A follow-up on quantitative and qualitative olfactory dysfunction and other symptoms in patients recovering from COVID-19 smell loss.

BACKGROUND: Sudden smell loss is a specific early symptom of COVID-19, which, prior to the emergence of Omicron, had estimated prevalence of ~40% to 75%. Chemosensory impairments affect physical and mental health, and dietary behavior. Thus, it is critical to understand the rate and time course of smell recovery. The aim of this cohort study was to characterize smell function and recovery up to 11 months post COVID-19 infection. METHODS: This longitudinal survey of individuals suffering COVID-19-related smell loss assessed disease symptoms and gustatory and olfactory function. Participants (n=12,313) who completed an initial survey (S1) about respiratory symptoms, chemosensory function and COVID-19 diagnosis between April and September 2020, were invited to complete a follow-up survey (S2). Between September 2020 and February 2021, 27.5% participants responded (n=3,386), with 1,468 being diagnosed with COVID-19 and suffering co-occurring smell and taste loss at the beginning of their illness. RESULTS: At follow-up (median time since COVID-19 onset ~200 days), ~60% of women and ~48% of men reported less than 80% of their pre-illness smell ability. Taste typically recovered faster than smell, and taste loss rarely persisted if smell recovered. Prevalence of parosmia and phantosmia was ~10% of participants in S1 and increased substantially in S2: ~47% for parosmia and ~25% for phantosmia. Persistent smell impairment was associated with more symptoms overall, suggesting it may be a key marker of long-COVID illness. The ability to smell during COVID-19 was rated slightly lower by those who did not eventually recover their pre-illness ability to smell at S2. CONCLUSIONS: While smell ability improves for many individuals who lost it during acute COVID-19, the prevalence of parosmia and phantosmia increases substantially over time. Olfactory dysfunction is associated with broader persistent symptoms of COVID-19, and may last for many months following acute COVID-19. Taste loss in the absence of smell loss is rare. Persistent qualitative smell symptoms are emerging as common long-term sequelae; more research into treatment options is strongly warranted given that even conservative estimates suggest millions of individuals may experience parosmia following COVID-19. Healthcare providers worldwide need to be prepared to treat post COVID-19 secondary effects on physical and mental health.

Age-related differences in network structure and dynamic synchrony of cognitive control.

Cognitive trajectories vary greatly across older individuals, and the neural mechanisms underlying these differences remain poorly understood. Here, we investigate the cognitive variability in older adults by linking the influence of white matter microstructure on the task-related organization of fast and effective communications between brain regions. Using diffusion tensor imaging and electroencephalography, we show that individual differences in white matter network organization are associated with network clustering and efficiency in the alpha and high-gamma bands, and that functional network dynamics partly explain individual differences in cognitive control performance in older adults. We show that older individuals with high versus low structural network clustering differ in task-related network dynamics and cognitive performance. These findings were corroborated by investigating magnetoencephalography networks in an independent dataset. This multimodal (fMRI and biological markers) brain connectivity framework of individual differences provides a holistic account of how differences in white matter microstructure underlie age-related variability in dynamic network organization and cognitive performance.

Factors associated with glycaemic control in adults with Type 1 diabetes: a registry-based analysis including 7601 individuals from 34 centres in Norway.

AIMS: To use data from the Norwegian Diabetes Registry for Adults and Statistics Norway to assess factors associated with glycaemic control in type 1 diabetes. METHODS: The analyses included all individuals aged ≥18 years who had a type 1 diabetes duration of >2 years and a recorded value in the registry between 2013 and 2015 (n=7601). Predicted mean HbA1c levels for subgroups of participants were assessed using linear regression analysis. RESULTS: Young age (18-25 years), low education levels, smoking, living alone, exercising infrequently, monitoring glucose infrequently, high insulin requirements, low frequency of symptomatic hypoglycaemia, history of ketoacidosis and a BMI <18.5 kg/m2 were associated with a 2-12-mmol/mol (0.2-1.1%) higher HbA1c level. Those with 10-15 years of diabetes duration had 5-mmol/mol (0.5%) higher HbA1c level than those who had a diabetes duration of 2-5 years. Sex, participation (ever) in a diabetes education course, or ever experiencing serious hypoglycaemia were not associated with glycaemic control. CONCLUSIONS: We present representative national data on factors that were associated with glycaemic control. A better understanding and awareness of these factors, together with technological advances in diabetes management, could lead to more personalized management strategies, better glycaemic control and a lower risk of diabetes complications.

