An age-related decline in melatonin secretion is not altered by food restriction.

Melatonin has been found to exhibit youth-maintaining and disease-preventing properties. The current study examined whether the age-retarding regimen of chronic food restriction (FR) slowed the decline in melatonin secretion reported to occur with age. Total nocturnal melatonin secretion was assessed by radioimmunoassay of the primary metabolite, 6-sulphatoxymelatonin (6-S-OH-MLT), in urine. Measurements were made through adulthood (70 to 765 days) on male Wistar rats maintained on the FR regimen (60% of the normal intake) with the control animals fed ad libitum (AL). The data of animals exhibiting gross pathology were excluded. Analyses of covariance found the FR regimen had no effect on either the levels or pattern of decline observed in 6-S-OH-MLT excretion through adulthood. However, the FR body-weight-indexed metabolite measures were approximately double those of the AL (p = .06). The possibility that this result may reflect unusually high melatonin peaks in the FR tissues is discussed.

Late summer and fall (March-May) pollen allergy and respiratory disease in Northern New South Wales, Australia.

BACKGROUND: Many people in the subtropical Northern Rivers area of New South Wales, Australia, blame the pollen of Tibouchina tree, which flowers at the same time as ragweed, Bahia grass and Bermuda grass, for hayfever and asthma exacerbations during fall between March and May. OBJECTIVES: To determine whether Tibouchina pollen is allergenic. To determine whether airborne ragweed pollen is present in this region for sufficient length of time and concentration to cause fall respiratory symptoms, and to determine if Bahia grass and Bermuda grass are associated with fall respiratory symptoms. METHODS: Pollen and Alternaria spores were monitored using a Burkard 7-day spore trap. Two hundred and six volunteers in the Northern Rivers area filled in questionnaires before skin prick tests (SPT) were performed with a panel of skin testing extracts. RESULTS: One hundred fifty-three (74.3%) subjects were atopic and reacted to one or more aeroallergens. Seventy were SPT positive to ragweed, OR 3.36 (CI 1.03 to 12.15) and 11 to Tibouchina (OR incalculable). Fifty of the 70 ragweed-positive subjects had fall hayfever or exacerbations of hayfever and/or asthma, OR 23.4 (CI 8.90 to 64.00). Eleven subjects were SPT positive to Tibouchina extract. There was a statistical association between Bermuda grass and hayfever, but not asthma OR 13.44 (CI 1.85 to 27.04). CONCLUSIONS: Ragweed pollen was present for a sufficient length of time and concentration to sensitize and provoke fall hayfever and asthma exacerbations. Tibouchina pollen is an aeroallergen causing mild-to-moderate allergic symptoms in a few people. There is an association between Bahia grass and asthma in children, and between Bermuda grass and allergic rhinitis in adults.

Preliminary criteria for the definition of allergic rhinitis: a systematic evaluation of clinical parameters in a disease cohort (II)

BACKGROUND: This study is a development of the work done in 'Preliminary criteria for the definition of allergic rhinitis: a systematic evaluation of clinical parameters in a disease cohort (I)'. OBJECTIVE: We sought to develop criteria which could be routinely used to define allergic rhinitis. METHODS: A total of 47 allergic rhinitis and 23 normal subjects were evaluated with a detailed questionnaire and history, physical examination, serum total immunoglobulin (Ig) E, skin prick tests and serum EAST's. RESULTS: Based on this data, we developed a preliminary scoring system by which a reliable diagnosis of allergic rhinitis can be made, which is based on the prevalence adjusted strength of association delta ( partial differential) rank values described in this report. Cumulative frequency histograms were constructed for allergic rhinitis and normal subjects in a variety of configurations. CONCLUSION: A simple scoring system which can be used to diagnose allergic rhinitis on a routine basis was developed in this study.

Molecular cloning and characterization of a cDNA encoding the human leucocyte vacuolar protein sorting (h1Vps45).

