Staphylococcal toxin-induced T cell proliferation in atopic eczema correlates with increased use of superantigen-reactive Vbeta-chains in cutaneous lymphocyte-associated antigen (CLA)-positive lymphocytes

Staphylococcal superantigens have been implicated in the pathogenesis of atopic dermatitis (AD). This may occur through superantigenic activation of T lymphocytes and their subsequent induction of the skin homing receptor CLA on activated cells. We investigated the proliferative responses of peripheral blood mononuclear cells (PBMC) from 10 patients with an infective exacerbation of AD and six normal controls to the staphylococcal superantigens, staphylococcal enterotoxin A and B (SEA, SEB) and toxic shock syndrome toxin-1 (TSST-1), and the mitogens phytohaemagglutinin (PHA) and concanavalin A (Con A). We also assessed CLA and T cell receptor (TCR) Vbeta-chain expression by immunofluorescence and flow cytometry before and after stimulation. PBMC from AD patients showed two-fold increased proliferation to SEA and SEB (P < 0.01) compared with normals, whereas the response to mitogenic stimulation was identical. Analysis of (TCR) Vbeta-chain expression demonstrated increased use of superantigen-reactive Vbeta families in freshly isolated PBMC in AD patients compared with controls. This pattern of Vbeta-chain expression was only observed in the CLA+ but not the total population of T cells. Furthermore, there was a positive correlation between the enhanced PBMC proliferative response and increased expression of superantigen-reactive Vbeta families in atopic patients. These data support the concept that superantigens are important in the pathogenesis of this common condition, and also provide evidence that the increased use of certain Vbeta families in circulating, CLA+, skin homing lymphocytes is of functional significance.     

MDR1 P-glycoprotein is expressed by endothelial cells of newly formed capillaries in human gliomas but is not expressed in the neovasculature of other primary tumors.

The expression of human MDR1 P-glycoprotein (Pgp) in the capillary endothelial cells of the central nervous system has been demonstrated. The brain capillary endothelial cells maintain the structure and function of the blood-brain barrier. Recently, the human MDR1 Pgp (and its mouse homologue MDR1a Pgp) has been shown to function as an important part of this barrier, pumping out xenobiotics from endothelial cells into the lumen of capillaries resulting in the protection of the brain parenchyma. To examine whether the endothelial cells of the newly formed capillaries during neoangiogenesis within malignant human brain tumors express MDR1 Pgp, 35 adult surgical brain tumor specimens (29 gliomas and 6 tumors metastatic to the brain) were obtained from previously untreated patients and studied by a new immunohistochemical sandwich method developed in our laboratory using the JSB-1 monoclonal antibody. JSB-1 is specific for the Pgp product of the human MDR1 (and not MDR3) gene. This sensitive method allows the detection of Pgp in capillary endothelial cells of normal brain in conventional paraffin sections after formalin fixation. The endothelial cells of the newly formed capillaries in 25 of 29 gliomas (86%) and 3 of 6 metastatic tumors, immunostained positive for MDR1 Pgp. The tumor cells in 7 of 35 cases were also positive for Pgp. In the 35 brain tumor cases investigated, the endothelial cells were Pgp positive in the tumor-brain border and in the brain further from the tumor. Capillary endothelial cells of neovasculature in 137 malignant tumors (non-brain) obtained from previously untreated patients showed no MDR1 Pgp expression. These results demonstrated that MDR1 Pgp is expressed not only in the capillaries of normal brain but also in the majority of the newly formed capillaries of brain tumors. Multidrug resistance of brain tumors may result not only from the expression of resistance markers in neoplastic cells but also from the MDR1 Pgp expression in endothelial cells of tumor capillaries. Pgp in this special localization can exclude chemotherapeutic agents from tumor cells that are located around the capillaries. The therapeutic benefit and selectivity of chemotherapeutic agents in combination with a Pgp-reversing agent should be evaluated.     

The development of respiratory syncytial virus-specific IgE and the release of histamine in nasopharyngeal secretions after infection

We studied the development of respiratory syncytial virus (RSV)-specific IgE and the release of histamine in nasopharyngeal secretions from 79 infants with various forms of respiratory illness due to RSV. RSV-IgE was measured by an enzyme-linked immunosorbent assay; specificity was confirmed by appropriate blocking experiments. Histamine content in the secretions was determined by fluorimetric methods. RSV-IgE was detectable in only one of 19 patients with RSV infection without wheezing, but was detectable in the majority of 60 patients with wheezing (P less than 0.01). Titers of RSV-IgE were significantly higher in patients with wheezing (P less than 0.05). Histamine was detectable in secretions of some patients with all forms of illness but was detected significantly more often (P = 0.05) and in higher concentrations in patients with wheezing. Peak titers of RSV-IgE and concentrations of histamine correlated significantly with the degree of hypoxia (P less than 0.001). Formation of RSV-specific IgE and release of histamine may adversely affect the outcome of RSV infection.     

