Reactivity of T lymphotropic retrovirus antibody (12/1-2) in man: comparison of epidermis with other epithelial cells.

The reactivity of a monoclonal antibody against human T lymphotropic retrovirus (antibody 12/1-2, recognising the HTLV-1 p19 internal core viral protein) with benign and malignant cutaneous biopsy specimens was examined and compared with results obtained on normal skin, on various other human cells and tissues, and on immunoblotted extracts of tonsil squamous epithelium. In keeping with previous studies, 12/1-2 labelled a proportion of the thymic epithelial stroma and the entire layer of basal cells in stratified non-keratinized and keratinized epithelium. Furthermore, antibody 12/1-2 reacted with basal cell carcinomas and showed an essentially identical staining pattern in normal skin, cutaneous T cell lymphomas, and a range of benign dermatoses. The dot blot preparations showed that 12/1-2 recognised an antigen associated with keratin intermediate filaments. These data indicate that antibody 12/1-2 forms a useful marker for subsets of epithelial cells, which presumably participate in T cell education, and that a range of cutaneous disorders of widely different aetiology show no abnormalities in epithelial expression of this antigen.     

Immunocytochemical staining of cells in pleural and peritoneal effusions with a panel of monoclonal antibodies.

A panel of seven monoclonal antibodies was applied to smears of cell deposit from 70 pleural and peritoneal fluids, using an immunoalkaline phosphatase (IAP) procedure. The cases were chosen to show typical cytological patterns, both benign and malignant, and in this way the diagnostic value of the method could be assessed. The antibodies used were 2D1 (anti-leucocyte), Ca 1, HMFG-2 (anti-milk fat globule membrane), LE61 and M73 (both anti-intermediate filament antibodies), anti-CEA, and K92 (anti-keratin). The anti-leucocyte antibody was found useful for distinguishing lymphoma from carcinoma. Anti-CEA gave positive reactions in 80% of carcinoma cases and was not seen to react with any other cell types. Ca 1 was positive with some cells in 95% of carcinoma cases, but mesothelial cells reacted with it in two cases. A strong reaction with the anti-milk fat globule membrane antibody was very constant in carcinoma but was also seen in mesothelial cells in 30% of benign effusions. The anti-keratin reacted with malignant cells in only a small proportion of cases. The antibodies against epithelial intermediate filaments reacted equally strongly with benign mesothelial cells and carcinoma cells, but gave negative reactions with lymphoma cells. It is concluded that a suitably chosen panel of monoclonal antibodies can be of great value in identifying neoplastic cells in serous effusions.     

Localization of binding sites for calcitonin gene-related peptide in rat brain by in vitro autoradiography

The distribution of binding sites for calcitonin gene-related peptide (CGRP) in rat brain were studied using in vitro autoradiography. In a radioreceptor assay using [125I]human calcitonin gene-related peptide as the radioligand, with cerebellar cortical membranes, rat calcitonin gene-related peptide had a binding affinity constant of 1.16 +/- 0.23 X 10(10) M-1 and a site concentration of 43.4 +/- 3.4 fmol/mg protein. In this system, human calcitonin gene-related peptide had a binding affinity constant of 3.9 +/- 0.7 X 10(9) M-1 whereas salmon calcitonin was very weak with a binding affinity constant of only 6.8 +/- 4.0 X 10(5) M-1. CGRP binding localized by in vitro autoradiography, using [125I]rat calcitonin gene-related peptide, had a characteristic distinct distribution in the rat brain. There were high concentrations of binding found over the accumbens nucleus, the organum vasculosum of the lamina terminalis, ventral caudate putamen, median eminence, the arcuate nucleus, lateral amygdaloid nucleus and lateral mammillary nucleus, the superior and inferior colliculi, pontine nuclei, molecular and Purkinje cell layers of the cerebellar cortex, the nucleus of the solitary tract, the inferior olivary nuclei, hypoglossal complex and the vestibular and cochlear nuclei. The distribution of these binding sites suggests multiple roles for CGRP in the central nervous system including auditory, visual, gustatory and somatosensory processing, and in neuroendocrine control.     

Evaluation of four methods for detection of group B streptococcal colonization.