We have isolated a novel cDNA clone from human leucocyte cDNA library, encoding a Sec1p-like vacuolar protein sorting (h1Vps45) which is believed to be implicated in vesicular transportation. Although the deduced amino acid (AA) sequence of this cDNA has revealed 97% identity to other known mammalian vacuolar protein sorting, there is an extensive variation in nucleotide sequence in comparison to that of three previously reported human (hVps45), rat (rVps45) and mouse (mVps45) vacuolar protein sorting (Vps45) cDNAs [1-3]. At the nucleotide sequence level h1Vps45 demonstrated 90% homology to the hVps45 and rVps45 and 89% identity to mVps45 with no significant homology in their noncoding regions. The 2.4 Kb mRNA corresponding to the h1Vps45 clone is widely distributed in a variety of human tissues expressing highest levels in peripheral blood mononuclear cells (PBMC), neutrophils, heart, spleen, and testis. The chromosomal mapping studies have demonstrated that the h1Vps45 is localized to long arm of human chromosome 1 at q21-q22. Our data indicates that we have isolated, characterized and mapped a novel cDNA encoding h1Vps45, which may play an important role in protein trafficking as well as have clinical significance in the release of inflammatory mediators e.g. histamine, bradykinin and cytokine release.

Purification and characterization of a human bradykinin binding protein from inflammatory cells.

Bradykinin (BK) is a potent mediator with a broad spectrum of pharmacological and inflammatory actions which are exerted through cell surface receptors. We report here the affinity chromatographic purification of a novel 14 kDa BK binding protein from human blood neutrophils and also peripheral blood mononuclear cells (PBMC), 80% of which are lymphocytes. Radioreceptor crosslinking experiments using bifunctional crosslinkers and radiolabelled BK identified a 14 kDa protein in these cell types both on the cell surface, in glycerol purified plasma membranes and in detergent solubilized cell extracts. Purification by BK affinity chromatography from a variety of BK responsive human cell types i.e. CCD-16Lu lung fibroblasts, HL60 promyelocytes, U937 myelomonocytes and Jurkat T lymphocytes also demonstrated a 14 kDa protein. Purified material obtained from three different BK affinity columns all demonstrated three major proteins at 190, 50 and 14 kDa when eluted with either excess BK or mild acid. Neutrophil fractions from detergent solubilized cell extracts contained an additional 150 kDa protein when eluted with mild acid. Neutrophil and PBMC crude plasma membrane BK affinity column purifications yielded only a single 14 kDa protein. Radioreceptor dot assays of the purified neutrophil eluates containing the 14 kDa protein revealed specific binding to [125I]-BK with a 160 fold excess signal ratio over the original membrane extract. Our data indicates that we have successfully isolated a 14 kDa novel human BK specific binding protein expressed on the surface of inflammatory cells.

High affinity bradykinin binding to human inflammatory cells.

Specific direct bradykinin (BK) binding and competitive inhibition was detected in human neutrophil and peripheral blood mononuclear cell (PBMC) detergent solubilized extracts and purified plasma membranes using in vitro radioreceptor ligand binding. Scatchard analyses of [125I]-BK binding revealed an equilibrium dissociation constant (Kd) of 2.9 x 10(-11) M for neutrophils and 5.6 x 10(-11) M for PBMC using [des-arg9]-BK a B1 agonist; 2.6 x 10(-11) M for neutrophils, 6.2 x 10(-11) M for PBMC with BK a B2 agonist; 5.4 x 10(-11) M for PBMC using Lys-BK a B2 agonist. The number of binding sites (Bmax) was calculated to be 0.113 fM/microgram protein (720 receptors per cell) for neutrophils and 0.200 fM/microgram protein (1289 receptors per cell) for PBMC with the B1 agonist while with the B2 agonists the values were 0.128 fM/microgram protein (818 receptors per cell) for neutrophils and 0.157 fM/microgram protein (1005 receptors per cell) for PBMC with BK, and 0.293 fM/microgram protein (1870 receptors per cell) with Lys-BK for PBMC. In a competitive binding inhibition assay using neutrophil and PBMC glycerol purified plasma membranes, high affinity binding in the nanomolar range was detected to Lys-BK and BK but with [des-arg9]-BK a 10-100 fold lower order affinity was observed this being indicative of pharmacologically defined B2 characteristics.

