Glucose appearance rate following protein ingestion in normal subjects. The fate of amino acids deaminated following protein ingestion is uncertain. Presumably, the majority of the carbon skeletons of the amino acids are converted into glucose in the liver. In the present study, tritiated glucose dilution tracer studies have been used to determine the effect of a protein meal on the glucose appearance rate in plasma. Five normal male subjects ingested 50 g of protein in the form of cottage cheese. The glucose appearance rate was determined using a constant infusion of 3H-glucose, and compared to the glucose appearance rate following the ingestion of just water in the same subjects over an 8-hour period. The total amount of protein deaminated and converted to urea also was quantitated. Urea production could account for the metabolism of 29.3 g of protein ingested, or 58.5%. Glucose appearing in the circulation as a result of amino acid metabolism determined by tracer methodology was 9.68 +/- 5.7 g. Based on the gluconeogenic potential of cottage cheese (42.3 g of glucose from 50 g of cottage cheese protein), this could only account for at most 43% of protein metabolized, or 23% of the total amount of protein ingested. The fate of the remaining amino acids metabolized remains to be determined.


Weight loss, body composition and risk factors for cardiovascular disease in obese children: long-term effects of two treatment strategies. Two treatment strategies were compared to determine their effects on weight loss, body composition and risk factors for cardiovascular disease (CVD) in 32 obese children (relative weight > 120%) aged 6-15 years. Modes of therapy included individual (Group I) or group treatment (Group II). The children were treated for 1 year and observed during the second year. Relative body weight decreased by 16.6% in Group I (p < 0.001) and by 15.8% (p < 0.01) in Group II during the first year; in both groups the lower relative body weight was maintained during the year of observation. No changes were observed in linear growth or lean body mass. Weight reduction was accompanied by increased high-density lipoprotein cholesterol (HDL-C) levels, increased ratio of HDL-C to total cholesterol, and reduced triglyceride concentrations in both groups. Fasting plasma insulin concentrations decreased significantly in Group I. In conclusion, intensive treatment produced significant weight loss, improved serum lipid profile and reduced hyperinsulinemia. These changes, if sustained, may reduce the risk of CVD later in life.


The relationship of weight-height indices of obesity to body fat content. The measurement called desirable body weight (DBW) was derived by actuaries to indicate that weight which is associated with the lowest mortality. Percent deviation from DBW has become a standard measure of fatness. A different obesity index, body mass index (BMI), is weight in kilograms divided by the square of height in meters. Many workers consider both measures inferior to the measurement of body fat content (BFC). We compared the three measures of fatness in 40 men aged 18-50 and 48 women aged 21-47, ranging from nonobese to extremely obese. Total BFC was determined by isotope dilution of 3H-labeled water. DBWs used were those listed in the US Air Force Examination Manual of 1971; these approximate the midpoint of the range of medium-frame values in the 1959 Metropolitan Life Insurance Tables, but have the advantage of providing a single value for each height. We found nearly perfect correlation (r = 0.99, p < 0.001) between BMI and percent deviation from DBW in both men and women ranging from 14% below to 305% above DBW. Correlations between percent deviation from DBW and total BFC were extremely high: 0.95 (p < 0.001) for the men and 0.94 (p < 0.001) for the women, essentially the same as correlations between BMI and BFC, which were 0.96 (p < 0.001) for the men and 0.95 (p < 0.001) for the women. It appears that the two technically simple weight-height indices, BMI and percent deviation from DBW, give just as accurate a measurement of fatness as the technically complex measurement of total BFC.(ABSTRACT TRUNCATED AT 250 WORDS)


Sucrose ingestion following exercise: selected cardiovascular, hormonal, renal, and metabolic effects. Carbohydrates, frequently consumed following exercise for glycogen resynthesis, have been shown to have other systemic effects in resting men. We examined the effects of postexercise sucrose (a disaccharide carbohydrate) ingestion on the renal, cardiovascular, and sympathetic nervous systems. Eight men consumed 1 l of water (W) or 1 l of a 200 g sucrose solution (S) following 1 hour of bicycle exercise at 70% heart rate reserve. Measurements were made during 2 hours of recovery. Heart rate and systolic blood pressure were elevated following S as compared to W (p < 0.009, p < 0.04, respectively). Diastolic blood pressure was lower after S (p < 0.04) and mean blood pressure did not differ between beverages. Plasma and urinary catecholamines decreased similarly after exercise regardless of treatment. After S insulin (p = 0.0019) and glucose (p = 0.0036) were increased but serum aldosterone (p = 0.0083) and potassium (p = 0.0285) responses were lower. No differences were observed for plasma renin activity. Urine volume and kaliuresis were less after S (p = 0.03, p = 0.03). A 24% increase in metabolic rate (p = 0.002) and increased respiratory exchange ratio (p = 0.02) after S were observed. Systemic effects of sucrose ingestion following exercise include cardiovascular, renal, endocrine, and metabolic changes.


Use of a modified food frequency questionnaire during pregnancy. The purpose of this study was to examine the usefulness of the self-administered modified Willett food frequency questionnaire (mWFFQ) relative to duplicate 24-hour recall interviews as a dietary assessment tool during pregnancy. Fifty healthy pregnant women living in the upper Midwest were recruited for the study. At gestational weeks 16 and 21 (second trimester) and 30 and 35 (third trimester) the women were interviewed at home; they provided oral responses concerning their food and beverage consumption during the previous 24 hours. At weeks 21 and 35, immediately following the interviews, the women were asked to complete an optically scannable food frequency questionnaire, modified to evaluate daily food consumption during a 2-month time period (mWFFQ). Kilocalories and seven nutrients were computed from all dietary data, based on USDA Handbooks 8 and 456. Regression analyses were performed between nutrient intake from the 24-hour recall averages and the nutrient data generated by the mWFFQ, with the timesters serving as independent time periods. According to our preset criterion, correlation coefficients indicated that the assessment tools were comparable during the third but not the second trimester. During both trials, the mWFFQ provided consistently higher kilocalorie and nutrient values than did the 24-hour recall with the exception of fat intake. Based on our findings we conclude that the mWFFQ is a useful tool for assessing nutrient intake of groups of pregnant women, but that familiarity with the characteristics of the participants and the instrument may be important to data interpretation.


Intolerance to piroxicam in patients with adverse reactions to nonsteroidal antiinflammatory drugs. To evaluate the tolerance to piroxicam in patients with urticaria induced by analgesic and/or nonsteroidal antiinflammatory drugs (NSAIDs), we carried out a 2-year study in an outpatient clinic. All the patients referred to the clinic for study entered a protocol for evaluation of intolerance to one or more drugs. If patients were allergic to at least two different NSAIDs they were allocated to group A, but if patients were allergic to only one they were considered as having selective intolerance (group B). Either piroxicam or placebo was administered under controlled conditions to both groups. In group A, five out of 18 patients had a positive response to piroxicam. In group B, in all the 25 cases studied a good tolerance to piroxicam was shown. These results indicate that in the group with intolerance to NSAIDs piroxicam induced a positive reaction in 27% of the cases, and that this drug should be administered with caution and with a previous controlled challenge in this type of patient. Piroxicam was well tolerated in the group with selective intolerance, indicating that mechanisms other than interference with the prostaglandin synthesis and release of inflammatory mediators participate in allergic reactions to NSAIDs.


Allergenicity of peanut and soybean extracts altered by chemical or thermal denaturation in patients with atopic dermatitis and positive food challenges. Peanuts and soybeans are two of the six most common foods to cause food hypersensitivity reactions in children. We used the serum of 10 patients with atopic dermatitis and positive double-blind, placebo-controlled, food challenges to peanut and two patients with atopic dermatitis and positive double-blind, placebo-controlled, food challenges to soybean to investigate the change in IgE-specific and IgG-specific binding to these proteins altered by either chemical or thermal denaturation. We used IgE- and IgG-specific ELISA-inhibition analyses to compare these effects on the crude peanut and crude soy extracts, as well as on the major allergenic fractions of both proteins. Heating the soy proteins at various temperatures and time intervals did not significantly change the IgE- or IgG-specific binding of the soy positive pooled serum. When the peanut proteins were subjected to similar heating experiments, the IgE- and IgG-specific binding did not change. When these same proteins were treated with enzymes in the immobilized digestive enzyme assay system used to mimic human digestion, the binding of IgE to the crude peanut and crude soy extracts was reduced; 100-fold for peanut and 10-fold for soybean. Therefore it appears that thermal denaturation of peanut and soybean protein extracts does not enhance or reduce IgE- and IgG-specific binding activity. Chemical denaturation appears to minimally reduce the binding of these proteins.


Complementary DNA cloning and expression in Escherichia coli of Aln g I, the major allergen in pollen of alder (Alnus glutinosa). Previous data showed that the major pollen allergens from trees of the order Fagales, in particular alder, birch, hazel, and hornbeam, are highly interrelated. As only the complete amino acid sequence of Bet v I, the major allergen from birch, has been known, it was of interest to obtain the primary structure of other major allergens of this group, to attribute IgE-binding properties to certain features of the amino acid sequences of those allergens. cDNA was synthesized from alder pollen mRNA, sequence-specifically amplified by polymerase chain reaction and cloned into plasmid bluescript. Comparison of the deduced amino acid sequences of Aln g I and Bet v I revealed a 86.8% homology. The Aln g I encoding cDNA was subcloned into pKK223-3 and expressed in Escherichia coli as a full-length nonfusion protein. The recombinant Aln g I bound IgE from tree pollen-allergic patients and was shown to share IgE-epitopes with Bet v I by inhibition studies with recombinant Bet v I. Computer-aided calculations predicted epitopes in both Aln g I and Bet v I at the same position; the Bet v I molecule was predicted to possess two additional epitopes near the N-terminus of the molecule.


Increased contraction and inositol phosphate formation of tracheal smooth muscle from hyperresponsive guinea pigs. Tracheal smooth muscle from guinea pigs with documented airway hyperresponsiveness in vivo after multiple antigen challenges produced 30% to 50% greater force than tracheas from control guinea pigs, when stimulated with carbachol, histamine, or leukotriene D4. When cultured smooth muscle cells were incubated with myo[2-3H]inositol, basal uptake of [3H]inositol was similar in cells from normal and hyperresponsive guinea pigs, but when these cells were stimulated with contractile agonists, there was increased uptake of inositol in hyperresponsive cells. Analysis of inositol phosphates by column chromatography and high-performance liquid chromatography revealed the presence of inositol-1,4,5-triphosphate, inositol-1,3,4-trisphosphate, inositol-1,4-bisphosphate, and inositol-1-monophosphate. The release of inositol-1,4,5-triphosphate, inositol-1,4-biphosphate, and inositol-1-monophosphate by smooth muscle cells stimulated with carbachol, leukotriene D4, or histamine was 20% to 40% greater in cells derived from hyperresponsive animals than cells from normal animals. These data demonstrate that the increased muscle contraction of hyperresponsive guinea pig tracheas is associated with increased inositol phosphate metabolism in these cells. Delineating the mechanisms of airway smooth muscle contraction should provide new pharmacologic targets for the inhibition of bronchoconstriction in asthma.