Variation in the achievement of HbA1c , blood pressure and LDL cholesterol targets in type 2 diabetes in general practice and characteristics associated with risk factor control.

AIMS: To identify population, general practitioner, and practice characteristics associated with the achievement of HbA1c , blood pressure and LDL cholesterol targets, and to describe variation in the achievement of risk factor control. METHODS: We conducted a cross-sectional survey of 9342 people with type 2 diabetes, 281 general practitioners and 77 general practices in Norway. Missing values (7.4%) were imputed using multiple imputation by chained equations. We used three-level logistic regression with the achievement of HbA1c , blood pressure and LDL cholesterol targets as dependent variables, and factors related to population, general practitioners, and practices as independent variables. RESULTS: Treatment targets were achieved for HbA1c in 64%, blood pressure in 50%, and LDL cholesterol in 52% of people with type 2 diabetes, and 17% met all three targets. There was substantial heterogeneity in target achievement among general practitioners and among practices; the estimated proportion of a GPs diabetes population at target was 55-73% (10-90 percentiles) for HbA1c , 36-63% for blood pressure, and 47-57% for LDL cholesterol targets. The models explained 11%, 5% and 14%, respectively, of the total variation in the achievement of HbA1c , blood pressure and LDL cholesterol targets. Use among general practitioners of a structured diabetes form was associated with 23% higher odds of achieving the HbA1c target (odds ratio 1.23, 95% confidence interval (CI) 1.02-1.47) and 17% higher odds of achieving the LDL cholesterol target (odds ratio 1.17, 95% CI 1.01-1.35). CONCLUSIONS: Clinical diabetes management is difficult, and few people meet all three risk factor control targets. The proportion of people reaching target varied among general practitioners and practices. Several population, general practitioner and practice characteristics only explained a small part of the total variation. The use of a structured diabetes form is recommended.

Population, general practitioner and practice characteristics are associated with screening procedures for microvascular complications in Type 2 diabetes care in Norway.

AIMS: To assess population, general practitioner (GP) and practice characteristics associated with the performance of microvascular screening procedures and to propose strategies to improve Type 2 diabetes care. METHODS: A cross-sectional survey in Norway (281 GPs from 77 practices) identified 8246 people with a Type 2 diabetes duration of 1 year or more. We used multilevel regression models with either the recording of at least two of three recommended screening procedures (albuminuria, monofilament, eye examination) or each procedure separately as dependent variable (yes/no), and characteristics related to the person with diabetes, GP or practice as independent variables. RESULTS: The performance of recommended screening procedures was recorded in the following percentages: albuminuria 31.5%, monofilament 27.5% and eye examination 60.0%. There was substantial heterogeneity between practices, and between GPs within practices for all procedures. Compared with people aged 60-69 years, those aged < 50 years were less likely to have an albuminuria test performed [odds ratio (OR) 0.75, 95% CI 0.61 to 0.93] and eye examination (OR 0.79, 95% CI 0.66 to 0.95). People with macrovascular disease had fewer screening procedures recorded (OR 0.68, 95% CI 0.59 to 0.78). Use of an electronic diabetes form was associated with improved screening  (OR 2.65, 95% CI 1.86 to 3.78). GPs with high workload recorded fewer procedures (OR 0.59, 95% CI 0.39 to 0.90). CONCLUSIONS: Performance of screening procedures was suboptimal overall, and in people who should be prioritized. Performance varied substantially between GPs and practices. The use of a structured diabetes form should be mandatory.

