Cow's milk protein-specific T-helper type I/II cytokine responses in infants with necrotizing enterocolitis.

Enteral feeding, in particular with formula feeds, is associated with necrotizing enterocolitis (NEC). In this study, we have examined, in the systemic and mucosal immune compartments, for evidence of bovine milk antigen sensitization in infants with NEC. Eleven newborns with Bell's staging 2-3 NEC [median post-conceptional age 31 wk (range 27-41 wk)], 21 neonatal controls [33 (28-40) wk] and 15 infants undergoing intestinal resection or mucosal biopsy for non-inflammatory conditions [39 (34-42) wk] were studied. Spontaneous and antigen or mitogen elicited interferon-gamma (IFN-gamma) [T-helper type I (Th1)], interleukin (IL)-4 and IL-5 [T-helper type II (Th2)] responses were enumerated using single-cell enzyme-linked immunospot (ELISPOT) assay in peripheral blood (PBMC) or lamina propria mononuclear cells. NEC infants, compared with controls, showed a significant elevation in baseline PBMC cytokine secreting cells, vigorous mitogen responses (20- to 120-fold increase) for IFN-gamma, IL-4 and IL-5 (p < 0.001), strong responses to beta-lactoglobulin (betalg) (IFN-gamma > IL-4/IL-5, p < or = 0.001), and somewhat smaller casein responses. Similarly, in the lamina propria, a small but significant increase in spontaneous cytokine-secreting cells was detected in NEC infants (p < 0.01), with an IFN-gamma/IL-4 predominant phytohemagglutinin (PHA)/concanavalin-A (ConA) response. Three of nine NEC infants (but no controls) also showed a positive ELISPOT response to betalg (IFN-gamma only) but none to casein. We have thus demonstrated significant cow's milk protein (CMP) sensitization in NEC, at least in the systemic compartment (mixed Th1/Th2), with minimal mucosal activation in some cases. These novel findings provide a potential mechanism for a direct contributory role of CMP in the pathogenesis of NEC.

The HMG domain protein SSRP1/PREIIBF is involved in activation of the human embryonic beta-like globin gene.

The human embryonic beta-like globin (epsilon-globin) gene is expressed in primitive erythroid cells of the yolk sac during the first few weeks of development. We have previously shown that developmental stage-specific expression of the epsilon-globin gene is mediated by multiple positive and negative regulatory elements upstream of the start of transcription. Of particular interest is one positive regulatory element, PRE II, that works together with other elements (PRE I and PRE V) to confer developmental stage- and/or tissue-specific expression on a minimal promoter. An approximately 85- to 90-kDa PRE II binding factor (PREIIBF) was identified in the nuclei of erythroid cells and shown to bind specifically to a novel 19-bp region within PRE II; binding of this protein to PRE II resulted in bending of the target DNA and was required for promoter activation. In this report, we present the cDNA expression cloning of PREIIBF. The cDNA encodes a previously identified member of the HMG domain family of DNA binding proteins termed SSRP1. By a number of biochemical and immunological criteria, recombinant SSRP1 appears to be identical to the PREII binding factor from erythroid nuclei. A hallmark of HMG domain proteins is their ability to bend their target DNAs; therefore, as we speculated previously, DNA bending by SSRP1/PREIIBF may contribute to the mechanism by which PRE II synergizes with other regulatory elements located upstream and downstream. In contrast with reports from other investigators, we demonstrate that SSRP1 binds DNA with clear sequence specificity. Moreover, we show that SSRP1/PREIIBF lacks a classical activation domain but that binding by this protein to PRE II is required for activation of a minimal promoter in stable erythroid cell lines. These studies provide the first evidence that SSRP1 plays a role in transcriptional regulation. SSRP1/PREIIBF may serve an architectural function by helping to coordinate the assembly of a multiprotein complex required for stage-specific regulation of the human epsilon-globin gene.

Methaemoglobin formation due to nitrite, disulfiram, 4-aminophenol and monoacetyldapsone hydroxylamine in diabetic and non-diabetic human erythrocytes in vitro.

Nitrite, monoacetyl dapsone hydroxylamine, 4-aminophenol and disulfiram-mediated methaemoglobin formation was studied in human diabetic and non-diabetic erythrocytes in vitro. Diabetic intact erythrocytes were significantly less sensitive compared with those of non-diabetics to haemoglobin oxidation caused by the hydroxylamine, nitrite and 4-aminophenol, but not disulfiram. In haemolysates, differential sensitivity did occur with disulfiram and was partially retained with 4-aminophenol and nitrite. The differences were lost with 4-aminophenol, nitrite and disulfiram in the presence of haemoglobin purified from the respective erythrocyte types. Diethyl maleate reduced methaemoglobin formation in non-diabetic intact erythrocytes with 4-aminophenol, the hydroxylamine and disulfiram, but not with nitrite. Overall, the differential sensitivity to methaemoglobin formation seen in diabetic compared with non-diabetic erythrocytes, is probably linked to differences in the respective cells' cytosolic anti-oxidant systems.