Natural history of occupational asthma: relevance of type of agent and other factors in the rate of development of symptoms in affected subjects. It is unknown whether factors such as the nature of the agent, gender, age, atopy, smoking habits, continuous or noncontinuous exposure, and pattern of asthmatic reaction can influence the rate of development of symptoms in subjects with occupational asthma. We compared several clinical and functional parameters among three groups of subjects with occupational asthma caused by Western red cedar (group 1, n = 433), isocyanates (group 2, n = 107), and high molecular weight agents acting through an IgE-mediated mechanism (group 3, n = 121). Survival analysis showed that the three curves relating years of exposure before onset of symptoms to the proportion of subjects without symptoms were significantly different in two respects: (1) almost 40% of subjects in groups 1 and 2 as compared with 20% of subjects in group 3 became symptomatic within 1 year of exposure; (2) after 5 years of exposure, the rate of sensitization was slower for subjects in groups 2 and 3 as compared with those in group 1. Having a nonimmediate reaction at the time of specific inhalation challenges, being continuously exposed and being younger slightly increased the risk at each time point on the curve of developing symptoms in subjects with occupational asthma. These data suggest that the natural history for onset of occupational asthma is different depending on the sensitizing agent. Factors such as age, type of exposure, and pattern of reaction on exposure to the agent also modulate the rate of development of this condition.


Identification and characterization of a second major peanut allergen, Ara h II, with use of the sera of patients with atopic dermatitis and positive peanut challenge. Peanuts are frequently a cause of food hypersensitivity reactions in children. Serum from nine patients with atopic dermatitis and a positive double-blind, placebo-controlled, food challenge to peanut were used in the process of identification and purification of the peanut allergens. Identification of a second major peanut allergen was accomplished with use of various biochemical and molecular techniques. Anion exchange chromatography of the crude peanut extract produced several fractions that bound IgE from the serum of the patient pool with positive challenges. By measuring antipeanut specific IgE and by IgE-specific immunoblotting we have identified an allergic component that has two closely migrating bands with a mean molecular weight of 17 kd. Two-dimensional gel electrophoresis of this fraction revealed it to have a mean isoelectric point of 5.2. According to allergen nomenclature of the IUIS Subcommittee for Allergen Nomenclature this allergen is designated, Ara h II (Arachis hypogaea).


Evaluation of the Multi-Test device for immediate hypersensitivity skin testing. Test-to-test reproducibility, user variability, and the effect of positive reactions on adjacent negative sites were evaluated for the Multi-Test skin testing device. Twenty-five subjects had skin tests with 24 histamine (1 mg/ml), four glycerosaline controls, and four "dry" controls on two testings 7 days apart. To assess reproducibility from large and smaller histamine reactions and/or their effect on adjacent negative controls, 10 of the 25 subjects were retested once with the same testing format, but histamine at 10 mg/ml was substituted. To determine whether large allergen reactions affect adjacent negative controls differently than histamine reactions, 24 additional patients were retested on arms and back with negative controls adjacent to allergens to which they had prior 3+ to 4+ skin test reactions. Conclusions from 2688 skin tests on 49 patients are as follows: large (mean > 10 mm) histamine reactions reproduced better than smaller (mean < 7 mm) responses--coefficients of variation were 12.3 and 21.4 respectively. A 14% user variability occurred when comparing mean wheal sizes from histamine produced by each nurse and 1.2% when comparing their coefficients of variation. Neither small histamine reactions nor large reactions from histamine and allergens affected adjacent negative controls. We conclude that Multi-Test is a highly reliable skin testing technique that provides good reproducibility of results and low user variability.


Comparison of the sensitivity and precision of four skin test devices. Twenty volunteers were skin tested with seven concentrations of histamine phosphate and a glycerosaline control to determine the relative sensitivity and precision of four skin test devices: Greer Pen (GP), Greer DermaPIK (DP), Center Multi-Test (MT), and Morrow Brown needle (MB). The end points of the study were (1) wheal and flare response of each device, with a dose-response curve, (2) the time required to apply each set of eight tests, and (3) the volunteers' subjective assessment of each device. On a different day, 10 of the volunteers were tested to determine the precision of each device. Dose-response curves for half-log dilutions of histamine phosphate were produced with a glycerosaline control. The DP and GP induced wheal and flare responses discernible from that of the glycerosaline control at a lower concentration of histamine phosphate than the MB and MT. The DP took a shorter time to apply eight samples than any other device. The MB was preferred by the most volunteers, but any device tested on the upper half of the back was usually preferred over that tested on the lower half. When 5 mg/ml histamine phosphate was used, coefficients of variation for each device demonstrated that for wheals the precision of the DP, GP, and MT was similar (mean, 21.1%, 23.1%, and 24.5%, respectively). The MB was larger (mean, 59.9%). For flares, the precision of GP and DP was similar (mean, 22.0% and 23.5%, respectively), with the MT and MB larger (mean, 35.5% and 58.2%, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)


Sensory, parasympathetic, and sympathetic neural influences in the nasal mucosa. Neural mechanisms contribute to many nasal symptoms and syndromes. Sensory nerve stimulation by irritants, mast cell products, and inflammatory mediators leads to sneezing and other systemic reflexes. Parasympathetic reflexes and sensory axon responses combine to increase nasal blood flow, fill venous sinusoids (which thickens the mucosa and reduces nasal patency), induce plasma extravasation, and stimulate glandular secretion of mucous and serous cell products. These putative roles for nerves and neuropeptides in pathologic events open new therapeutic avenues. Anticholinergic agents, peptide neurotransmitter agonists and antagonists, drugs to reduce or modulate sensory or parasympathetic nerve function, potent topically applied glucocorticosteroids, and agents to inactivate inflammatory, secretory, or vascular cells may be of use. Ablation of sensory nerves by topical application of the chili pepper neurotoxin capsaicin has been successful in reducing the symptoms of refractory vasomotor rhinitis.


Response of nasal mucosa to histamine or methacholine challenge: use of a quantitative method to examine the modulatory effects of atropine and ipratropium bromide. We have developed a new technique for the direct local administration of test solutions to the nasal mucosa and for quantification of nasal secretory responses. This technique, a variation on several published reports of filter paper use, allows simple and rapid determination of drug effects and facilitates the analysis of ipsilateral and contralateral responses to local challenge of the nasal mucosa. We have used this technique to investigate the secretory responses of the nasal mucosa to methacholine and histamine and to determine the effects of atropine and ipratropium bromide (Atrovent nasal spray) on these secretory responses.


Intranasal anticholinergic therapy of rhinorrhea. This article reviews the role of anticholinergic therapy for the rhinorrhea that occurs in various rhinopathies, including irritant reactions, perennial nonallergic rhinitis, viral infection rhinitis, allergic rhinitis, and temperature-induced rhinitis. The use of a topical anticholinergic medication, ipratropium bromide, and its ability to inhibit methacholine and rhinitis-induced hypersecretion is emphasized. Ipratropium bromide appears to be both safe and effective in reducing this troublesome symptom.


The use of intranasal anticholinergic agents in the treatment of nonallergic perennial rhinitis. Nonallergic perennial rhinitis is a heterogenous disorder. Although the underlying cause is unknown, there is evidence of an autonomic imbalance with parasympathetic hyperreactivity in affected persons. We review this evidence and the role of ipratropium bromide, an anticholinergic drug with topical activity, in the management of nonallergic perennial rhinitis. The pharmacologic features, safety, and efficacy of ipratropium bromide are reviewed.


The anticholinergic treatment of allergic perennial rhinitis. Anticholinergic agents have been used for nonallergic rhinitis expressly to control rhinorrhea. In allergic rhinitis, rhinorrhea can be extremely troublesome and unresponsive to traditional pharmacotherapeutic rhinitis treatments. Anticholinergic agents, through their specific ability to decrease nasal secretory response, should have beneficial effects for allergic rhinitis. In a recent trial ipratropium bromide at concentrations of 0.03% and 0.06% reduced rhinorrhea in allergic subjects without any demonstrable rebound effect. Therefore anticholinergic therapies may be a useful adjunct in controlling the rhinorrhea associated with allergic rhinitis.


A double-blind, placebo-controlled study of the safety and efficacy of ipratropium bromide nasal spray versus placebo in patients with the common cold. Ipratropium bromide (IB) has been found to reduce secretions in the upper respiratory tract; this is accomplished through competitive inhibition of acetylcholine at muscarinic receptors that control rhinorrhea production. This study compared the safety and efficacy of IB with placebo in the symptomatic relief of rhinorrhea in patients with the common cold. Human subjects with symptoms of a common cold, primarily rhinorrhea, were enrolled and treated with either IB (84 micrograms/nostril) or placebo; each was administered as two sprays per nostril, four times a day, for 4 days. Primary efficacy analyses were in-clinic measurements of nasal discharge weights over a 3-hour period after administration on days 1 and 2 and assessment of rhinorrhea symptoms by use of a subjective patient-completed visual analog rating scale. IB significantly reduced rhinorrhea an average of 18% over placebo for days 1 and 2 (p = 0.01). Visual analog scale scores showed an average improvement in rhinorrhea of 22% over placebo (p = 0.001). When patients with relatively minor rhinorrhea (baseline weight of nasal discharge < or = 1.0 gm) were excluded, IB produced an average reduction in nasal discharge of 23% over placebo for days 1 and 2 (p = 0.003).


Physical and mammographic diagnosis of breast cancer and initial work-up. This article briefly reviews breast cancer examination and discusses a variety of physical changes that may occur in the breast suffering from breast cancer. The selected topics include evaluation of breast masses, nipple discharge, nipple changes other than discharge, and a discussion of inflammatory breast cancer and its differentiation from mastitis. Breast cancer appearing as a solitary axillary mass is also discussed. A brief discussion is also undertaken of aspiration cytology, mammographically directed needle localized open biopsies, and mammographically directed or stereotactic needle aspirations for cytology.