HIV-1 induction of CD40 on endothelial cells promotes the outgrowth of AIDS-associated B-cell lymphomas.

Human immunodeficiency virus (HIV)-1 infection is associated with the development of aggressive extranodal B-cell non-Hodgkin's lymphomas. Using microvascular endothelial cell (MVEC)-enriched bone marrow stromal cultures, HIV infection of stromal MVECs from lymphoma patients induced the outgrowth of malignant B cells. MVECs were the only HIV-infected cells in the stroma, and purified brain MVECs also induced a phenotype supportive of neoplastic B-cell attachment and proliferation. HIV infection of MVECs stimulated surface expression of CD40 and allowed preferential induction of the vascular cell adhesion molecule VCAM-1 after CD40 triggering. B-lymphoma cells expressed the CD40 ligand (CD40L), and blocking of CD40-CD40L interactions between HIV-infected MVECs and B-lymphoma cells inhibited B-cell attachment and proliferation. These observations suggest that HIV promotes B-lymphoma cell growth through facilitating attachment of lymphoma cells to HIV-infected MVECs and represent a novel mechanism through which viruses may induce malignancies.

Correction of interleukin-2 production in patients with systemic lupus erythematosus by removal of spontaneously activated suppressor cells.

Interleukin-2 (IL-2) production in vitro is depressed in systemic lupus erythematosus (SLE) patients. It is not known whether this abnormality is caused by a defect in the producer lymphocytes or by excessive suppression. We report that removal of OKT8 (Leu 2a)+ cells increased the IL-2 production by in vitro-stimulated lymphocytes to normal or above normal levels in 19 of 21 SLE patients. This increase was more apparent in those patients with clinically inactive disease and/or receiving less than 7.5 mg of prednisone. Removal of OKT8+ cells from normals did not significantly increase IL-2 activity. SLE, but not normal, OKT8+ cells decreased IL-2 production when added back to autologous OKT8-depleted cells. In some experiments, OKT8+ cells from normal donors also suppressed IL-2 production in SLE. This result suggests that the defect in IL-2 production is complex and may involve multiple cell interactions. Three lines of evidence suggest that the SLE OKT8+ cells actively inhibit the production of IL-2 rather than passively absorb this lymphokine: (a) only 3.2% of SLE lymphocytes expressed IL-2 receptors as detected with anti-Tac; (b) freshly prepared SLE lymphocytes did not absorb IL-2; and (c) cell-free supernatants from SLE OKT8+ cells inhibited IL-2 production, but not IL-2 activity. Double-labeling studies by flow cytometry revealed that 19.3% of SLE OKT8+ cells were also Ia-positive, and approximately 33% co-expressed the natural killer cell marker, HNK-1 (Leu 7). Removal of Leu 7+ cells also significantly elevated IL-2 production in SLE. These studies suggest that one or more circulating mononuclear cell subsets in SLE patients can suppress IL-2 production and that one subset may possibly belong to a non-T, non-B "third mononuclear population."

1,25-Dihydroxyvitamin D3 receptor measurement in primary renal cell carcinomas and autologous normal kidney tissue.

Recently it was reported that 1-alpha,25-dihydroxyvitamin D3 [1,25-(OH)2D3] inhibited cell growth in a cell line derived from a metastasis from renal cell carcinoma. We have examined samples from 23 primary renal cell carcinomas for 1,25-(OH)2D3 receptor content, and compared it with the concentrations in autologous normal kidney tissue. Nineteen of 23 (83%) renal cell carcinomas had detectable (above 1 fmol/mg protein) 1,25-(OH)2D3 receptor levels, and 15 of 23 (65%) had levels above 5 fmol/mg protein. Mean value for the renal cell carcinomas was 8.2 fmol/mg protein (range, 0-28 fmol/mg protein), and the mean value for autologous normal kidney tissue was 23.1 fmol/mg protein (range, 6.6-53.7 fmol/mg protein). The 1,25-(OH)2D3 receptor levels in the renal cell carcinomas were significantly lower than in the autologous normal kidney tissue (P less than 0.001). The 1,25-(OH)2D3 receptor was characterized by sucrose gradient analysis and DNA-cellulose chromatography. The features found for renal cell carcinoma were similar to the 1,25-(OH)2D3 receptor in normal human tissue. No correlation of 1,25-(OH)2D3 receptor levels to clinical parameters was found. This study shows that carcinomas originating from the kidney, the major vitamin D regulating organ, usually contain the 1,25-(OH)2D3 receptor. The receptor may have a cellular function in the transformed cell.