A reappraisal of frog muscle chloride conductance-voltage relations at pH 9

Voltage clamp experiments in muscle show that the steady state chloride conductance at pH 9 remains rather independent of [Cl^–]_o, for [Cl^–]_o in the range 165–265 mM. The steady state conductance-voltage relation has a maximum near V _rest — 20 mV. The initial-conductance — voltage relation obtained when the voltage is stepped away from a constant conditioning value (e.g. the resting potential) approaches an asymptotic maximum for hyperpolarizing steps and a minimum for depolarizing steps. The conductance declines with time if the test voltage is more negative than the resting potential, but remains constant if it is more positive. When the conditioning voltage is varied and the test kept constant the initial conductance at the step is also seen to be sigmoidal, if the test step is hyperpolarizing: for large negative conditioning steps the conductance at the test potential approaches the same asymptotic value as does the steady state relation, independent of the test voltage. At positive conditioning voltages it approaches a maximum asymptote which is dependent on the test voltage. When the test step is positive-going the initial conductance at the step is weakly dependent on the conditioning voltage and for large negative conditioning potentials is larger than predicted from the steady state relation. In summary, during hyperpolarizing voltage steps the chloride conductance seems to decline due to a gating phenomenon, but openstate conductance seems to be voltage-dependent, and at membrane potentials more positive than the resting it rapidly (within the settling time of the clamp) assumes a value almost independent of any preceding (conditioning) voltage.     

Effect of therapeutic dosages of prednisone on human immunoglobulin G metabolism

In order to evaluate factors possibly responsible for the increased incidence of infections in patients treated with corticosteroids, immunoglobulin G (IgG) metabolism was studied in 8 patients before and during the administration of prednisone in a therapeutic range (30 mg. per day). The survival of intravenously administered IgG was determined, and total circulating IgG, fractional catabolic rate, and synthetic rate were calculated. There was no significant decrease in the measured levels of circulating IgG, but all patients had a shortened half-time (5 to 45 percent) and an increased fractional catabolic rate (p < 0.01). Since corticosteroid therapy was the only variable in these paired studies, it appears that corticosteroids can cause a hypercatabolism of IgG in some patients. Therefore, the maintenance of normal levels of IgG must be the result of an increased synthetic rate.     

The role of the autonomic innervation in the control of glucagon release during hypoglycaemia in the calf

1. The extent to which the autonomic innervation to the pancreas is implicated in the control of glucagon release during hypoglycaemia has been investigated in calves 3-6 weeks after birth.2. A pronounced rise in plasma glucagon concentration occurred in normal conscious calves in response to hypoglycaemia following administration of insulin (0.1 u./kg). Prior treatment with atropine caused no significant change in the hypoglycaemic response to insulin in these animals but the rise in plasma glucagon concentration was delayed.3. Section of both splanchnic nerves produced no significant change in the tolerance of conscious calves to this small dose of insulin and the changes in plasma glucagon concentration in these animals were within the normal range.4. In contrast, the same dose of insulin produced severe hypoglycaemia, accompanied by convulsions, in atropinized calves with cut splanchnic nerves. In spite of the intensity of the hypoglycaemic stimulus the rise in plasma glucagon concentration was both delayed and diminished in these animals.5. Administration of atropine alone (0.2 mg/kg) to normal fasting calves produced a significant fall in the mean plasma concentrations of both glucose and glucagon (P < 0.01) within 30 min, without affecting that of insulin.6. A significant increase in plasma glucagon concentration also occurred in response to stimulation of the peripheral ends of the thoracic vagi in adrenalectomized calves with cut splanchnic nerves under barbiturate anaesthesia. A rise in mean plasma glucose concentration was also observed in these experiments and found to be significantly correlated with the glucagon response.7. It is concluded that changes in either sympathetic or parasympathetic efferent activity may modify plasma glucagon concentration in the conscious calf, but that only the latter mechanism is likely to be implicated in the response to changes in plasma glucose concentration within the physiological range.     