Early detection of breast cancer.

Early detection of breast cancer. Breast cancer is the most common malignancy in North American women. The incidence rate is increasing by about 2% annually, while the mortality rate has remained stable for 50 years. Screening by physical examination and mammography can decrease the mortality rate in women over 50 by 30%. The screening process is rewarding, but meticulous attention to detail is required in both the physical and mammographic assessment in order to define significant abnormalities.


Mammography quality assurance.

Mammography quality assurance. A poor-quality mammogram is worse than no mammogram. This is particularly true in the screening situation. Quality assurance in mammography is defined as all of the practices that monitor and improve the delivery of mammographic services, including technical performance of the equipment, personnel qualifications, patient interactions, communication, and follow-up. Quality control is the component of quality assurance focusing on the technical performance of mammography equipment. The effectiveness and success of screening mammography depend on consistent production of high-resolution, low-dose mammographic images and their accurate interpretation. This article describes the American College of Radiology's Mammography Accreditation Program as it relates to mammography quality assurance.


Psychological impact of the diagnosis of breast cancer on the patient and her family. The diagnosis of breast cancer creates emotional distress for patients as well as family members. This article reviews studies on the psychological adjustment of women and their family members during the diagnosis, hospitalization, and early convalescence from breast surgery. Studies indicate that the diagnostic phase is an extremely stressful time for women, marked by high anxiety, uncertainty, and difficulty making decisions. The hospital phase is especially difficult for spouses, who must juggle work responsibilities with added home responsibilities and also spend time at the hospital supporting their wives. In the convalescent phase, patients and family members need to adjust to changes in family roles, cope with fears about recurrence, and learn to balance the needs of all family members. In order to provide high quality health care to breast cancer patients and their family members, physicians and nurses need to address the emotional as well as the physical aspects of recovery.


The in situ carcinomas of the breast.

The in situ carcinomas of the breast. In situ carcinoma of the breast is being diagnosed with greater frequency on breast biopsies. Of the two types of CIS, lobular carcinoma in situ is considered a marker for increased risk of developing breast cancer in either breast, and treatment options are based on different philosophies of careful follow-up vs preventive surgery. Ductal carcinoma in situ is a direct precursor to invasive carcinoma and the variety of treatments available reflect the need to completely excise this lesion. Several trials are now in progress to define the roles of surgery, radiation, and hormonal manipulation in the treatment of both types of CIS.


Reconstructive options following mastectomy. Reconstructive breast surgery is an option a woman may choose following breast loss due to disease, trauma, or congenital malformation. In choosing breast reconstruction a woman must decide on timing and type of procedure. Recent decisions by the Food and Drug Administration have severely limited the type of reconstructive procedure that can be performed. This article discusses all of the options recently available in order to give information to those treating patients who have had these procedures as well as to be encyclopedic in the event that these techniques become available again.


Post-stimulus depression of reflex changes in circular muscle activity in the guinea pig small intestine. The extent and time course of depression of successive reflex responses recorded with intracellular microelectrodes from the circular smooth muscle of the guinea pig small intestine were determined. Two stimuli were used, distension and distortion of the mucosa by compression; these were applied either at the same or at different sites. Excitatory responses oral and inhibitory responses anal to the stimuli were recorded. Post-stimulus depression of both ascending excitatory and descending inhibitory reflexes occurred, but the extent of depression was slightly less for the descending inhibition. A conditioning distension lasting 9 s depressed the excitatory response to a test distension applied 2 s later at the same site by 90%. After 30 s the depression was 50% and test responses were normal if inter-stimulus intervals were increased to 2 min. Increasing the duration of the conditioning stimulus increased the depression. Post-stimulus depression was less for compression stimuli than for distension stimuli and prior mucosal compression had almost no effect on responses to subsequent distension. The post-stimulus depression was greater if conditioning and test stimuli were at the same rather than different sites. For different sites, conditioning stimuli at 15 mm from the recording site (near) depressed responses to stimuli at 30 mm (far) to a greater extent than far stimuli depressed responses to near stimuli. If the conditioning stimulus at 15 mm was maintained until after the far test stimulus was applied, depression of the test response did not occur. It is concluded that the major sites of post-stimulus depression are at the synapses between primary sensory neurons and the first interneurons of reflex pathways, and that post-stimulus depression also occurs at other places in the pathway, presumably at synapses between interneurons or between interneurons and motor neurons.


The pathology of breast cancer: staging and prognostic indicators. The natural history of breast cancer is complex and the treatment modalities need to be adjusted to this heterogeneous disease. Several prognostic indicators have been described for breast cancer, including the extent of axillary nodal metastasis, the size of the primary tumor mass, various histopathologic characteristics, estrogen and progesterone receptor content, tumor proliferation index, detection of oncogenes, tumor suppressor genes, loss of heterozygosity, and growth factors. Although no single parameter or combination of parameters can definitively predict the outcome of the disease, combined criteria such as tumor estrogen receptor content, cell proliferative index, and lymph node status are relevant for identifying subsets of breast cancer patients that may require different therapeutic modalities. Detection of oncogenes, tumor suppressor genes, and growth factors need further evaluation to determine their usefulness as prognostic factors.


Actions of carotid chemoreceptors on subretrofacial bulbospinal neurons in the cat. Thirty-one barosensitive bulbospinal neurons were recorded from the subretrofacial (SRF) nucleus in eight chloralose-anaesthetised, paralysed cats. Close arterial injections of CO2-saturated saline were used to stimulate carotid body chemoreceptors. Seven neurons were abruptly excited and six neurons abruptly inhibited by chemoreceptor stimuli: these were primary responses, independent of changes in blood pressure or central respiratory drive (monitored from the phrenic nerve). A further eight neurons responded only indirectly to chemoreceptor stimuli, showing enhanced modulation of their activity coupled to the enhanced central respiratory drive. The distinction between primary and secondary responses was shown more clearly when central respiratory drive was inhibited by superior laryngeal nerve stimulation. The remaining ten neurons showed no clear response to chemoreceptor stimuli. SRF bulbospinal neurons thus show the same range of responses to chemoreceptor stimuli as the sympathetic neurons they are believed to drive.


Systemic therapy of breast cancer in early and advanced disease. Despite substantial advances in breast cancer treatment, mortality from this disease remains high. While adjuvant chemo-hormonal therapy has significantly improved survival, breast cancer still claims the lives of more than 45,000 American women each year. This article is a brief review of various modalities used in the management of breast cancer, including adjuvant and neoadjuvant therapy, treatment for advanced disease, dose intensification, and autologous bone marrow transplantation.


The amplitude and periodicity of synchronized renal sympathetic nerve discharges in anesthetized cats: differential effect of baroreceptor activity. We applied a computerized peak detection algorithm to recordings of synchronized sympathetic nerve discharges from anesthetized cats to retrieve information about the characteristics of renal nerve activity (RNA) during changes in baroreceptor activity. The algorithm scanned the series of RNA voltages for significant increases followed by significant decreases in a small cluster of voltage values. Once each synchronized RNA peak had been detected, its corresponding amplitude, width and peak-to-peak interval were calculated. The peak-to-peak interval periodicity showed two modes of synchronized discharge, one between 200-500 ms accounting for 47% of intervals, and a higher 20-180 ms frequency (49% of intervals). Baroreceptor stimulation decreased the occurrence of the high frequencies while increasing the probability of the lower frequency components. The overall occurrence of synchronized peaks per second fell linearly to zero with increases in blood pressure. The peak amplitude of RNA was unimodally distributed and was not affected by baroreceptor stimulation until an increase in mean arterial pressure reached a threshold (mean 142 +/- 5 mmHg) whereupon it fell quickly to zero. Sino-aortic vagal denervation did not affect the distribution of peak height. The width of synchronized discharges was also unimodal, mean 82 +/- 1 ms, and was almost unchanged during baroreflex stimulation acting in parallel with changes in the peak amplitude and decreasing at high blood pressures. Sino-aortic vagal denervation did not affect the synchronized width. There was no relationship between the periodicity and amplitude or width of synchronized discharges under all conditions. The results indicate that the periodicity and amplitude of renal synchronized discharges appear to be independent of each other and are differentially affected by baroreceptor input.


Patient and family caregiver reaction to new and recurrent breast cancer. Exploratory data on the mental health (depression), symptoms, and functional status of breast cancer patients and the mental health (depression) and reaction to care of their caregivers are examined in this paper. How both are influenced by new and recurrent disease is also reported. Patients and family caregivers were followed over a six-month period to determine whether new or recurrent disease status altered their reaction to the cancer experience. The data suggest that psychological distress may be more marked in the family member than in the patient. Differences in caregiver reaction were not due to new or recurrent disease status, health status of the patient, or the care process of the caregiver. Future research is needed to isolate the cause of variations in mental health of women and their family caregivers experiencing new or recurrent breast cancer.


Psychosocial adjustment of the family to breast cancer: a longitudinal analysis. The purpose of the current study was to assess the impact of the mother's breast cancer on her family during rehabilitation and to determine the processes families use to adjust to the illness over time. Data were obtained from 111 child-rearing mothers with breast cancer on three occasions at four-month intervals using standardized measures of psychosocial adjustment. Results revealed that over time the families experienced significantly lower levels of illness-related demands and the marriages became better adjusted. Levels of depressive mood in the women, however, remained stable. This negative mood negatively affected the quality of the marriage, which, in turn, caused the family to cope less frequently with its problems and to function less well. Results strongly suggest the importance of formally assessing the woman's level of depressed mood and channeling women with stable depressive mood and their families into more intensive support services than those typically provided. Evidence from the current study is that, when not attended to, this depressive mood has sustained, deleterious effects on how well the family does during rehabilitation.


Effect of asphyxia on the frequency and amplitude modulation of synchronized renal nerve activity in the cat. A computerized peak detection algorithm was used to retrieve new information about changes in the characteristics of renal nerve activity (RNA) with asphyxia in anesthetized cats. The algorithm scanned the series of RNA voltages for significant increases followed by significant decreases in a small cluster of voltage values. Once each synchronized RNA peak had been detected, its corresponding amplitude, width, and peak-to-peak interval were calculated. The peak-to-peak interval showed two rhythms of synchronized discharge: one between 200-500 ms accounting for 38 +/- 3% of intervals and a higher 20-180 ms frequency (52 +/- 5% of intervals). Asphyxia did not change the periodicity distribution despite increases in arterial pressure. The peak amplitude of RNA, reflecting the number of active fibers, was unimodally distributed and was increased 39 +/- 9% with asphyxia. The shape of the distribution was unchanged. The width of the synchronized activity was also unimodally distributed, mean 79 +/- 3 ms, and increased by asphyxia to 99 +/- 5 ms. The results indicate that the control of the periodicity and amplitude of synchronized discharge appear to be independent and are differentially affected by chemoreceptor input.