Effect of dexamethasone on hexamethylene bisacetamide-induced Friend cell erythrodifferentiation.

Friend erythroleukemic cells can be induced to differentiate by chemicals such as dimethyl sulfoxide and hexamethylene bisacetamide (HMBA) in a dose-dependent manner. Others have shown that dexamethasone and other steroid hormones can inhibit the differentiation induced by dimethyl sulfoxide. We show here that dexamethasone has a dual mode of action on the HMBA-induced differentiation of a Friend cell line, DS19. At concentrations above 10(-10) M, dexamethasone inhibits the HMBA-induced differentiation in DS19 cells. We further found that as the concentration of HMBA is reduced, the amount of dexamethasone required to inhibit differentiation increases. Such inhibition seems to act primarily by decreasing the probability that a cell will become committed to differentiate. Besides, dexamethasone does not inhibit hemoglobin synthesis of cells which are committed in its absence. In addition, we show that sensitivity to the inhibition by dexamethasone is inversely related to the inducibility of cell populations. The second action of dexamethasone, observed at concentrations between 10(-10) and 10(-13) M, is to increase the proportion of hemoglobin-containing cells relative to that for cells cultured in its absence. The degree of this synergistic effect for induced differentiation is inversely related to the level of induction in the absence of dexamethasone and thus is best observed in the cells cultured at low levels of HMBA. We present evidence that this synergistic effect may be due to an increased viability of the induced cells and possibly an increase in the proliferative capacity of these cells.

Augmentation of natural killer cell activity after arterial embolization of renal carcinomas.

Preoperative embolization of the renal artery has been reported to improve the survival of patients with advanced renal carcinomas compared to operative treatment only. To investigate possible immunological consequences of tumor embolization, natural killer (NK) cell activity in peripheral blood was investigated immediately before and at different time intervals after occlusion of the renal artery by insertion of a metal coil. A slight increase in NK activity could be observed 24 hr postembolization while a marked augmentation was seen after 48 hr. The high NK activity persisted up to 96 hr after embolization, the last time period included in the study. Two patients undergoing the same procedure but in whom embolization was unsuccessful showed no alteration in NK activity. It is suggested that interferon produced by macrophages activated by the necrotizing tumor might be responsible for the augmentation of NK activity.

Beta-carotene in HIV infection: an extended evaluation.

OBJECTIVE: Several small short-term intervention studies have suggested that beta-carotene supplementation in HIV-infected patients can increase the number of various immune cells including CD4 cells. This prospective double-blinded study was designed to investigate whether beta-carotene supplementation would result in this immuno-enhancement in a larger number of patients over a longer time period. METHODS: HIV-positive patients were randomly assigned to receive either 60 mg beta-carotene orally three times daily or a matched placebo. In addition, all patients received a multivitamin supplement. Patients were evaluated at baseline, 1 month, and 3 months for T-cell quantitative subsets, natural killer cells, HIV p24 antigen, beta-carotene levels, complete blood counts and chemistry batteries. Body weights and Karnofsky scores were evaluated at each visit. RESULTS: Seventy-two patients signed informed consent forms and entered the study. Except for serum beta-carotene concentration, there were no statistically significant differences (P < 0.05) between the treatment (60 mg beta-carotene three times daily and multivitamins) and placebo (placebo and multivitamins) groups at baseline or after either 1 or 3 months of treatment. DISCUSSION: Earlier studies suggesting that beta-carotene supplementation increased levels of immune cells in HIV-infected patients were not replicated in this study. The addition of a multivitamin supplement to both arms of this study may have masked any difference between the two groups. However, on the basis of the results of this study, we would not recommend supplementation with high doses of beta-carotene for HIV-infected patients.

