Declining incidence of keratinocyte carcinoma in organ transplant recipients.

BACKGROUND: All organ transplant populations are predisposed to increased rates of keratinocyte carcinoma (KC). Since this increased risk was first appreciated, immunosuppressive regimens have changed and organ transplant recipients (OTRs) have been aggressively screened for KC. There is a perception that these measures have impacted on KC incidence but there is a paucity of population-based studies on post-transplant rates of basal cell carcinoma (BCC). OBJECTIVES: To identify trends in incidence rates for KC following solid organ transplantation over the past two decades. METHODS: This nationwide, population-based study included all solid OTRs transplanted between 1994 and 2014. Patient data were matched to national cancer registry data to determine the standardized incidence ratio (SIR) of KC in solid OTRs compared with the general population. RESULTS: In total 3580 solid OTRs were included. The total follow-up time was 28 407 person-years (median follow-up 7·11 years). The overall SIRs for squamous cell carcinoma (SCC) and BCC were 19·7 and 7·0, respectively. Our study documents a progressive fall in the SIRs for SCC and BCC from peak SIRs (95% confidence intervals) in 1994-1996 of 26·4 (21·5-32·4) and 9·1 (7·4-11·3) to 6·3 (2·3-16·7) and 3·2 (1·4-7·1) in 2012-2014, respectively. The ratio of SCC to BCC has remained at 3 to 1 over the last two decades. CONCLUSIONS: Our study is the first to demonstrate a significant reduction over the past two decades in the incidences of both SCC and BCC following solid organ transplantation. The SCC-to-BCC ratio was maintained, demonstrating that both are reducing equally. This trend coincided with temporal changes in immunosuppressive protocols and the introduction of skin cancer prevention programmes. What's already known about this topic? Prior studies have shown that the risk of cutaneous squamous cell carcinoma (SCC) has declined over recent decades following solid organ transplantation. It is not known whether the risk of basal cell carcinoma (BCC) has reduced in line with this. What does this study add? Our study documents a progressive fall in the risk of SCC and BCC following solid organ transplantation over the last two decades. The SCC-to-BCC ratio was maintained, demonstrating that both are reducing equally. The trends observed in our study coincided with temporal changes in immunosuppressive protocols and the introduction of cancer prevention programmes, suggesting that these factors have positively impacted on the risk of keratinocyte carcinoma in this cohort.

Autosomal dominant tubulointerstitial kidney disease (ADTKD) in Ireland.

Introduction: Autosomal dominant tubulointerstitial kidney disease (ADTKD) is a rare genetic cause of renal impairment resulting from mutations in the MUC1, UMOD, HNF1B, REN, and SEC61A1 genes. Neither the national or global prevalence of these diseases has been determined. We aimed to establish a database of patients with ADTKD in Ireland and report the clinical and genetic characteristics of these families. Methods: We identified patients via the Irish Kidney Gene Project and referral to the national renal genetics clinic in Beaumont Hospital who met the clinical criteria for ADTKD (chronic kidney disease, bland urinary sediment, and autosomal dominant inheritance). Eligible patients were then invited to undergo genetic testing by a variety of methods including panel-based testing, whole exome sequencing and, in five families who met the criteria for diagnosis of ADTKD but were negative for causal genetic mutations, we analyzed urinary cell smears for the presence of MUC1fs protein. Results: We studied 54 individuals from 16 families. We identified mutations in the MUC1 gene in three families, UMOD in five families, HNF1beta in two families, and the presence of abnormal MUC1 protein in urine smears in three families (one of which was previously known to carry the genetic mutation). We were unable to identify a mutation in 4 families (3 of whom also tested negative for urinary MUC1fs). Conclusions: There are 4443 people with ESRD in Ireland, 24 of whom are members of the cohort described herein. We observe that ADTKD represents at least 0.54% of Irish ESRD patients.

Reduced pro-inflammatory responses to Staphylococcus aureus bloodstream infection and low prevalence of enterotoxin genes in isolates from patients on haemodialysis.