Sympathetic and sensory neurons projecting into the cervical sympathetic trunk in the chicken. The cell bodies of the sensory and sympathetic pre- and postganglionic neurons projecting into the cervical sympathetic trunk were retrogradely labeled with horseradish peroxidase in the chicken. Preganglionic neurons were located in the spinal segments T1-T6 (maximum T2), postganglionic neurons in the paravertebral ganglia T1-T3 (maximum T1) and sensory neurons in the dorsal root ganglia T1-T4 (maximum T1). Labeled preganglionic neurons were widely distributed across the intermediate gray matter and lateral funiculus, but the majority of them were located in the intermediomedial area dorsolateral to the central canal. The short and long axis diameters of labeled preganglionic neurons in this area decreased caudally. From the data of the present study, it is estimated that about 4190 preganglionic, about 450 postganglionic and about 390 sensory neurons project into the cervical sympathetic trunk cranial to the paravertebral ganglion T1 in the chicken.


Ultrastructural localization of angiotensin II-like immunoreactivity (A II-LI) in the vegetative networks of the spinal cord of the guinea pig. The localization of angiotensin II-like immunoreactivity (A II-LI) has been studied at an ultrastructural level in the thoracic and sacral spinal cord vegetative regions of male and female guinea pigs. Most of the AII-LI structures are axon terminals and fibers localized in both vegetative networks which connect bilaterally and cranio-caudally and/or caudo-cranially the preganglionic vegetative nuclei in the thoracic and sacral spinal cord intermediate zone. The A II-LI product is localized predominantly in large granular and small vesicles of axon terminals and varicosites which form axo-somatic, axo-dendritic and axo-axonal synapses. Occasionally A II-LI is observed over cysterns of the endoplasmatic reticulum and throughout the cytoplasm of astrocytes which are neighbouring on blood vessels and are in contact with neuronal processi. Immunoreactive terminals are observed in direct contacts with endothelial cells too. These observations confirm recently described light-microscopic localization of Ang II-LI in the studied spinal cord areas, as well as its suggested role as transmitter and/or modulator of the neuronal function and regulatory role over the cardiovascular function and blood circulation.


Seasonal variations in acetylcholine content and the levels of cholinergic enzymes in the alimentary tract and heart of Rana esculenta L. The acetylcholine (ACh) content and the relative levels of choline-acetyltransferase (ChAT) and acetylcholinesterase (AChE) were measured in the stomach and intestinal musculature and in the heart muscles of frogs (Rana esculenta L.) during hibernation (January-stage 1), low activity (April-stage 2) and high activity period (July-stage 3). The ACh content doubled in the stomach from stage 1 to 2, then little change occurred. In the intestine, the ACh content changed little during the stages, while slightly decreased levels were found in the heart from stage 1 to 3. A significant increase in the level of ACh was found in the brain during the entire period. The level of ChAT increased in all tissue samples during the time. The AChE level was not significantly different during the stages in the stomach and intestine. In the heart, a 2.5 fold increase was measured in April compared with January, followed by a sharp decrease to half of the April level by July. In the brain, the activity of this enzyme increased continuously from January to July. The results show that these important cholinergic parameters change differentially in different organs in this poikilotherm animal during the year, presumably resulting in a fine control of the viscera, probably in cooperation with other autonomic transmitter systems.


Responses of single units in the midline medulla to stimulation of the rostral ventrolateral medulla. Single units in the midline medulla were characterized as sympathoexcitatory (SE), sympathoinhibitory (SI) or serotonin (5-HT) neurons. Post-stimulatory changes in the firing patterns of sympathoexcitatory, sympathoinhibitory and 5-HT units were observed during single shock stimulation of the pressor area of the rostral ventrolateral medulla (RVLM). Excitation, inhibition, or no change in cell firing patterns were observed for each cell type, but each cell showed only one type of response to stimulation. No midline neurons were antidromically activated by stimulation of the rostral ventrolateral medulla. These results are discussed in relation to neuronal pathways between the RVLM and the midline medulla involved in the generation of sympathetic activity.


Diabetic photographic eye screening using a mobile unit in Tayside, Scotland. Early detection and hospital referral of patients with diabetic changes (retinopathy) is essential if laser photocoagulation is to be effective. The British Diabetic Association (BDA) have therefore funded several diabetic mobile eye screening units and donated them to areas throughout the country, the aim being to screen patients for diabetic retinopathy in those areas. This article describes the use and findings of such a unit in the Tayside area.


Use of unsharp masks with high-contrast retinal nerve fibre layer photographs. Retinal nerve fibre layer (RNFL) photography is an established and integral tool in the management of ocular hypertensives and glaucoma suspects, and in assessing the management of glaucoma in an ophthalmic practice. When RNFL negatives are viewed for analysis, high-contrast negatives yield the most information. When positive prints are subsequently requested, the RNFL negatives are difficult to print. The use of an unsharp mask facilitates the transfer of all the high-contrast negative density range to the print. The use of an unsharp mask has been simplified so that it is quick and efficient and can be adapted for high-contrast negatives other than RNFL images.


The use of video in assessing and illustrating abnormal eye movements in young children. Eye movement studies can be useful in neuro-ophthalmological investigations of infants and young children. In our laboratory we use a combination of an electro-oculogram and video to record eye movements. A composite video image is created consisting of an image of the electro-oculographic eye movement trace superimposed on an image of the patient's eyes and face. This permits the qualitative clinical appearance of the case to be illustrated simultaneously with the quantitative eye movement trace.


Some simplified methods for the photography of equipment and apparatus. A simplified approach to the photography of equipment and apparatus is described. A number of examples are shown of the use of portable flash equipment, fluorescent light boxes and 'north-light' (daylight from North-facing windows), as light sources.


Blast: injury patterns and their recording. Exposure to explosive blast results in a variety of injuries dependent on the degree of blast loading, the relative contribution of secondary fragments and the interaction of a casualty with the surroundings. The internal and more covert injuries peculiar to blast are easily overlooked in the haste to treat the obvious external wounds which are usually much more dramatic. This paper describes the pathogenesis of blast injury and suggests how best it may be recorded.


Translocation of aged cyclodiene insecticide residues from soil into forage crops and pastures at various growth stages under field conditions. Field trials were conducted to measure translocation of pesticides by summer and winter forage/pasture species from soil containing aged residues of heptachlor and, to a lesser extent, dieldrin. Substantial amounts of heptachlor epoxide, and lesser amounts of gamma-chlordane were translocated to plants from contaminated soil. Residue levels varied with crop species and stage of plant development. In summer crops residues were higher in soybean > cowpeas > lab-lab > Sorghum > millet > sweet saccaline at the grazing and mature stages. Compared to glasshouse studies undertaken previously, residues in crops grown under field conditions were much lower. This apparently reflects the lower soil moisture levels and the reduced rates of translocation. Heptachlor residues in winter crops were highest in Saia oats > Berseem clover > Haifa clover > Cassia oats > Tetila ryegrass > Schooner barley > Shaftal clover > Hunter river lucerne at the grazing stage. There were no detectable levels in barley and oats at the mature stage. No dieldrin residues were translocated into the various crop species.


Comparative metabolism of phenanthrene in the rat and guinea pig. In the present study the biotransformation of phenanthrene in the rat and guinea pig was investigated. 14C-labelled phenanthrene was administered by gavage in corn oil to Sprague-Dawley rats (10 mg/kg b.w./day) and guinea pigs (10 mg/kg b.w./day). Urine and feces were separately collected for the determination of the radioactivity content, and pooled urine was used for the analysis of metabolites. Phenanthrene was metabolized by the rat and guinea pig to free hydroxylated phenanthrenes and their conjugates. The percentages of conjugates, expressed as the total urinary radioactivity, were 39% glucuronides, 24% sulfates and 18% cysteinylglycine for rats; and 39% glucuronides, 23% sulfates and 28% cysteinylglycine for guinea pigs. Enzymatic hydrolysis of glucuronides and sulfates resulted in the formation of free 1,2-, 3,4- and 9,10-dihydrodiols of phenanthrene and 1-, 2-, 3-, and 4-hydroxyphenanthrene in both species.


Hexachlorophene induced alterations in brain carbohydrate metabolism of Wistar rat. Effect of repeated oral administration of hexachlorophene (HCP) on glycolytic and oxidative pathways was studied in the rat brain. The rats were divided into three batches of six in each batch. The first batch was treated with paralytic dose (60 mg.kg-1.day-1) of HCP for 7 days. The second batch of animals was treated with sublethal dose (18 mg.kg-1.day-1) for 7 days. The third batch of animals was served as the age matched controls which received vehicle (corn oil) only. The glycolytic and oxidative metabolism of carbohydrates was significantly inhibited in the brain of rat during HCP treatment and the inhibition was more pronounced in paralytic dose treatment as compared to sublethal dose treatment. The inhibition of NADP-isocitrate dehydrogenase coupled with glucose 6-phosphate dehydrogenase indicates reduced generation of NADPH2 and pentoses for the synthesis of fatty acids and nucleotides.


Cholecystokinin, a satiety signal in newborn infants? This study was undertaken in order to describe circulating glucose and cholecystokinin (CCK) concentrations in relation to the spontaneous feeding behavior of the human newborn infant. Eighty-three, healthy, 3-days-old infants were studied in connection with breast feeding. Blood samples from the infants were cross-sectionally collected before feeding, 5 and 10 min after the start of sucking, and after the infants had sucked ad libitum. Before feeding, the infants presented a typical "hunger behavior", which changed in connection with breast feeding into a pattern associated with satiety. A significant increase in the plasma CCK concentration was found, 5 min (P = 0.004) and 10 min (P = 0.02) after the start of sucking, as well as after feeding (P = 0.04). Furthermore, a positive correlation between the CCK concentration and the volume of ingested milk was found 10 min after the start of sucking, when 91% of of the volume of milk had been ingested; Rs = 0.51, n = 19, P < 0.02. However, no change was found in the glucose concentration in connection with breast feeding. It is concluded that CCK may be important as a satiety factor in the regulation of food intake in the newborn infant.