An enzyme immunoassay for the flow cytometer (FEIA).

This report describes the use of a fluorescent activated cell sorter (FACS) in combination with an enzyme immunosorbent assay (EIA). This combination of techniques expands the versatility of the flow cytometer. It introduces a new set of fluorescent enzyme products for antigen detection. These highly substantive fluorescent compounds permit the flow cytometer to quantify cell-EIA reactions and also to delineate subpopulations of cells with different quantities of surface antigens. Because the FEIA product is colored, as well as fluorescent, a simple light microscope may also be used to define the distribution of the label on the cell surface. These techniques have been applied to the examination of antigens on human erythrocytes, human T cell lymphoma cells (H9), and to surface markers on the tissue culture cell line K562.

Quantification of DNA binding to cell-surfaces by flow cytometry.

DNA binding to cell-surfaces has been documented in several studies. The interaction of DNA with cells has been shown to have therapeutic potential as a non-viral form of gene delivery and DNA vaccination. Recently, bacterial DNA binding and internalization has been demonstrated in some cells to trigger secretion of cytokines and cell activation. Previous studies to quantify DNA binding to cells have used radiolabeled DNA. Here we report a non-radioactive assay for quantification of cell-surface DNA binding based on the isoparametric analysis of flow cytometric data as described by Chatelier et al., Embo J., 5 (1986) 1181. This assay has the advantage over previously used procedures in not employing radioactive material and being able to discriminate viable from non-viable cells that bind DNA. With the importance of understanding the interaction of DNA with cells, this assay may have application for the identification and characterization of reagents designed to either enhance or inhibit DNA binding to cells.

Lymphokine mRNA expression in the spinal cords of Lewis rats with experimental autoimmune encephalomyelitis is associated with a host recruited CD45R hi/CD4+ population during recovery.

To evaluate CD4+ T cell subpopulations involved in the induction and recovery from experimental autoimmune encephalomyelitis (EAE), the CD45R phenotype and lymphokine mRNA profile was evaluated for encephalitogenic CD4+ T cell lines in vitro and compared to CD4+ T cells isolated from the spinal cord of Lewis rats with EAE. All of the myelin basic protein (MBP)-specific T cell lines and clones that adoptively transferred EAE were > 90% CD4+ and > 90% CD45R lo. A time course of EAE disease progression was monitored as a function of the percentage of CD45R hi/CD4+ T cells isolated from the spinal cords of diseased animals. The majority of CD4+ T cells found in the central nervous system during the early phase of passive EAE were CD45R lo (the same as the encephalitogenic lines/clones). A large increase of the CD45R hi/CD4+ T cells (up to 45%) was observed during the peak and recovery phases of EAE. Lymphokine mRNA production was analyzed from antigen-stimulated MBP-specific lines, and from spinal cord lymphocytes isolated from rats with EAE. The BP-specific lines produced Th1 lymphokines (IL-2, IFN-gamma, and TNF-alpha), while the spinal cord lymphocytes produced the same Th1 lymphokines as well as IL-4 and IL-10. The CD45R hi/CD4+ T cells isolated from the spinal cords were larger and expressed more lymphokine RNA per cell than the CD45R lo/CD4+ T cells. The encephalitogenic cells (CD45R lo) were detected in the spinal cords of rats with a fluorescent dye and by allelic transfers and all of the CD45R hi/CD4+ T cells were found to be host recruited. Thus, it appears that the CD45R hi/CD4+ lymphocytes found in the spinal cord represent a host-recruited, activated cellular infiltrate that increased in number in the recovery phase of EAE and synthesized both Th1 and Th2 lymphokines.

Cellular basis for the elevated gallium-67 computed lung index in a rheumatoid lung patient.