High resolution autoantibody detection by optimized protein G-horseradish peroxidase immunostaining in a western blotting assay.

We report a procedure for optimisation of Western blotting using protein G-horseradish peroxidase (protein G-HRP) which avoids the false positive reactions often caused by second antibodies and increases the detection of autoantibodies by protein G conjugate. A number of modifications were investigated. Higher concentrations of serum and protein G-HRP at 1:5 to 1:10 and 1:100, respectively, increased the detection to the same order as that obtained with second antibody systems and gold staining with silver enhancement. The role of various detergents in the procedure was established. 3-[(3-Cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) in incubation with protein G-HRP increased the binding between protein G and immunoglobulin G. Addition of Tween-20 for blocking produced little background so that protein blockers could be avoided. Prolonged incubation with serum increased markedly the sensitivity of the procedure when compared with the recommended 2 h incubation period. Polyvinylidene difluoride membrane provided better transfer effect, lower background and higher mechanical strength than nitrocellulose membrane. The utilization of only one antibody-specific ligand increased the simplicity, reliability, economy, efficiency and specificity of the method. These modifications make this method significantly better for detection and screening for autoantibodies.

Polymorphism of IgE gene in chronic urticaria.

Our aim was to determine whether heterogeneity of the IgE (C epsilon) gene could be demonstrated in patients with chronic urticaria (CU). We performed Southern blots on DNA extracted from peripheral blood leucocytes of 20 patients with CU and 20 normal controls. Using a human C epsilon gene probe containing four exons of the constant segment of the IgE heavy chain, we showed the presence of a restriction fragment length polymorphism of the C epsilon gene segment in four of 20 patients with CU, but in none of 20 normal subjects. Family studies of two propositi revealed the presence of this C epsilon gene polymorphism in other family members. Our data show that a proportion of patients with CU have a polymorphism of the constant segment of the C epsilon gene. Further studies of this polymorphic gene fragment indicated that it was derived from duplication of the 3rd and 4th exons of functional C epsilon gene and was very likely to be located close to this gene. It raises the possibility that polymorphism of the functional C epsilon gene may affect expression of this gene. This could possibly lead to dysfunctional IgE-receptor interaction with consequent alteration in mediator release.

Peripheral blood lymphocyte subset distribution and function in patients with Alzheimer's disease and other dementias.

BACKGROUND: Until recently, new data on immune aspects of Alzheimer's disease (AD) have suggested that some facets of AD pathogenesis may be immune related. However, the effects of dementia itself on immune function have not been considered. AIM: To compare the distribution of peripheral blood lymphocyte subsets and their function in patients with AD and other dementias. METHODS: Peripheral blood lymphocyte numbers, T cell subset distribution, proliferative responses to mitogens and suppressor cell assay were studied in a well characterised group of patients with AD, and compared to patients with other forms of dementia. Age and sex matched elderly controls were screened to exclude dementia, and young controls were medical, paramedical and laboratory staff. Analysis of variance (ANOVA) and student's test were used for statistical analysis. RESULTS: The CD8+ lymphocyte population was reduced in AD and in other forms of dementia, when compared with non-demented elderly and young controls. Concanavalin A induced lymphocyte transformation was reduced in all dementia groups and in elderly compared with young controls. The changes in T cell numbers and function were not specific for Alzheimer's disease, but were found also in other forms of dementia.

A preparative method for sequencing proteins and peptides: in situ gel staining with subsequent passive elution onto polyvinylidine difluoride membranes.

A preparative method for obtaining both N-terminal and internal peptide amino acid sequences from purified proteins is reported. The methodology reliably yields high fidelity signal from between 14 to 30 residues per purified protein or peptide, with low backgrounds on amino acid analysis. The procedure relies on the use of in situ staining of proteins during preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the utilisation of microconcentrators to repeatedly concentrate small amounts of proteins onto a small polyvinylidene difluoride (PVDF) disc until sufficient amounts have been adsorbed so as to give a strong sequencing signal. The protein elution and subsequent adsorption can be monitored visually with a dye and the final product, a PVDF disc with the adsorbed protein or peptide, can be directly inserted into the automated amino acid sequencer.