The effect of 10% O2 on the continuous breathing induced by O2 or O2 plus cord occlusion in the fetal sheep. Although the administration of 100% O2 alone or combined with umbilical cord occlusion induces continuous breathing and arousal in the fetal sheep (Baier, Hasan, Cates, Hooper, Nowaczyk & Rigatto, 1990a), the individual contribution of O2 and cord occlusion to the response have not been determined. We hypothesized that if O2 is an important factor in the induction of continuous breathing, administration of O2 low enough (10%) to bring fetal arterial PO2 to about 20 torr while the fetus is breathing continuously should reverse these changes. Thus we subjected 12 chronically instrumented fetal sheep to 10% O2 for 10 minutes after the establishment of continuous breathing by O2 (4 fetuses; 137 +/- 1 days) or by O2 plus umbilical cord occlusion (8 fetuses; 134 +/- 1 days). Arterial PO2 decreased from about 250 torr to 20 torr during 10% O2. This induced a significant decrease in breathing output (EMGdi x f) related primarily to a decrease in frequency (f). In 3/5 experiments in 4 fetuses, with O2 alone, apnoea developed within 4 +/- 0.6 min; in 12/13 experiments in 8 fetuses, with added cord occlusion it developed at 5 +/- 0.6 min. With the decrease in PaO2, electrocortical activity (ECoG) switched from low to high-voltage within 6 minutes in 5/5 experiments (O2 alone) and in 11/13 (O2 plus cord occlusion). The findings suggest that umbilical cord occlusion alone is not sufficient to maintain breathing continuously and an increased PaO2 is needed. We speculate that in the fetus there is a vital link between PaO2, breathing and ECoG with low PaO2 inhibiting and high PaO2 favouring breathing and arousal.


Fetal breathing is not initiated after cord occlusion in the unanaesthetized fetal lamb in utero. We investigated the role of cord occlusion in the initiation of breathing at birth using an extracorporeal membrane oxygenator system to control fetal blood gases independently of the placenta in 12 chronically instrumented fetal lambs. In group IA (n = 9; exp = 12) PaCO2 was kept constant (5.62 +/- 0.21 to 5.70 +/- 0.23 kPa) during cord occlusion. Group IB (n = 7; exp = 8) were cord occlusion experiments from group IA in which no fetal breathing movements had occurred; CO2 flow to the membrane was increased and fetal PaCO2 rose significantly (5.45 +/- 0.24 to 8.27 +/- 0.56 kPa). In group II (n = 7; exp = 12) PaCO2 was allowed to increase from 5.98 +/- 0.24 kPa to 8.09 +/- 0.48 kPa after cord occlusion. Within 5 min of cord occlusion, FBM did not occur in 11 out of 12 experiments in group IA or in 11 out of 12 experiments in group II. In contrast in group IB breathing did occur in 5 out of 8 experiments. When they occurred, fetal breathing movements were always associated with low voltage electrocortical activity. Our results do not support the hypothesis that the initiation of breathing within 5 minutes of birth is dependent on an inhibitory factor of placental origin. Furthermore these data suggest an association between the presence of breathing and a substantial rise in PaCO2.


Effects of hyperoxia on the metabolic response to cold of the newborn rat. We asked what effects hyperoxia may have on the metabolic response to cold of the newborn rat. Whole body gaseous metabolism (VO2 and VCO2) was measured in 2-day old rats by open flow respirometry at ambient temperatures (Tamb) between 40 and 20 degrees C, changed at a rate of 0.5 degrees C/min during normoxia and hyperoxia (100% O2 breathing). In normoxia, the thermoneutral range was very narrow, at Tamb = 33-35 degrees C. A decrease in Tamb at first stimulated VO2; a further drop in Tamb below 28 degrees C reduced metabolic rate. The metabolic response to cold was not sufficient to maintain body temperature (Tb). In hyperoxia average values of VO2 were above the normoxic values at all Tamb, but the difference was mostly apparent at low Tamb; at 20 degrees C, hyperoxic VO2 averaged 73% more than in normoxia. This metabolic increase determined a significant but small rise of Tb. We conclude that in the 2-days-old rat hyperoxia has a stimulatory effect on metabolism which is Tamb-dependent, being much more apparent in the cold. This supports the concept that the normoxic VO2 of the newborn is limited by the supply of O2. However, the fact that in the cold, even in hyperoxia, VO2 did not reach very high values, and Tb was not maintained, suggests that not only O2 availability, but also the rate of O2 utilization limits the aerobic metabolic response of the newborn.


The vit gene maps to the mi (microphthalmia) locus of the laboratory mouse. The murine model for human vitiligo (the vit/vit mouse) develops progressive depigmentation of the pelage, skin, and eyes. The vit gene is inherited as an autosomal recessive. We have used classical breeding and isozyme marker analysis to map this vit gene that produces a vitiligo-like condition in the mouse. Crossbreeding the C57BL/6J-vit/vit mice with C57BL/6J mice carrying the Miwh and/or miws alleles at the microphthalmia locus resulted in mutant phenotypes, demonstrating absence of complementation. When vit is heterozygous with the Miwh allele, a "blotched" pigment pattern results. When it is heterozygous with the miws allele, a novel expression of the vitiliginous phenotype results. Further mating analysis of these crossbred populations demonstrates allelic inheritance between vit and the alleles at the microphthalmia locus. Other breeding studies using alleles at the agouti, belted, brown, dominant spotting, extension, mahogany, patch, and piebald loci did not demonstrate pigmentation explainable by allelic inheritance with the vit gene. Also, vit was tested for linkage with isozyme markers located on chromosomes 1, 4, 5, 7, 9, and 11, and results were negative. Therefore, the vit (vitiligo) gene of the laboratory mouse has been mapped to the mi (microphthalmia) locus on chromosome 6. The gene properly should be designated as mivit.


Selection for canalization at extra dorsocentral and scutellar bristles in Drosophila melanogaster. Direct artificial selection for a specific pattern, in the number and position of extra bristles, was carried out in a wild-type population of Drosophila melanogaster to canalize (estimated by probit width) the selected phenotypes. From the same population, independent lines were selected for extra dorsocentral bristles (lines D3 and D4) and for extra scutellar bristles (lines E2, E3, and E4). Differences at canalization between both dorsocentral and scutellar systems were detected. Results fit an independent control hypothesis for canalization, at two symmetrical extra bristles, in the main regions in which extra bristles appear.


Identification of an autosome to X chromosome translocation in the domestic cow. Chromosomes were harvested from two fibroblast lines derived from a phenotypically normal cow and her albino daughter, both known to be heterozygous for an X/autosome translocation. QFH-banding after early BrdU incorporation identified the translocated autosomal material as chromosome 23 bands 13-25, and revealed that the centromeric portion of the translocated chromosome 23 had been retained. Fluorescent in situ hybridization of a BoLA Class I cDNA probe to the normal chromosome 23 and to the translocated autosomal material confirmed the identity of the translocation, and allowed a more precise sublocalization of the MHC in cattle than that previously reported.


Blepharitis.

Blepharitis. Blepharitis is an acute or chronic inflammatory process involving the eyelids that is frequently associated with conjunctivitis. In its many clinical forms, blepharitis is one of the most common diseases seen by ophthalmologists; yet it remains a diagnostic and therapeutic challenge. This article reviews the clinical presentation, classification, diagnosis, etiology and pathogenesis, and treatment of blepharitis.


Infectious conjunctivitis.

Infectious conjunctivitis. This article is designed to aid the clinician in the diagnosis of bacterial and viral conjunctivitis by evaluating a wide variety of infectious causes of conjunctivitis. The work-up, including techniques of examination and pertinent laboratory studies, is discussed. Clinical features of each type are covered at length and specific therapy for each is included.


Conjunctivitis of the newborn.

Conjunctivitis of the newborn. Infectious conjunctivitis of the newborn is caused by a wide variety of microorganisms. The ocular findings may be part of a widespread systemic infection. Clinical presentations are not diagnostic of the cause, and a microbiologic work-up with cytology, cultures, and microbial sensitivities is mandatory. The selection of specific antimicrobial therapy is based on the findings of laboratory studies. Prophylaxis with silver nitrate solution, 1.0% tetracycline, or 0.05% erythromycin ointment is effective for the prevention of gonococcal and chlamydial conjunctivitis in the newborn.


Bacterial keratitis.

Bacterial keratitis. Bacterial keratitis is an urgent problem requiring recognition, laboratory evaluation, and administration of therapy. The recognized risk factors are outlined. The likely bacterial causes may vary among regions of the country over a period of time. Topical fortified antibiotic eyedrops are the mainstay of therapy; indications for other modalities of therapy are discussed. Specific antibiotic regimens are offered.


Fungal keratitis.

Fungal keratitis. As Jones has emphasized, the successful management of microbial keratitis, fungal or otherwise, necessitates five steps: (1) clinical suspicion and clinical diagnosis; (2) performing the proper laboratory procedures; (3) initiating antimicrobial therapy based on the results of laboratory studies; (4) modifying the initial therapy based on the clinical response; and (5) deciding correctly when and how to terminate therapy. Keratomycosis poses special diagnostic and therapeutic challenges for ophthalmologists because of its low incidence, and its resistance to treatment, because of the lack of antifungal agents with good penetration into the eye, and because of the difficulties in obtaining meaningful in vitro drug susceptibility results for fungal isolates. We believe, however, that ophthalmologists are now diagnosing keratomycosis earlier and treating it more effectively.


Infectious endophthalmitis.

Infectious endophthalmitis. The internist or family medicine consultant can have an important role in the management of endophthalmitis. Even though intraocular antimicrobial therapy is the most effective method of treatment, the consultant can assist in the selection of systemic antibiotic agents and in the monitoring of systemic side effects of the treatment. In addition, the consultant can be called on to evaluate patients with endogenous endophthalmitis for systemic involvement. The joint efforts of the ophthalmologist and consultant should allow optimal treatment in an attempt to improve the visual prognosis for these devastating ocular infections.


Viral infections of the choroid and retina. DNA (herpes viruses) and RNA (rubella, rubeola) group viruses are recognized causes of viral retinitis. Severe damage is often the result, not only because the viruses have a cytologic effect but also because the antigens may cause immune complex deposition that results in vasculitis. Most of the viral retinitides are associated with systemic disease; immune-compromised individuals are more susceptible than healthy individuals. A distinct clinical entity, acute retinal necrosis, affects the eye only in healthy individuals and is associated with the herpes family of viruses.