A patient with high levels of serum rheumatoid factor and an open lung biopsy which showed high-grade interstitial pneumonia with large numbers of lymphocytes and plasmocytes had intense gallium uptake in the lungs. Lymphocytes and/or plasmocytes might be responsible for the gallium uptake even though neutrophils are usually credited with high-level uptake. Differential cell counts demonstrated plasmocyte and lymphocyte preponderance, but neutrophil paucity. In vitro cell cultures of purified neutrophils, monocytes, leukemic plasmocytes, and resting and stimulated lymphocytes with 67Ga showed that plasmocytes take up comparatively low levels of 67Ga, but that activated lymphocytes take up levels that approach neutrophils. It is probable that both rheumatoid lung plasmocytes and activated lymphocytes are responsible for the pulmonary 67Ga concentration in this patient.

T-cell receptor variable beta genes show differential expression in CD4 and CD8 T cells.

Studies in transgenic and inbred strains of mice have shown that the critical molecular interactions controlling positive selection involve major histocompatibility complex (MHC), T-cell receptor (TCR), and CD4 or CD8 coreceptor molecules. Correlations have been established between MHC gene products and the percentage of CD4 or CD8 T cells that express specific variable (V) beta-gene products as part of the alpha beta heterodimer. These studies have important implications regarding potential mechanisms of HLA-linked autoimmune diseases in humans. If similar interactions are required for positive selection in humans, one would predict that the TCR repertoire expressed by mature, peripheral blood CD4 and CD8 T cells would vary. To test this hypothesis the expression of specific TCR V beta-region genes by CD4 and CD8 T cells from healthy individuals was compared using both triple-color flow cytometry and polymerase chain reaction based experimental approaches. The results show that the TCR repertoire does vary as a function of CD4 and CD8 T-cell subsets. Among unrelated individuals certain V beta genes were consistently overrepresented in the CD4 population (V beta-5.1, -6.7a, and -18); some were skewed to the CD8 population (V beta-14) while others showed variable patterns (V beta-12 and -17). Deletion of entire V beta gene families was not observed suggesting that this is a rare event in humans. Attempts to correlate the expressed TCR repertoire in humans with HLA alleles will require consideration of these differences in expression as a function of subset.

Immunization of BALB/c mice with a monoclonal anti-DNA antibody induces an anti-idiotypic antibody reactive with a cell-surface DNA binding protein.

DNA binds to cell-surface proteins on human and murine leukocytes and induces secretion of the cytokine interleukin 6 (IL-6). Cell-surface DNA binding molecules have been shown to serve as target antigens for the production of autoantibodies in patients with systemic lupus erythematosus (SLE), and in lupus-prone mice. Recent studies have demonstrated that a subset of anti-anti-DNA antibodies, isolated from patients with SLE, are idiotypically related to antibodies reactive with a cell-surface DNA binding molecule. We now report that immunization of normal mice with a murine monoclonal anti-DNA antibody induces an anti-idiotypic response which has reactivity with a cell-surface DNA binding molecule. An anti-idiotypic anti-DNA monoclonal antibody (LB17) was isolated from the spleen of an immunized mouse. This monoclonal antibody blocked the binding of DNA to murine splenocytes and mimicked the functional effect of DNA by stimulating the secretion of IL-6. These experiments provide further evidence for an idiotypic connectivity between antibodies to cell-surface DNA binding proteins and anti-DNA antibodies. It is hypothesized that this idiotypic system is part of the network of natural autoantibodies and that its perturbation may give rise to pathogenic antibodies.

Inhibition of phytohaemagglutinin-induced lymphoproliferation by soluble annexin II in sera from patients with renal cell carcinoma.