Four methods (streak plate, pour plate, selective broth, and direct fluorescent-antibody staining) were evaluated for their ability to detect group B streptococcal colonization in parturient women and their offspring. When colonization was defined as a positive culture by any method from any site, selective broth was the most sensitive method, detecting 100% of colonized mothers and infants at birth and 48 h of age. This method failed to detect only one colonized individual (infant at 24 h of age). The other three methods detected from 20 to 56% of colonized individuals.     

Calcium signals in single T cells on activation by lectin

Succinylation of concanavalin A (Con A) reduces its oligomer size while retaining its mitogenicity, and provides a probe of T cell activation. We have observed responses of cytosolic ionized calcium to succinyl Con A in suspensions of Jurkat and rat lymph node (LN) cells, using a fluorimeter, and in single cells settled on glass, using a dual wavelength video imaging system. In the fluorimeter a mitogenic level of succinyl Con A (30 micrograms/ml) produced only a 15-30 nM rise in average cell calcium in the suspended Jurkat or rat cells whereas the use of quantitative video imaging produced asynchronous 250-1000 nM pulses of free calcium in 35% of Jurkat cells and 300-850 nM pulses in 45% of rat LN cells. In Jurkat cells these pulses were sometimes repetitive, giving rise to apparent oscillations. In the fluorimeter 30 micrograms/ml of native Con A (a supra-mitogenic concentration) produced a 300 nM rise in average cell calcium in suspended Jurkat cells, and a 100 nM rise in rat LN cells; when major histocompatibility complex class II-bearing cells were removed the response rose. Mitogenic Con A (3 micrograms/ml) produced a much lower rise in calcium. With video imaging the response seen was greater. Levels greater than 30 micrograms/ml Con A caused 700-5000 nM pulses synchronously in 94% of Jurkat cells and 250-1000 nM pulses in 73% of rat LN cells. At 3 micrograms/ml Con A produced asynchronous 300-1100 nM pulses in 36% of rat LN cells. We conclude that the absence of a calcium signal in the fluorimeter can conceal asynchronous calcium responses in individual cells and that brief asynchronous cytosolic calcium pulses are sufficient for lectin to activate rat T cells.     

Effects of heparin on platelet aggregation and release and thromboxane A2 production.

Heparin, when added to citrated platelet-rich plasma (PRP), caused potentiation of platelet aggregation and the release reaction induced by the aggregating agents adenosine diphosphate (ADP), arachidonic acid, collagen, and epinephrine. At low concentrations (4.7 x 10(-5) M) arachidonic acid failed to cause aggregation of platelets in citrated PRP. However, in the presence of heparin, the same concentration of arachidonic acid caused aggregation. Examination of PRP for the presence of thromboxane A2 (TxA2) by use of a bioassay revealed that heparin also stimulated release of TxA2. This finding indicated that platelets released more TxA2 when they were challenged by low concentrations of arachidonic acid in the presence of heparin than in its absence. Platelets were labeled with 3H-arachidonic acid and 14C-serotonin, and attempts were made to determine whether heparin stimulated the platelet release reaction first with subsequent increased production of TxA2, or alternatively, whether heparin stimulated TxA2 production first with subsequent enhancement of the release reaction. In view of the demonstrated simultaneous release of 14C-serotonin and 3H-arachidonic acid metabolites, it appeared that either release of 14C and 3H occurs concurrently or, even if one of these events is dependent on the other, both events take place in rapid succession. Timed sequential studies revealed that in the presence of arachidonic acid, the addition of heparin hastened the apparently simultaneous release of both 14C and 3H.     

High rates of clustering of strains causing tuberculosis in Harare, Zimbabwe: a molecular epidemiological study