A gentle method for linking Tris to amino acids and peptides.

A gentle method for the addition of Tris to the carboxyl group of amino acids or peptides for the purpose of altering their solubility and/or for providing sites for further derivatization is described. Under mild alkaline conditions and in high concentrations of aqueous dimethylformamide, the amino group of Tris cleaves simple esters of N-protected amino acids or peptides to form an amino acid-Tris linkage. The effects of pH, temperature and dimethylformamide concentration on the rate and the efficiency of the reaction of Tris with benzyloxycarbonyl-alanine methyl ester were examined and the general applicability of the method demonstrated on a range of amino acid and small peptide substrates. Side-chain protection was not required and the degree of racemization was found to be lower than with conventional chemical coupling.

In vivo production of tumour necrosis factor-alpha and interleukin-6 in BALB/c mice inoculated intranasally with a high dose of respiratory syncytial virus.

Intranasal administration of an inoculum of 10(7) focus-forming units (FFU) of respiratory syncytial (RS) virus induced disease in BALB/c mice with signs of anorexia, cachexia, ruffled fur, and pneumonia. Mice displayed mild signs of illness on day 1 postinoculation (PI), followed by a transient recovery phase of 3 days. Disease rapidly reappeared on day 5 PI and worsened on subsequent days, with mortalities by day 7 PI. Mice inoculated with 5 x 10(6) FFU exhibited milder signs of disease, while those inoculated with 2 x 10(6) FFU and control mice given only Hep-2c cell suspension exhibited no noticeable signs of illness. High levels of bioactive tumour necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) were detected in both lungs and sera of mice inoculated with 10(7) FFU of virus. Peak levels of both cytokines were detected at day 1 PI but remained detectable throughout the 7 day period studied postinoculation. Cytokine levels were much lower or were undetectable in control mice. These results suggest that the macrophage is stimulated in vivo to produce inflammatory cytokines in response to RS virus infection.

Outbreak of botulism in north west England and Wales, June, 1989.

The clinical features of 27 patients identified in an outbreak of botulism in Lancashire, England, and North Wales are reviewed. All but 1 of the patients (age range 14 months to 74 years) were admitted to hospital: 12 were treated in intensive care units, and 8 received positive pressure ventilation. 1 patient died with an aspiration pneumonia. The clinical presentations contained several unusual features, with evidence of segmental demyelination in some patients and drowsiness, sore throats, and fever in others. The widely dispersed source of intoxication with patients presenting singly to several hospitals added to the difficulties of diagnosis. Successful clinical management depends on full and early recognition both of the dangers of impaired oropharyngeal function and of the rapid neurological changes in botulism.

A microtiter plate assay for the characterization of serine proteases by their esterase activity.

The action of serine (and cysteine) proteases on peptide esters proceeds, as a generalization, orders of magnitude faster than the corresponding enzymatic hydrolysis of peptide bonds or peptide amides. Esterolysis liberates an alcohol while generating a free carboxyl group on the peptide; the proton produced can be detected by the use of an appropriate indicator. The action of trypsin on benzyloxycarbonylalanylarginine methyl ester was used as a model for the development of a simple microtiter plate assay procedure that takes advantage of the speed of these reactions and the ease of detection afforded by the color change of the indicator. A family of ester substrates of the form benzyloxycarbonylalanyl-X-methyl ester, in which X is one of the 20 common amino acids, was synthesized to allow the determination of the primary specificity profiles of serine proteases. Using a 96-well microtiter plate the specificity profiles of four enzymes with all 20 substrates can be carried out in approximately 4 h per enzyme, including setting up and data processing. The primary substrate preferences of trypsin, chymotrypsin, thrombin, pancreatic elastase, alpha-lytic protease, subtilisin, and proteinase K were determined to demonstrate the method and were found to be in good general agreement with reported specificities established by more conventional means.

Substrate preference profiles of proteases released by allergenic pollens.