Patients with end-stage renal failure undergo regular haemodialysis (HD) and often develop episodes of Staphylococcus aureus bloodstream infection (BSI), which can re-occur. However, clinically, patients on HD, with S. aureus BSI, respond well to treatment, rarely developing overt signs of sepsis. We investigated the contributions of bacterial virulence and cytokine responses to the clinical course of S. aureus BSI in HD and non-HD patients. Seventy patients were recruited, including 27 (38.6 %) patients on HD. Isolates were spa-typed and virulence and antimicrobial resistance gene carriage was investigated using DNA microarray analysis. Four inflammatory cytokines, IL-6, RANTES, GROγ and leptin, were measured in patient plasma on the day of diagnosis and after 7 days. There was no significant difference in the prevalence of genotypes or antimicrobial resistance genes in S. aureus isolates from HD compared to non-HD patients. The enterotoxin gene cluster (containing staphylococcal enterotoxins seg, sei, sem, sen, seo and seu) was significantly less prevalent among BSI isolates from HD patients compared to non-HD patients. Comparing inflammatory cytokine response to S. aureus BSI in HD patients to non-HD patients, IL-6 and GROγ were significantly lower (p = 0.021 and p = 0.001, respectively) in HD patients compared to other patients on the day of diagnosis and RANTES levels were significantly lower (p = 0.025) in HD patients on day 7 following diagnosis. Lowered cytokine responses in HD patients and a reduced potential for super-antigen production by infecting isolates may partly explain the favourable clinical responses to episodes of S. aureus BSI in HD patients that we noted clinically.

Pre-transplant histology does not improve prediction of 5-year kidney allograft outcomes above and beyond clinical parameters.

Pre-implant kidney biopsy is used to determine suitability of marginal donor kidneys for transplantation. However, there is limited data examining the utility of pre-implant histology in predicting medium term graft outcome. This retrospective study examined kidney transplants over a 10-year period at a single center to determine if pre-implant histology can identify cases of eGFR ≤35 ml/min/1.73m2 at 5 year follow up beyond a clinical predictive logistic regression model. We also compared outcomes of dual kidney transplants with standard single kidney transplants. Of 1195 transplants, 171 received a pre-implant kidney biopsy and 15 were dual transplants. There was no significant difference in graft and patient survival rates. Median eGFR was lower in recipients of biopsied kidneys compared with standard kidney transplants (44 vs. 54 ml/min/1.73m2, p < .001). Median eGFR of dual transplant and standard kidney transplants were similar (58 vs. 54 ml/min/1.73m2, p = .64). Glomerular sclerosis (p = .05) and Karpinski Score (p = .03) were significant predictors of eGFR at 5-years in multivariate analysis but did not improve discrimination of eGFR ≤35 ml/min/1.73m2 at 5-years beyond a clinical prediction model comprising donor age, donor hypertension and terminal donor creatinine (C-statistic 0.67 vs. 0.66; p = .647). Pre-implant histology did not improve prediction of medium-term graft outcomes beyond clinical predictors alone. Allograft function of dual transplant kidneys was similar to standard transplants, suggesting that there is scope to increase utilization of kidneys considered marginal based on histology.

Twenty-year survivors of kidney transplantation.

There have been few studies of patients with renal allografts functioning for more than 20 years. We sought to identify clinical factors associated with ultra long-term (>20 year) renal allograft survival and to describe the clinical features of these patients. We performed a retrospective analysis of the Irish Renal Transplant Database and included 1174 transplants in 1002 patients. There were 255 (21.74%) patients with graft function for 20 years or more. Multivariate analysis identified recipient age (HR 1.01, CI 1.01-1.02), gender (male HR 1.25, CI 1.08-1.45), acute rejection (HR 1.26, CI 1.09-1.45) and transplant type (living related donor vs. deceased donor) (HR 0.52, CI 0.40-0.66) as significantly associated with long-term graft loss. Median serum creatinine was 115 µmol/L. The 5-year graft survival in 20-year survivors was 74.7%. The mean age at death was 62.7 years (±10.6). The most common causes of death were cardiovascular disease and malignancy. The two major causes of graft loss were death (with function) and interstitial fibrosis/tubular atrophy. Comorbidities included skin cancer (36.1%), coronary heart disease (17.3%) and other malignancies (14.5%). This study identifies factors associated with long-term allograft survival and a high rate of morbidity and early mortality in long-term transplant recipients.

Gender and immunosuppression impact on Merkel cell carcinoma diagnosis and prognosis. A population based cohort study.

Background: Merkel cell carcinoma (MCC), a rare cutaneous neuroendocrine endocrine tumour is increasing in incidence, and continues to carry a poor prognosis. Objectives: The objectives of this study were to examine all Irish cases of MCC from 1 January 1994 over 2 decades, focusing on gender and organ transplantation recipients (OTRs). Cases were identified from the National Cancer Registry of Ireland. Covariates of interest included age, body site, period of diagnosis, deprivation-status and history of non-melanoma skin cancer (NMSC). Results: In total 314 MCC cases were identified. A female predominance was noted (53.8%). Comparison between age-standardised rates between the earliest period (1994-1996) with the latest period (2012-2014) showed an increase of 105% in total. The trend in age-standardised incidence rates were noted to be increasing significantly (p = 0.0004). Average age at diagnosis was 77.6 years (male 75.1 years, female 79.7 years). Overall, the majority of MCC cases presented on the head and neck (n = 170, 54.1%). Differences in anatomical location of MCCs were noted between genders. Males were found to be more likely to have a history of previous NMSCs (males n = 73 [57.9%], females n = 53 [42.1%]). Thirty-one percentage of patients died from MCC, average survival was 3.5 years in those who died of this malignancy. Ten organ transplant recipients developed MCC. OTR who developed MCC were diagnosed at a younger average age of 65.1 years. Standardized incidence ratio for MCC in OTR was 59.96. A higher proportion of OTR died from MCC (70%), with a shorter median survival of 0.14 years. In competing risks regression, gender was not significantly associated with risk of dying, females having a non-significantly higher hazard of dying. Organ transplant recipients and patients from less deprived areas were at greater risk of dying from MCC. Conclusions: This population based study provides epidemiological, clinical and outcome data for MCC over a 20-year period.