Other infections of the choroid and retina. Toxoplasmosis, histoplasmosis, Lyme disease, syphilis, tuberculosis, and ocular toxocariasis. The major clinical infectious syndromes involving the retina and choroid are discussed. Means for successful diagnosis and treatment are presented, as well as the major complications. This work emphasizes the need to identify the infectious nature of these syndromes in order to avoid the all-too-common pitfall of just treating all cases of uveitis with steroids alone.


Human immunodeficiency virus and opportunistic ocular infections. Opportunistic ocular infections are a common and important complication of the acquired immunodeficiency syndrome. Cytomegalovirus retinitis is by far the most frequent infection, but other viral, bacterial, and parasitic infections of the posterior segment of the eye may be seen. Infections of the anterior segment are less common but may cause visual loss if not treated. Prompt recognition of these diseases is essential because ocular involvement may be an early sign of systemic dissemination.


Infections of the lacrimal system.

Infections of the lacrimal system. Deficiency of the tears or obstruction to the free flow of tears predispose to infections of the lacrimal system. The relevant anatomy of the lacrimal system and the pathologic conditions leading to obstruction are outlined. The clinical manifestations of infections of the lacrimal gland, the canaliculus, and the lacrimal sac are described, and the basis for medical and surgical treatment is discussed.


Preseptal and orbital cellulitis.

Preseptal and orbital cellulitis. The patient with a tender, erythematous, swollen eyelid represents a complex clinical challenge to the physician, who must arrive at a correct diagnosis from numerous differential possibilities. Knowledge of the anatomy of the orbit and surrounding structures and proper clinical and radiologic examination are necessary to accurately diagnose these conditions. Proper selection of antibiotic therapy and timely surgical intervention, directed at both the orbital infection and the underlying condition, are essential to avoid serious morbidity from these infections. This article reviews in detail preseptal cellulitis, Hemophilus influenzae type B infection in children, orbital infection related to paranasal sinusitis, orbital infection from other causes, and mycotic infections of the orbit.


Ocular leprosy.

Ocular leprosy. Leprosy, also known as Hansen's disease, affects millions of people worldwide and is largely confined to the developing world. Although Hansen's disease is disfiguring, crippling, and even blinding in its later stages, it is not commonly lethal but gradually steals sensory input from the digits and the visual pathways. In this article, the epidemiology and pathogenesis of leprosy are reviewed, with an emphasis on ocular involvement, treatment, and prevention.


Onchocerciasis.

Onchocerciasis. Onchocerciasis is a parasitic disease with blinding consequences infecting approximately 18 million persons worldwide. In hyperendemic areas, nearly half the population are likely to become blind before they die. Blindness reduces the life expectancy of those affected and has an enormous socioeconomic impact on these communities. Ivermectin, given as a single oral dose of 150 micrograms/kg, can control the manifestations of the disease and can be safely distributed on a mass scale in endemic areas. This drug provides hope for the control of what has been until now an untreatable and devastating disease.


T cell recognition of QA-1b antigens on cells lacking a functional Tap-2 transporter. MHC class Ia H chains and beta 2-microglobulin assemble with appropriate peptides to form stable cell surface molecules that serve as targets for Ag-specific CTL. The structural similarities of class Ia and the less polymorphic Q/T/M (class Ib) molecules suggest that class Ib molecules also play a role in antigen presentation, although the origin of the peptides they present remains mostly unclear. The cell line RMA-S has a defect in class I Ag presentation, presumably due to a mutation in a peptide transporter gene. This defect can be overcome by transfection of RMA-S cells with the Tap-2 gene (formerly Ham-2) that encodes an ATP-binding transporter protein. We now show that a substantial portion of alloreactive CTL specific for Qa-1 class Ib molecules recognize Qa-1b on RMA-S cells and thus differ from most class Ia specific CTL. Those anti-Qa-1b CTL that do not recognize untransfected RMA-S do lyse RMA-S transfected with Tap-2. We also examine the effects of Qdm, a gene that maps to the D region and alters recognition of Qa-1. Qdm(k) strains lack an epitope(s) recognized by some (Qdm dependent) anti-Qa-1 CTL whereas Qdm+ strains express this epitope. Thus, Qdm-dependent CTL do not recognize Qa-1 on Qdm(k) targets whereas Qdm-independent CTL recognize Qa-1 epitopes in all strains. Although Qdm-independent CTL varied as to whether they recognized RMA-S vs RMA, all nine Qdm-dependent clones only recognized Qa-1b on RMA and not RMA-S. This result is consistent with Qdm encoding a peptide dependent upon the TAP transporter for cell membrane expression.


Direct binding of peptides to MHC class I molecules on living cells. Analysis at the single cell level. To directly assess the binding of exogenous peptides to cell surface-associated MHC class I molecules at the single cell level, we examined the possibility of combining the use of biotinylated peptide derivatives with an immunofluorescence detection system based on flow cytometry. Various biotinylated derivatives of the adenovirus 5 early region 1A peptide 234-243, an antigenic peptide recognized by CTL in the context of H-2Db, were first screened in functional assays for their ability to bind efficiently to Db molecules on living cells. Suitable peptide derivatives were then tested for their ability to generate positive fluorescence signals upon addition of phycoerythrin-labeled streptavidin to peptide derivative-bearing cells. Strong fluorescent staining of Db-expressing cells was achieved after incubation with a peptide derivative containing a biotin group at the C-terminus. Competition experiments using the unmodified parental peptide as well as unrelated peptides known to bind to Kd, Kb, or Db, respectively, established that binding of the biotinylated peptide to living cells was Db-specific. By using Con A blasts derived from different H-2 congenic mouse strains, it could be shown that the biotinylated peptide bound only to Db among > 20 class I alleles tested. Moreover, binding of the biotinylated peptide to cells expressing the Dbm13 and Dbm14 mutant molecules was drastically reduced compared to Db. Binding of the biotinylated peptide to freshly isolated Db+ cells was readily detectable, allowing direct assessment of the relative amount of peptide bound to distinct lymphocyte subpopulations by three-color flow cytometry. While minor differences between peripheral T and B cells could be documented, thymocytes were found to differ widely in their peptide binding activity. In all cases, these differences correlated positively with the differential expression of Db at the cell surface. Finally, kinetic studies at different temperatures strongly suggested that the biotinylated peptide first associated with Db molecules available constitutively at the cell surface and then with newly arrived Db molecules.


Role of neuroendocrine hormones in murine T cell development. Growth hormone exerts thymopoietic effects in vivo. Growth hormone (GH) and other neuroendocrine mediators have been implicated previously in T cell development. We therefore examined thymic development in DW/J dwarf mice. DW/J mice lack acidophilic anterior pituitary cells and consequently are totally deficient in the production of GH, as well as other neuroendocrine hormones. DW/J dwarf mice had greatly hypoplastic thymi that continued to decrease in size as the mice aged. Characterization of the different T cell subpopulations within the thymi of dwarf mice indicated a deficiency in CD4+/CD8+ double-positive thymocytes. This deficiency of progenitor cells became more complete as the mice aged culminating in the total disappearance of this subpopulation in some dwarf mice > 3 mo of age. Analysis of the lymph nodes indicated that a population of double-positive (CD4/CD8) T cells appeared in some mice concurrent with the disappearance of double-positive cells in the thymus suggesting that these thymocytes may have migrated to the periphery. However, peripheral T cells from dwarf mice did exhibit Ag-specific responses indicating that these mice have functional T cells. Treatment of the mice with recombinant human GH, which binds both murine growth hormone receptors and murine prolactin receptors, or ovine GH, which binds murine growth hormone receptors but not murine prolactin receptors, resulted in an increase in thymic size and the reappearance of the CD4+/CD8+ double-positive cells within the thymus. Additionally, after GH treatment, the double-positive cells disappeared from the lymph nodes. The thymi of mice treated with GH failed to attain normal size but did develop a normal distribution of T cell progenitors. Thus, GH exerts significant thymopoietic effects in vivo. Neuroendocrine hormones may be important for normal T cell differentiation to occur within the murine thymus.


Systemic suppression of delayed-type hypersensitivity by supernatants from UV-irradiated keratinocytes. An essential role for keratinocyte-derived IL-10. Exposing murine keratinocyte cultures to UV radiation causes the release of a suppressive cytokine that mimics the immunosuppressive effects of total-body UV exposure. Injecting supernatants from UV-irradiated keratinocyte cultures into mice inhibits their ability to generate a delayed-type hypersensitivity reaction against allogeneic histocompatibility Ag, and spleen cells from mice injected with supernatant do not respond to alloantigen in the in vitro MLR. A unique feature of the immunosuppression induced by either total-body UV-exposure or injecting the suppressive cytokine from UV-irradiated keratinocytes is the selectivity of suppression. Although cellular immune reactions such as delayed-type hypersensitivity are suppressed antibody production is unaffected. Because the selective nature to the UV-induced immunosuppression is similar to the biologic activity of IL-10, we examined the hypothesis that UV exposure of keratinocytes causes the release of IL-10. Keratinocyte monolayers were exposed to UV radiation and at specific times after exposure mRNA was isolated or the culture supernatant from the cells was collected. IL-10 mRNA expression was enhanced in UV-irradiated keratinocytes. The secretion of IL-10 by the irradiated keratinocytes was determined by Western blot analysis. A band reactive with anti-IL-10 mAb was found in supernatants from the UV-irradiated but not the mock-irradiated cells. IL-10 biologic activity was determined by the ability of the supernatants from the UV-irradiated keratinocytes to suppress IFN-gamma production by Ag-activated Th 1 cell clones. Anti-IL-10 mAb neutralized the ability of supernatants from UV-irradiated keratinocytes to suppress the induction of delayed-type hypersensitivity in vivo. Furthermore, injecting UV-irradiated mice with antibodies against IL-10 partially inhibited in vivo immunosuppression. These data indicate that activated keratinocytes are capable of secreting IL-10 and suggest that the release of IL-10 by UV-irradiated keratinocytes plays an essential role in the induction of systemic immunosuppression after total-body UV exposure.