BACKGROUND: Pollens are important triggers for allergic asthma and seasonal rhinitis. We have recently reported that proteases released by major allergenic pollens can injure airway epithelial cells in vitro. Disruption of epithelial integrity by proteases released following deposition of pollens on mucosal surfaces could promote sensitization and induce inflammation. OBJECTIVE: To compare protease activities released by allergenic pollens of various genera. METHODS: We used a rapid microassay which quantifies cleavage of dipeptide ester substrates to characterize the substrate preference profiles of serine proteases in diffusates of the pollens of perennial ryegrass (Lolium perenne), Kentucky blue grass (Poa pratensis), Bermuda grass (Cynodon dactylon), Western ragweed (Ambrosia spp.), white birch (Betula spp.) and Sydney golden wattle (Acacia longifolia). RESULTS: Comparison of the profiles revealed notable differences as well as similarities between serine protease activities released by these pollens. Diffusates of Kentucky blue grass pollen exhibited very high substrate preference for arginine and lysine. For other pollens, cleavage of the cysteine substrate was usually the most rapid and was associated with marked preference for leucine and methionine. There was considerable variation between these pollens in the rates of cleavage of the histidine substrate. In addition, we observed high rates of cleavage of arginine and lysine substrates by Acacia pollen diffusate. CONCLUSION: At least two dominant patterns of substrate preference are identifiable in the mixtures of proteases released by hydrated pollens. Purification of the proteases responsible for these patterns of activity will facilitate investigation of their role in airway epithelial injury and allergic disease.

Alterations in blood leucocyte adhesion molecule profiles in HIV-1 infection.

CD4 and CD8 lymphocyte numbers in the gut lamina propria are grossly altered in HIV-1 infection, out of proportion to alterations in the circulation. Such alterations in lymphocyte counts in the tissues may be due to altered leucocyte migration from the blood. One factor affecting leucocyte migration is adhesion molecule expression. Levels of adhesion molecule expression on peripheral CD4 and CD8 lymphocytes, monocytes and neutrophils from HIV-1-infected (AIDS and non-AIDS) and low-risk control individuals were compared. CD11a, CD62L, CD44, CD49d and beta7 integrin expression were examined by FACS analysis of fresh whole blood. Significant alterations in adhesion molecule expression were detected in HIV infection. The most striking alterations were observed in the CD8 lymphocyte population. CD11a expression was increased and CD62L and CD44 decreased. The CD4 lymphocyte population followed a similar, though less striking, pattern of alteration in adhesion molecule expression. Neutrophils displayed significantly reduced expression of both CD11a and CD62L, but only after onset of AIDS. Monocytes from infected individuals without AIDS displayed a different pattern of altered adhesion molecule expression compared with individuals with AIDS. These findings suggest that in HIV infection, leucocyte functions, such as migration, which require adhesion molecules are abnormal.

Testicular endocrine function in paraplegic men.

The responses of FSH and LH to the injection of LHRH were studied in fifteen paraplegic men and ten normal controls. The basal FSH levels in the paraplegic men were elevated in eight cases, basal LH being raised in nine cases. The responses of FSH were exaggerated in fourteen patients, and LH responses were exaggerated in nine patients. Only one patient was normal in respect of both hormones. Subnormal plasma testosterone was found in three patients. This depressed testicular function could not be explained as being due to spread of urinary tract infection and a direct neurological effect is the probable explanation.

A new library of alpha-lytic protease S1 mutants generated by combinatorial random substitution.

Four positions in the S1 site of alpha-lytic protease (positions 190, 192, 213 and 218) were subjected to targeted random mutagenesis. In the resulting library we found active mutant proteases whose cleavage preferences could be grouped into three distinct families. Some potentially useful enzymes (such as one that prefers to cleave at Asn and Cys residues) were identified. In addition, we discuss instances where it was possible to relate changes in substrate specificity to alterations in the structure of the substrate binding site.

Eradication of cryptosporidia and microsporidia following successful antiretroviral therapy.

OBJECTIVES: Incidence of opportunistic protozoal infections causing diarrheal illnesses in patients with HIV has decreased since the introduction of highly active antiretroviral therapy (HAART). The objective of this study was to determine whether the parasites, cryptosporidia, and microsporidia were effectively eradicated or only suppressed following treatment. DESIGN: Six HIV-positive patients with diarrheal symptoms caused by cryptosporidia or microsporidia were prospectively followed up with stool samples and duodenal biopsies. Samples were taken before HAART, between 1 to 3 months, and 6 months post-HAART. METHODS: Duodenal samples were analyzed using routine histology and transmission electron microscopy. Stool samples were analyzed by both light microscopy and polymerase chain reaction (PCR) techniques. RESULTS: Patients who responded successfully to HAART eradicated both cryptosporidial and microsporidial organisms. Symptoms improved within 1 month of therapy but complete eradication of the organisms was only observed after 6 months of treatment. CONCLUSIONS: AIDs-related cryptosporidiosis and microsporidiosis can be cured following successful antiretroviral therapy.