Purification of histamine receptor proteins from detergent-solubilized human peripheral blood mononuclear cells.

Histamine is released from mast cells and basophils by either immunological or nonimmunological mechanisms. Histamine, which is the most potent short acting mediator released from these cells, exerts its diverse biological actions by binding to cell surface histamine receptors. We report the affinity purification of histamine receptor proteins from Triton X-100 solubilized peripheral human blood mononuclear cells which include lymphocytes and monocytes. Three different designs of histamine affinity columns were constructed; all three resulted in the same material being eluted. This consisted of bands which on SDS-PAGE after boiling and reduction had the following molecular weights: 193K, 84K, 58K, 48K, 37K, and 16K. The most abundant bands were of molecular weights 193K, 48K, and 16K, and these were disulfide bonded together to form a high molecular weight complex. (The 58K band was present in lower amounts than the others, and in only a few fractions. It had the same molecular weight as the dimeric form of histamine methyltransferase which is present in small amounts in mononuclear cells and may therefore have copurified.) The histamine binding proteins described in this report were purified by conventional affinity chromatography, rather than by an expression cloning approach which obviates the use of any protein chemistry. Consequently, we had the advantage of being able to verify the histamine binding specificity of our purified proteins directly and with several independent assays as follows. The histamine binding specificity of all three columns was established by specific elution with histamine, by preabsorption of crude cell extract with excess free histamine prior to column application, and by comparison with control columns. Independent determination of the binding specificity, using a radioreceptor dot blot assay, of the eluate containing only the 193K, 48K, and 16K disulfide-linked subunits confirmed that the purified material bound specifically to [3H]histamine and that a 300-500-fold degree of purification from tissue extract had been obtained. Following cell surface radioreceptor cross-linking of radiolabeled histamine to intact mononuclear cells, the 16K band was detected, indicating it to be the ligand-binding subunit for histamine. These same three proteins were purified from T lymphocyte and monocytoid cell lines, indicating that both lymphocyte and monocyte subsets of mononuclear cells express these proteins.(ABSTRACT TRUNCATED AT 400 WORDS)

Diagnosing allergies. The hows and whys.

Allergies are a common problem dealt with in family practice. This article reviews the importance of a proper management approach and details the essential features in establishing the diagnosis with special emphasis given to the importance of good clinical skills.

The interaction of a Huntington disease factor with receptors for the neurotoxin kainic acid.

A factor from mammalian and human brain, which inhibits the rate of migration of leukocytes obtained from sufferers from Huntington disease (Walls and Ruwoldt, 1984), inhibited the specific binding of the neurotoxin [3H]kainic acid to rat brain synaptic membranes. The factor was present in sucrose-particulate but not in soluble fractions from rat sub-cortical tissue, and was destroyed by tryptic digestion. Whereas an ammonium sulfate fraction of direct saline extracts of brain (Walls and Ruwoldt, 1984) gave poor chromatography on HPLC, prior separation of a sucrose-particulate fraction resulted in much improved chromatography. There was a good concordance between leukocyte migration inhibitory activity and [3H]kainic acid binding inhibitory activity. The factor may be an endogenous modulator of the kainic acid subset of receptors for the excitatory neurotransmitter glutamic acid.

Ocular cicatricial pemphigoid.

A series of 17 patients with ocular cicatricial pemphigoid (OCP) is described retrospectively. The importance of early recognition, especially of disease involving the medial canthus and caruncular region, diagnosis by biopsy, and adequate immunosuppressive and surgical therapy, are emphasised. A detailed grading scheme has been developed and this has enabled the authors to determine the success or failure of the therapy during the active treatment period. A combined ophthalmological and immunological approach to treatment can result in a successful visual outcome of this potentially blinding disease.