Increased avidity of mutant IgM antibodies caused by the absence of COOH-terminal glycosylation of the mu H chain. We have previously described the isolation of two hybridoma variants secreting higher avidity IgM (D5 and 7F5), starting from the E11 hybridoma cell line, which produces an antibody specific for the A Ag of the ABO blood group system. In order to explain at the molecular level this increased reactivity, cDNA encoding the H and L chains of the E11, D5, and 7F5 mAb were cloned and sequenced. Comparison of the nucleotide sequences showed a single point mutation in each of the two mAb produced by the hybridoma variants. The mutations were both located in the H chain C region and caused a Ser to Phe substitution at position 565 in the D5 mAb and a Asn to Tyr substitution at position 563 in the 7F5 mAb. Both substitutions modified the consensus glycosylation sequence (Asn-X-Ser/Thr) located in the tail piece of the secretory mu-chain. The absence of glycosylation at this site was confirmed by CNBr cleavage of the [14C]mannose-labeled mAb. The two single point mutations were solely responsible for the increased avidity of the antibodies, as confirmed by site-directed mutagenesis of the E11 mu-chain and serologic analysis of the mutated E11 antibodies. We conclude that the absence of glycosylation at Asn 563 is responsible for the increased avidity of the mutant, possibly by altering the quaternary structure of the IgM polymer. To our knowledge, this is the first report that point mutations in the H chain C region can influence the reactivity of IgM mAb.


Role of protein kinase C activity in tumor necrosis factor-alpha gene expression. Involvement at the transcriptional level. Primary rat astrocytes express TNF-alpha protein in response to various stimuli including a combined treatment with IFN-gamma and LPS, or IFN-gamma and IL-1 beta. This study was undertaken to further elucidate the mechanisms underlying TNF-alpha gene expression in the astrocyte, and to determine the intracellular signaling pathways involved in IFN-gamma/LPS and/or IFN-gamma/IL-1 beta induction of the TNF-alpha gene. We demonstrate that TNF-alpha mRNA is rapidly induced, and mRNA levels peak after 2 h of stimulation. De novo protein synthesis is not required for TNF-alpha expression because the inclusion of cycloheximide does not prevent expression of the gene and acts to superinduce TNF-alpha mRNA levels. IFN-gamma/LPS induces transcriptional activation of the TNF-alpha gene as assessed by nuclear run-on experiments. Cycloheximide acts to increase both transcription of the TNF-alpha gene and stability of TNF-alpha mRNA thereby resulting in increased TNF-alpha steady state mRNA levels. Two protein kinase C (PKC) inhibitors, H7 and staurosporine, abrogate IFN-gamma/LPS- and IFN-gamma/IL-1 beta-induced TNF-alpha expression in a dose-dependent manner. PKC activity is required for transcription of the TNF-alpha gene, and does not appear to be involved in TNF-alpha mRNA stabilization. Taken together, these data demonstrate that TNF-alpha gene expression in primary rat astrocytes is induced in a PKC-dependent manner.


Antibody engineering by codon-based mutagenesis in a filamentous phage vector system. A novel codon-based mutagenesis procedure is described that allows rapid and efficient modification of antibody amino acid sequences expressed as F(ab) fragments in M13. The procedure succeeded in generating a library of mutations in the complementarity-determining regions of chimeric L6, an antibody against a tumor-associated Ag. A set of anti-Id antibodies (anti-Id 1, 3, and 7) that bind near the L6 Ag-binding site served as model Ag. The goal was to select mutant antibody sequences that altered the L6 reactivity with the anti-Id in subtle ways, i.e., to eliminate the binding to one anti-Id while preserving other reactivities or to identify mutants with increased binding. A high frequency of variant M13 phage clones exhibiting altered specificity for the anti-Id were identified. Codon-based mutagenesis in conjunction with the M13 antibody expression and screening system should provide an efficient and general approach for redirecting the specificity and potentially improving the affinity of antibodies in vitro.


Application of a filamentous phage pVIII fusion protein system suitable for efficient production, screening, and mutagenesis of F(ab) antibody fragments. We describe the application of a novel filamentous phage vector system suitable for efficient screening and production of F(ab) antibody fragments. The vector system can concurrently produce free F(ab) fragments and F(ab) displayed on the surface of M13 bacteriophage via a VHCH1-pVIII fusion protein. When expressed in a supO (nonsuppressor) strain of Escherichia coli free F(ab) can be produced. Antibody F(ab) fragments are secreted into culture medium at concentrations up to 0.3 mg/liter and conveniently subjected to detailed analysis with little or no purification. Higher concentrations of F(ab) (approximately 10 mg/liter) were found to accumulate in the periplasmic space. In this report the vector system is shown to produce correctly folded and assembled F(ab) fragments of chimeric L6, a mAb against a tumor-associated Ag expressed by many human carcinomas.


L chain isotype regulation in horse. I. Characterization of Ig lambda genes. Analysis of 10 cDNA encoding lambda L chains of horse Ig indicated that this species may employ a relatively small number of variable region (V lambda) genes in the splenic B cell population. The V lambda sequences of all of the cDNA analyzed were closely related (> 88% identity at the nucleotide level) and were characterized by a deletion of the amino acid residue at position 3 compared with V lambda sequences so far described in other species. The 10 V lambda sequences could be grouped into three groups, V lambda 1 to V lambda 3, on the basis of a number of linked substitutions. Sequences within the groups showed the greatest divergence in the third cdr regions and at the V-J junctions. The junctional variation included amino acid substitutions on both sides of the V-J junction as well as the insertion or deletion of two to four amino acid residues. Four C lambda genes were identified in genomic blots of horse DNA, and three of these were found expressed in splenic cDNA. The fourth C lambda gene may represent a pseudogene, inasmuch as the associated J region possessed a defective heptamer joining sequence. Six of the nine possible V lambda-C lambda combinations were found in the cDNA analyzed, suggesting that genes belonging to groups V lambda 1 through V lambda 3 may rearrange to any one of three J lambda-C lambda genes. One V lambda germline gene was characterized and found to represent a distinct V lambda group (V lambda 4), not represented in the cDNA sequences analyzed. The number of germline V lambda genes was estimated to be 20 to 30, based on analysis of restriction fragments hybridizing with V lambda probes. On the basis of these data, we propose that the V lambda repertoire in horse may consist of relatively limited number of genes, of which only a few may be used at high frequency in the splenic B cell population. The results indicate that predominance of lambda-chains in horse Ig may not simply be due to the presence of a large germline V lambda gene repertoire.


Nucleotide sequence of messenger RNA encoding VHDJH and VKJK of a highly conserved idiotype-defined primary response anti-hapten antibody. The primary humoral immune response of mice to the hapten phthalate (Xmp) is focused upon two adjacent immunodominant negatively charged carboxyl groups on a benzene ring that are in positions meta and para to the azolinkage (i.e., Xmp) to the protein carrier keyhole limpet hemocyanin. A significant fraction of the anti-Xmp antibodies raised in several different inbred mouse strains (BALB/c, DBA/2, A/HeHa; C3H, and SM/J), and many wild mouse populations express a cross-reactive Id, CRIXmp-1. This CRIXmp-1 is conspicuously absent in C57BL/6 mice. In order to obtain a better understanding of the events and parameters that influence the selection and regulation of the primary response B cell repertoire, and to explore the structural basis of Ag binding, we have determined the nucleotide sequence of the entire V region gene complexes, which encode the H and L chains of these highly conserved and dominant CRIXmp-1+ antibodies. Our data establish that the H chain gene complex consists of a single VH germ-line gene that is identical to VH Oxazolone-1, encoding the H chain of another highly conserved and dominant cross-reactive Id family associated with the primary response to Oxazolone. In CRIXmp-1+ Xmp-specific hybridomas this gene is joined to a limited set of D region sequences that express a conserved amino acid motif-GLR. At least three of the five D regions examined are coded for by DFL16.2. This VHD complex can be utilized with one of three different JH region genes (JH1, JH2, and JH4) without any significant effect upon antibody fine specificity or Id. In spite of this lack of JH fidelity all of the CRIXmp-1+ hybridomas have precisely maintained the same length in the H chain CDR3 and FRW4 by altering either the length of the D segment or the length of JH. Nucleotide sequence analysis of the VL gene complex of CRIXmp-1+ anti-Xmp antibodies indicates that the L chain V region is also encoded by a single germ-line gene. The amino acid sequence predicted from the nucleotide sequence of the VKJK from Xmp-specific CRIXmp-1+ hybridomas is identical to the sequence of the anti-arsonate antibody 1210.7, which is the prototype of another Id family (CRI) that is conserved and dominant in BALB/c mice.


Genes encoding Xenopus laevis Ig L chains. Implications for the evolution of kappa and lambda chains. Xenopus laevis Ig contain two distinct types of L chains, designated rho or L1 and sigma or L2. We have analyzed Xenopus genomic DNA by Southern blotting with cDNA probes specific for L1 V and C regions. Many fragments hybridized to the V probe, but only one or two fragments hybridized to the C probe. Corresponding C, J, and V gene segments were identified on clones isolated from a genomic library prepared from the same DNA. One clone contains a C gene segment separated from a J gene segment by an intron of 3.4 kb. The J and C gene segments are nearly identical in sequence to cDNA clones analyzed previously. The C segment is somewhat more similar and the J segment considerably more similar in sequence to the corresponding segments of mammalian kappa chains than to those of mammalian lambda chains. Upstream of the J segment is a typical recombination signal sequence with a spacer of 23 bp, as in J kappa. A second clone from the library contains four V gene segments, separated by 2.1 to 3.6 kb. Two of these, V1 and V3, have the expected structural and regulatory features of V genes, and are very similar in sequence to each other and to mammalian V kappa. A third gene segment, V2, resembles V1 and V3 in its coding region and nearby 5'-flanking region, but diverges in sequence 5' to position -95 with loss of the octamer promoter element. The fourth V-like segment is similar to the others at the 3'-end, but upstream of codon 64 bears no resemblance in sequence to any Ig V region. All four V segments have typical recombination signal sequences with 12-bp spacers at their 3'-ends, as in V kappa. Taken together, the data suggest that Xenopus L1 L chain genes are members of the kappa gene family.


Effect of Haemophilus influenzae polysaccharide outer membrane protein complex conjugate vaccine on macrophages. Haemophilus influenzae type b polysaccharide-conjugate vaccines elicit protective antibody responses in young infants. One of these conjugates, polysaccharide linked to outer membrane protein complex (PRP-OMPC), is produced by linking the capsular polysaccharide to an outer membrane protein complex derived from group B Neisseria meningitidis. The outer membrane protein complex contains T cell carrier epitopes that elicit T cell-dependent antibody responses. OMPC also has been shown to increase the antibody response to other proteins administered concurrently that are not covalently linked (i.e., acts as an adjuvant). In this study PRP-OMPC immunized mice demonstrated significant increases in spleen size as well as in splenocyte number as compared to saline controls (p < 0.01, p < 0.001, respectively). No such increase was noted after immunization with another H. influenzae type b-conjugate vaccine, oligosaccharide linked to a variant of diphtheria toxin. By analytic flow cytometry, the mice immunized with PRP-OMPC demonstrated an increase in large splenocytes expressing the Ag Mac-1 (CD11b, CR3). Furthermore, the spleens on histologic examination were characterized by an increase in the red pulp area consisting predominantly of cells of macrophage morphology. By immunohistochemical staining, the cells were identified as macrophages due to expression of Mac-1 and p150,95 (CD11C) Ag. After PRP-OMPC immunization, severe combined immunodeficient mice also demonstrated significant splenomegaly with an increase in macrophages identified by expression of Mac-1 and MHC class II Ag. Thus PRP-OMPC vaccine resulted in T cell-independent splenomegaly with an increase number of macrophages. We propose that this unique property may confer increased immunogenicity to PRP-OMPC through macrophage activation and cytokine release. Furthermore, the effect on macrophages may explain the "adjuvant" capacity of OMPC.