Annexin II (AII) is a member of a family of glycoproteins which bind negatively charged phospholipids in a calcium-dependent manner. Annexins are membrane-associated proteins, expressed both in normal and malignant cells, but have also been detected as soluble molecules in serum and other body fluids. Because of their adhesive properties, it has been suggested that annexins play a role in the metastatic process. An ELISA was established for quantification of soluble AII. Within-run variation was 5.2-10.4% and run-to-run variation 12.4-15.6%. Soluble AII was detected in all sera studies. A strongly positive serum was arbitrarily given the value 100 AII units and used as reference serum. The mean level in sera from 20 normal blood donors was 49 (SE 5.6) AII units. Sera from peripheral blood of five patients with renal cell carcinoma and sera from blood obtained from the renal vein of the same patients contained 47 (SE 20) and 83 (SE 28) AII units, respectively. In two patients, AII levels were increased in renal vein serum as compared with peripheral blood serum. Interestingly, in both cases, and in none of the three remaining cases, phytohaemagglutinin-stimulated lymphoproliferation was suppressed by renal vein serum as compared with peripheral blood serum. Affinity absorption of AII from the renal vein sera with increased AII levels strongly reduced their immunosuppressive activity. Addition of affinity-purified AII to cell cultures suppressed lymphoproliferation. These data show that the level of AII is markedly increased in renal vein sera from some patients with renal cell carcinoma, suggesting that AII may be locally released in vivo. The study also demonstrates an immunosuppressive effect of soluble AII in vitro. We speculate that soluble AII released by the tumour has immunosuppressive properties. This study identifies soluble AII as a novel immunosuppressive factor in sera from patients with renal cell carcinoma. A further study including a larger number of patients is currently in progress, in order to investigate the pathological significance of this finding.

A simple and practical technic for detecting cancer cells in urine and urinary bladder washings by flow cytometry.

DNA measurement by flow cytometry has been demonstrated to be a potentially useful technic in the diagnosis of bladder cancer by detecting neoplastic cells in bladder washings and urine specimens. The authors' goal was to develop a simple and practical method utilizing the new generation of cytofluorographs designed for use in the clinical laboratory. This method combined direct fixation with cell lysis yielding fixed intact nuclei. Following RNase and pepsin digestion, the nuclei were separated from debris and aggregates on a sucrose barrier, stained with ethidium bromide, and analyzed with an argon laser analytic cytofluorograph. Urines and bladder washings from 14 patients with positive urinary cytology and histologically diagnosed bladder cancers were compared with specimens from patients without urothelial malignancies. DNA histograms clearly delineated aneuploid from diploid populations and often identified S, G2M, and G1 phase nuclei. Aneuploid populations have been detected in all tumor specimens with positive cytologies studied to date.

Interleukin-12 p40 m-RNA expression in human kidney allograft biopsies.

Interleukin-12 (IL-12) is a heterodimeric cytokine implicated in the early differentiation of naive T-lymphocytes into the Th1 subset. IL-12 is important for induction of the cellular immune response against viruses, intracellular parasites and neoplasms. Its role in alloresponsiveness has not been fully elucidated. Preliminary data in the literature point toward the prevalence of Th1 lymphocytes in processes of allograft rejection. In attempt to further investigate the expression of this cytokine during episodes of cellular rejection of renal allografts, we searched for IL-12 message in human kidney allograft biopsies using the reverse transcriptase-polymerase chain reaction technique. Twenty-three allograft core biopsies from 19 patients were obtained percutaneously for clinical indications in 18 cases, and as part of an investigational protocol in five cases. A portion of the tissue was used for RNA extraction using the guanidium-thiocyanide phenol-chloroform method. Histology was performed on the remaining core material. Ten mg of total RNA were used for reverse transcription. PCR of the c-DNAs was done for 40 cycles using primers for the p40 subunit of IL-12 and GAPDH which was used as a control. PCR products were photographed after electrophoresis, transferred to a nylon membrane and hybridized with a radiolabelled cloned human IL-12 p40 1 kb c-DNA fragment. Autoradiographies were developed after 20-min exposure. All samples were run in triplicate. IL-12 p40 m-RNA was expressed in all 17 biopsies showing acute cellular rejection as well as in all three biopsies showing focal interstitial fibrosis. No message was found in the presence of normal allograft histology. This is the first in vivo report of IL-12 p40 subunit m-RNA expression during renal allograft rejection in humans. The role of this Th1 cytokine in the alloresponse deserves further investigation.