We examined the pattern of tuberculosis (TB) transmission (i.e., reactivation versus recent transmission) and the impact of human immunodeficiency virus (HIV) infection in Harare, Zimbabwe. Consecutive adult smear-positive pulmonary TB patients presenting to an urban hospital in Harare were enrolled. A detailed epidemiological questionnaire was completed, and tests for HIV type 1 and CD4 cell counts were performed for each patient. Molecular fingerprinting of the genomic DNA recovered from cultures of sputum was performed by two molecular typing methods: spacer oligonucleotide typing (spoligotyping) and analysis of variable number of tandem DNA repeats (VNTRs). A cluster was defined as isolates from two or more patients that shared the same spoligotype pattern or the same VNTR pattern, or both. DNA suitable for typing was recovered from 224 patients. The prevalence of HIV infection was 79%. Of 187 patient isolates (78.6%) typed by both spoligotyping and analysis of VNTRs, 147 were identified as part of a cluster by both methods. By spoligotyping alone, 84.1% of patient isolates were grouped into 20 clusters. The cluster size was generally <8 patient isolates, although three large clusters comprised 68, 25, and 23 patient isolates. A total of 89.4% of the patient isolates grouped into 12 clusters defined by analysis of VNTRs, with 2 large clusters consisting of 127 and 13 patient isolates, respectively. Thirty-six percent of patient isolates with a shared spoligotype and 17% with a shared VNTR pattern were geographically linked within Harare, but they were not linked on the basis of the patient's home district. In a multivariate analysis, there were no independent predictors of clustering, including HIV infection status. Comparison with the International Spoligotype database (Pasteur Institute, Pointe a Pitre, Guadeloupe) demonstrated that our three largest spoligotype clusters are well recognized and ubiquitous in Africa. In this epidemiologically well characterized urban population with a high prevalence of HIV infection, we identified a very high level of strain clustering, indicating substantial ongoing recent TB transmission. Geographic linkage could be detected in a proportion of these clusters. A small group of actively circulating strains accounted for most of the cases of TB transmission.     

Postsynaptic fall in intracellular pH and increase in surface pH caused by efflux of formate and acetate anions through GABA-gated channels in crayfish muscle fibres.

H(+)-selective microelectrodes and a two- or three-microelectrode voltage clamp were used to examine the influence of weak-acid, carboxylate anions on the actions of GABA on postsynaptic intracellular pH, surface pH and on membrane potential in fibres of the crayfish leg opener muscle. Substitution of 30 mM Cl- by formate or acetate promoted a GABA-induced decrease in intracellular pH, which was coupled to an increase in surface pH and to a depolarization. Such effects were not seen in the presence of an equivalent amount of lactate, methanesulphonate or glucuronate. Both the GABA-induced depolarization and the fall in internal pH promoted by formate and acetate were blocked by picrotoxin, and the fall in pH was reversibly inhibited by a K(+)-induced depolarization. The rate of the fall in intracellular pH produced by GABA (0.2 mM) was about 0.02 pH units/min in the presence of formate and 0.03 pH units/min in the presence of acetate. Under steady-state conditions, both 30 mM formate and acetate (but not lactate) induced a positive shift in the reversal potential of GABA-activated current, which was accounted for by a relative permeability vs Cl- of formate and acetate of 0.5 and 0.15, respectively. The conductance sequence of the anions was identical to the permeability sequence, i.e. Cl- greater than formate greater than acetate greater than lactate approximately equal to 0. This sequence is strictly correlated to the Stokes diameter of the anions. The relative permeabilities of the anions indicate that the effective diameter of the GABA-gated channel is about 0.5 nm. The fact that the GABA-induced acidosis was slower in the presence of formate than in the presence of acetate suggests that, in the former case, the rate-limiting step in the fall in internal pH is the entry of non-dissociated formic acid. All the above results are consistent with a scheme where GABA induces a channel-mediated efflux of permeant weak-acid anions, which gives rise to an inward (depolarizing) current and to an intracellular acidosis. A comparison of the permeability properties of crayfish and vertebrate GABA-gated channels suggests that effects similar to those seen in this work are likely to occur in mammalian and other vertebrate neurons in the presence of permeant weak-acid anions.     

Lactoferrin in aspirates of odontogenic cyst fluid.

The possibility that the presence of lactoferrin in aspirates of odontogenic cyst fluid might be a useful preoperative diagnostic marker for odontogenic keratocyst was investigated. Using qualitative and quantitative immunodiffusion methods fluid from 29 of 29 dental (radicular) cysts, 12 of 14 dentigerous cysts and 27 of 31 keratocysts were found to contain lactoferrin. Although some of the highest concentrations of lactoferrin were detected in fluids from keratocysts, there was no significant difference between lactoferrin concentrations among the three groups. Neutrophil elastase was detected in 20 of 24 samples tested, 22 of which also contained lactoferrin. Immunocytochemical localisation of both lactoferrin and elastase was confined to neutrophils infiltrating cyst walls. These results suggest that lactoferrin in fluid from odontogenic cysts is derived from infiltrating neutrophils and that its presence in aspirated fluids is not a useful diagnostic marker for odontogenic keratocyst.