The actions of anandamide on rat superficial medullary dorsal horn neurons in vitro

Whole-cell patch-clamp recordings were made from neurons in the trigeminal nucleus caudalis and trigeminal ganglion, in vitro , to investigate the cellular actions of the endogenous cannabinoid, anandamide. Anandamide has been shown to act through both the cannabinoid receptor 1 (CB1) and the vanilloid receptor 1 (VR1). Anandamide (30 μ m ) caused a 54 % increase in the rate of miniature excitatory post-synaptic currents (mEPSCs), without affecting their amplitude. The effect of anandamide was blocked by the VR1 antagonist capsazepine (20 μ m ), but not by the CB1-specific antagonist AM251 (3 μ m ). Application of the VR1 receptor agonist capsaicin (300 n m ) caused a 4200 % increase in the mEPSC rate. In dissociated trigeminal ganglion neurons, both anandamide and capsaicin caused an outward current in neurons that were voltage clamped at +40 mV. The maximal outward current produced by anandamide (EC₅₀, 10 μ m ) was 45 % of that produced by capsaicin (10 μ m ). Co-application of the VR1 antagonist capsazepine (30 μ m ) completely reversed the effects of both capsaicin and anandamide. The anandamide transport inhibitor, AM404 (30 μ m ) caused a 40 % increase in mEPSC rate in the slice preparation and an outward current in dissociated neurons. The latter current was reversed by the VR1 antagonist iodoresiniferatoxin (1 μ m ). The fatty acid amide hydrolase (FAAH) inhibitors phenylmethylsulfonyl fluoride (PMSF) (20 μ m ) and OL53 (1 μ m ) did not enhance the effect of anandamide in either the slice or dissociated neuron preparations. These results suggest that within the superficial medullary dorsal horn, anandamide (30 μ m ) acts presynaptically to enhance the release of glutamate via activation of the VR1 receptor.     

Promoting dietary change: demonstrating reduction of dietary fat

This short demonstration accomplishes several goals. People see where fat comes from and learn amounts needed to be removed. They practice substituting complex carbohydrate foods for fat. Most important, they learn that changing is not an "all-or-nothing" proposition. They can reduce dietary fat and still eat many of their favorite foods. This demonstration is a useful tool for nutrition educators who want to promote dietary change.     

Return to alertness after brain-stem hemorrhage. A case with evoked potential and roentgenographic evidence of bilateral tegmental damage

After a 65-year-old man had received anticoagulation therapy for brain-stem ischemia, a large, bilateral pontomesencephalic hemorrhage developed in the ischemic region. He survived a period of being "locked in" to attain a limited functional recovery. When he first became alert, brain-stem auditory evoked potentials and short-latency somatosensory evoked potentials (SEPs) demonstrated bilateral brain-stem damage; computed tomography revealed a bilateral tegmental hematoma. Results of repeated studies changed little as clinical improvement occurred. Recovery from brain-stem hemorrhage is rare, and return of consciousness with bilateral tegmental involvement is even more rare. The short-latency SEPs are useful in defining the extent of brain-stem damage, but they evaluate structures distinct from those regulating consciousness and cannot predict a return to alertness.     

Rapid accurate absolute granulocyte count determination during severe leukopenia using the flow cytometer

The absolute granulocyte count (AGC) in 125 blood samples from patients with total white blood cell counts of less than 1,000/microliter was estimated using three different methods, which were then compared for efficiency and accuracy. The three methods were 25 cell differential counts using Wright's-stained blood smears, granulocyte percentage estimates from WBC counting chambers, and combined narrow- and wide-angle light-scatter characteristics determined on a flow cytometer. A survey of clinical laboratories at University Hospital Cancer Centers revealed that the smear differential was the most-often-used method in those laboratories even when less than 25 cells could be counted. Consequently the data obtained from the counting chamber and flow cytometer methods were compared to the smear differential "standard" using linear regression, and outliers were identified. There was good correlation between AGC determined by smear differential and WBC counting chamber (correlation coefficient .911) and excellent correlation between the AGC determined by smear differential and the flow cytometer method (correlation coefficient .970). The flow cytometer method used in this investigation required minimal specimen preparation, and test results were available at a rate of 60 seconds/sample. The ease of sample preparation, speed, and statistical reliability of test results makes the flow cytometer an attractive alternate method of determining granulocyte counts on leukopenic patients as compared to the stained blood smear differential.