Transfection of mouse cytotoxic T lymphocyte with an antisense granzyme A vector reduces lytic activity. Murine CTL have seven serine proteases, known as granzymes, in their lytic granules. Despite considerable effort, convincing evidence that these enzymes play an obligatory role in the lytic process has not been presented. To investigate the function of one of these proteases, granzyme A (GA), we utilized an antisense expression vector to lower the level of the enzyme in the cells. An expression vector containing antisense cDNA for GA and the gene for hygromycin B resistance was constructed and electroporated into the murine CTL line, AR1. Transfectants were selected based on resistance to hygromycin B, and a number of stable lines were developed. One of the antisense lines had greatly reduced levels of GA mRNA, when compared to the parental cells or to control lines transfected with the vector lacking the antisense DNA. The message levels for two other CTL granule proteins, granzyme B and perforin, were unaffected by the antisense vector. The amount of GA, as measured by enzymatic activity, was 3- to 10-fold lower in the transfectant. Most significantly, this line also consistently showed 50 to 70% lower ability to lyse nucleated target cells and to degrade their DNA. Furthermore, it exhibited 90 to 95% lower lytic activity to anti-CD3-coated SRBC. Conjugate formation with target cells, however, was normal. These data provide strong evidence that GA plays an important role in the cytolytic cycle, and that the quantity of enzyme is a limiting factor in these cytolytic cells.


Eosinophil transendothelial migration induced by cytokines. I. Role of endothelial and eosinophil adhesion molecules in IL-1 beta-induced transendothelial migration. IL-1 beta promotes adhesiveness in human umbilical vein endothelial cells (HuVEC) for eosinophils through expression of adhesion molecules including intercellular adhesion molecules-1 (ICAM-1), E-selectin, and vascular cell adhesion molecule-1 (VCAM-1). Using an in vitro endothelial monolayer system, we examined whether IL-1 beta or TNF-alpha can promote eosinophil transendothelial migration. We also evaluated the contributions of ICAM-1, E-selectin, VCAM-1, leukocyte adhesion complex (CD11/18), and very late Ag-4 (CD11b/18) (VLA-4) in this process using blocking mAb, and determined the changes in expression of CD11b and L-selectin on eosinophils that had undergone transmigration. IL-1 beta and TNF-alpha treatment of HuVEC (4 h, 5 ng/ml) induced significant transendothelial migration of eosinophils (a 4.1 +/- 0.4-fold (IL-1 beta) and 2.0 +/- 0.9-fold (TNF-alpha) increase from the spontaneous value of 3.2 +/- 0.3%). Increased CD11b expression and shedding of L-selectin were observed on eosinophils following IL-1 beta-induced eosinophil transendothelial migration. Studies with mAb revealed that blockade of either ICAM-1 or CD11/18 inhibited transmigration, while antibodies against VCAM-1 and VLA-4 had no inhibitory effect. Among antibodies which block beta 2 integrins, anti-CD18 mAb had the best inhibitory effect (88% inhibition). The combined inhibitory effect of anti-CD11a mAb and anti-CD11b mAb was roughly equal to that of anti-CD18, although anti-CD11a (31% inhibition) and anti-CD11b (52% inhibition) were less effective individually. Anti-ICAM-1 by itself inhibited IL-1 beta-induced eosinophil transendothelial migration (24% inhibition) whereas neither anti-E-selectin nor anti-VCAM-1 were effective inhibitors. Interestingly, the combination of anti-E-selectin and anti-VCAM-1 with anti-ICAM-1 inhibited IL-1 beta-induced eosinophil transendothelial migration significantly better (53% inhibition) than anti-ICAM-1 alone. These results suggest that although the initial attachment of eosinophils to IL-1 beta-activated endothelial cells involves VCAM-1, E-selectin, and ICAM-1, the subsequent transendothelial migration process relies heavily on ICAM-1 and CD11/18. Finally, the changes that eosinophils have been observed to undergo during infiltration in vivo, namely increased expression of CD11/18 and shedding of L-selectin, appear to take place as a direct result of the interaction between eosinophils and endothelial cells.


IL-10 stimulates monocyte Fc gamma R surface expression and cytotoxic activity. Distinct regulation of antibody-dependent cellular cytotoxicity by IFN-gamma, IL-4, and IL-10. T cell-derived cytokines IFN-gamma and IL-4 have different regulatory effects on two functionally important molecules on human monocytes: MHC class II Ag and the Fc receptor for monomeric IgG, Fc gamma RI (CD64). MHC class II Ag, and Fc gamma RI are both upregulated in the presence of IFN-gamma. IL-4 induces MHC class II Ag expression but reduces Fc gamma RI expression. Recently, we showed that the cytokine IL-10 also affects MHC class II Ag expression. Here, we demonstrate that in contrast to the down-regulation of MHC class II Ag expression, IL-10 stimulates Fc gamma RI expression on human monocytes comparable to the levels of Fc gamma RI expression induced by IFN-gamma. The IL-10-induced Fc gamma RI expression is specific because anti-IL-10 antibodies completely reverse the IL-10-induced surface expression of Fc gamma RI and correlate with an enhanced capacity to lyse anti-D-coated human rhesus-positive erythrocytes. IL-10 fails to induce the expression of Fc gamma RII (CD32) and Fc gamma RIII (CD16). Furthermore, we demonstrate that IL-10 is able to prevent down-regulation in surface membrane expression of all three Fc gamma R that can be found when monocytes are cultured in the presence of IL-4. In contrast to IFN-gamma, IL-10 does not restore the reduced antibody-dependent cellular cytotoxicity (ADCC) activity of IL-4-cultured monocytes. Together, these results show that, similar to IFN-gamma, IL-10 is capable of enhancing Fc gamma R expression and ADCC activity, and that IFN-gamma, IL-4, and IL-10 have different regulatory effects on both monocyte Ag-presenting capacity and ADCC activity.


Probing the human antibody repertoire to exogenous antigens. Characterization of the H and L chain V region gene segments from anti-hepatitis B virus antibodies. Structural studies of human antibody V regions have been largely limited to those involving the fetal repertoire, autoantibodies, and malignant cell rearrangements, leaving the "normal" repertoire relatively unexplored. In this study we describe the nucleotide sequences of the H and L chain V regions of four antibodies specific for the surface Ag of the hepatitis B virus. Monoclonal cell lines were derived from healthy individuals who received standard immunizations with the serum-derived or recombinant hepatitis B virus vaccines by fusion of PBL to a heterohybridoma cell line, SPAZ-4. We utilized the polymerase chain reaction to amplify the H and L chain V regions for cloning and sequencing. The four antibodies express the following V region combinations: VHIII/V lambda V, VHIII/V kappa II, VHIV/V kappa I, VHV/V lambda III. When compared to germline genes with the closest sequence homology, all of the V regions appear to have undergone somatic mutation, ranging from 3.4 to 11.3% for the H chain, and 5.1 to 9.2% for the L chain. Analysis of the mutations shows them to be typical for an Ag-driven immune response.


Cytotoxic T lymphocytes do not appear to select for mutations in an immunodominant epitope of simian immunodeficiency virus gag. Studies to date assessing HIV escape from CTL in vivo have yielded conflicting results. Previous studies have demonstrated that simian immunodeficiency virus of macaques (SIVmac)-infected rhesus monkeys expressing the MHC class I allele Mamu-A*01 reproducibly develop a gag-specific CTL response limited to a 9-amino acid epitope of the SIVmac gag protein (residues 182-190 within peptide 11C). To determine whether CTL have a role in selecting for AIDS virus mutants, we examined mutations in SIVmac proviral DNA encoding this gag CTL epitope in PBL of infected rhesus monkeys. Three Mamu-A*01+ rhesus monkeys were infected with SIVmac and assessed for gag- and peptide 11C-specific CTL responses. This specific CTL response was maintained in two monkeys, but lost in the third animal 2 yr after infection. The generation of proviral gag mutations was then determined by sequencing 500-bp proviral fragments amplified from fresh PBL obtained from the monkeys more than 2.5 yr after infection. Although numerous point mutations were characterized in 131 polymerase chain reaction-generated clones of SIVmac gag, only four mutations within the gag CTL epitope-coding region of the genome were identified. Comparison of synonymous and nonsynonymous nucleotide substitutions in the regions encoding peptide 11C (p11C) and the flanking gag protein indicated a lack of selective pressure for viral mutations in the CTL epitope coding region. Interestingly, a predominant gag mutant encoding a single amino acid change in p11C was found in a monkey which lost its CTL activity. However, even in this setting there was no evidence for selection of mutations in the CTL epitope coding region when compared with the flanking region. Furthermore, synthetic peptides corresponding to all naturally occurring variants in the gag epitope-coding region were recognized by cloned and bulk cultured effector cells of the infected monkeys with persistent CTL. These results indicate that SIVmac gag- and p11C-specific CTL do not select for mutations in the immunodominant epitope-coding region and that the naturally occurring mutants do not appear to escape CTL recognition.


IFN-gamma receptor-Ig fusion proteins. Half-life, immunogenicity, and in vivo activity. Two mouse IFN-gamma receptor (MoIFN gamma R)-Ig fusion proteins, which were constructed for the purpose of creating new efficient mouse IFN-gamma (MoIFN-gamma) inhibitor molecules, were studied in vivo to determine their plasma half-life and immunogenicity, and to show their biologic activity. The hybrid proteins show 40-h blood persistency. They do not provoke an antibody response when injected into mice, and they are biologically active in vivo, as demonstrated by the prevention of streptozotocin-induced diabetes. The two fusion proteins are efficient MoIFN-gamma antagonists and can be used in mouse models of human diseases to investigate the role of MoIFN gamma in these pathologic states.