observations implicate inv-spa-dependent effectors in mediation of Salmonella-induced enteritis in cattle. Clearly, however, other undefined serotype-specific virulence factors are also involved in Salmonella-induced enteritis.

Secreted effector proteins of Salmonella dublin act in concert to induce enteritis.

The ability of enteropathogenic salmonellae to recruit inflammatory cells and induce secretory responses in the infected ileum is considered to be a main feature in Salmonella-induced enteritis. Interactions between the pathogen and intestinal epithelial cells result in a variety of cellular responses mediating inflammation and fluid secretion. It is becoming apparent that proteins secreted by the Inv-Spa type III secretion system of Salmonella spp. play a key role in the induction of these responses. We have recently demonstrated that the SopB effector protein is translocated into eukaryotic cells via a Sip-dependent pathway and mediates inflammation and fluid secretion in infected ileal mucosa. However, SopB did not appear to be the only effector involved, as inactivation of the sopB gene only partially impaired enteropathogenicity. We suggested that at least some of such protein effectors are likely to be proteins of the same class as SopB, i.e., secreted effector proteins translocated into eukaroyotic cells via a Sip-dependent pathway. In this work, we identify SopD, another secreted protein belonging to the family of Sop effectors of Salmonella dublin. Using the cya reporter system we showed that SopD is translocated into eukaroyotic cells. We assessed the potential involvement of SopD in enteropathogenicity and found that inactivation of sopD has an additive effect in relation to the sopB mutation.

Salmonella enterica serovars Typhimurium and Dublin can lyse macrophages by a mechanism distinct from apoptosis.

Salmonella enterica serovars Typhimurium and Dublin lysed primary bovine alveolar macrophages and immortalized J774.2 macrophage-like cells in the absence of either the morphological changes or DNA fragmentation characteristic of apoptosis. Macrophage lysis was dependent on a subset of caspases and an intact sipB gene.

Mutation of waaN reduces Salmonella enterica serovar Typhimurium-induced enteritis and net secretion of type III secretion system 1-dependent proteins.

Mutation of waaN, a gene involved in lipid A biosynthesis, reduced enteropathogenic responses induced by Salmonella enterica serovar Typhimurium in bovine ligated ileal loops. However, the secretion of key virulence determinants was also reduced, and therefore the reduction in enteropathogenicity cannot be solely attributed to a reduction in biological activity of lipid A.

Identification of a pathogenicity island required for Salmonella enteropathogenicity.

Salmonella spp. interact with ileal mucosa and disrupt normal intestinal function, which results in an acute inflammatory cell influx, fluid secretion and enteritis. We have recently characterized SopB, a novel secreted effector protein of Salmonella dublin, and presented evidence that SopB is translocated into eukaryotic cells via a sip-dependent pathway to promote fluid secretion and inflammatory responses. Here, we show that sopB is located on a large DNA fragment unique to the Salmonella chromosome. This locus is conserved in Salmonella and maps at approximately 20 centisome of the S. typhimurium chromosome. Sequence analysis revealed that this Salmonella-specific DNA fragment is flanked by DNA sequences with significant sequence similarity to the Escherichia coli K-12 genes, tRNA1ser (serT) on one side and copS/copR on the other. Thus, this Salmonella-specific DNA fragment has features characteristic of 'pathogenicity islands' and, therefore, it was denoted SPI-5 (Salmonella pathogenicity island-5). SPI-5 was sequenced and was found to contain five novel genes, pipA, pipB, pipC, pipD (pathogenicity island-encoded proteins) and orf, in addition to sopB. The effect of mutations in pipA, pipB and pipD on the induction of fluid secretion and an acute inflammatory cell influx was assessed in bovine ligated ileal loops. The effect of mutations in SPI-5-encoded genes on systemic salmonellosis was assessed in mice. The results of these experiments suggest that SPI-5-encoded genes contribute to enteric but not to systemic salmonellosis.

Differential regulation of enteric and systemic salmonellosis by slyA.

Mutation of slyA, which reduces Salmonella typhimurium virulence in mice, caused only minor attenuation of S. typhimurium virulence in orally inoculated calves. This correlated with modest reductions in intestinal invasion and enteropathogenic responses in bovine ligated ileal loops. slyA appears to regulate virulence genes involved in systemic, but not enteric, salmonellosis.