Genetic polymorphism in Methylenetetrahydrofolate Reductase chloride transport protein 6 (MTHFR CLCN6) gene is associated with keratinocyte skin cancer in a cohort of renal transplant recipients.

Background: Renal transplant recipients (RTRs) are at increased risk of keratinocyte cancer (KC), especially cutaneous squamous cell carcinoma (cSCC). Previous studies identified a genetic variant of the Methylenetetrahydrofolate Reductase (MTHFR) gene, C677T, which conferred a risk for diagnosis of cSCC in Irish RTRs. Objective: We sought to find further genetic variation in MTHFR and overlap genes that may be associated with a diagnosis of KC in RTRs. Methods: Genotyping of a combined RTR population (n = 821) from two centres, Ireland (n = 546) and the USA (n = 275), was performed. This included 290 RTRs with KC and 444 without. Eleven single nucleotide polymorphisms (SNPs) in the MTHFR gene and seven in the overlap gene MTHFR Chloride transport protein 6 (CLCN6) were evaluated and association explored by time to event analysis (from transplant to first KC) using Cox proportional hazards model. Results: Polymorphism at MTHFR CLCN6 (rs9651118) was significantly associated with KC in RTRs (HR 1.50, 95% CI 1.17-1.91, p < 0.00061) and cSCC (HR 1.63, 95% CI 1.14-2.34, p = 0.007). A separate SNP, MTHFR C677T, was also significantly associated with KC in the Irish population (HR 1.31, 95% CI 1.05-1.63, p = 0.016), but not American RTRs. Conclusions: We report the association of a SNP in the MTHFR overlap gene, CLCN6 and KC in a combined RTR population. While the exact function of CLCN6 is not known, it is proposed to be involved in folate availability. Future applications could include incorporation in a polygenic risk score for KC in RTRs to help identify those at increased risk beyond traditional risk factor assessment.

Induction of a non-encephalitogenic type 2 T helper-cell autoimmune response in multiple sclerosis after administration of an altered peptide ligand in a placebo-controlled, randomized phase II trial. The Altered Peptide Ligand in Relapsing MS Study Group.

In this 'double-blind', randomized, placebo-controlled phase II trial, we compared an altered peptide ligand of myelin basic protein with placebo, evaluating their safety and influence on magnetic resonance imaging in relapsing-remitting multiple sclerosis. A safety board suspended the trial because of hypersensitivity reactions in 9% of the patients. There were no increases in either clinical relapses or in new enhancing lesions in any patient, even those with hypersensitivity reactions. Secondary analysis of those patients completing the study showed that the volume and number of enhancing lesions were reduced at a dose of 5 mg. There was also a regulatory type 2 T helper-cell response to altered peptide ligand that cross-reacted with the native peptide.

Warfarin use in hemodialysis patients: what is the risk?

BACKGROUND: There is a paucity of data concerning the risks associated with warfarin in hemodialysis (HD) patients. We compared major bleeding episodes in this group with HD patients not receiving warfarin and with a cohort of non-HD patients receiving warfarin. METHODS: A retrospective review of 141 HD patients on warfarin (HDW), 704 HD patients not on warfarin (HDNW) and 3,266 non-dialysis warfarin patients (NDW) was performed. Hospital admissions for hemorrhagic events and ischemic strokes were examined as was hospital length of stay and blood product use. INR variability was also assessed. RESULTS: The incidence rates for major hemorrhage per 100 patient years was 10.8 in the HDW group as compared to 8.0 in the HDNW (p = 0.593) and 2.1 in the NDW (p < 0.001) groups. Mean units of red blood cell transfusions required was higher in patients on dialysis with no significant difference between HDW and HDNW groups. The risk of ischemic stroke per 100 patient years was 1.7 in the HDW group as compared to 0.7 in the HDNW groups (p = 0.636) and 0.4 in the NDW (p = 0.003). The HDW group had higher inter-measurement INR variability compared to the NDW group (p = 0.034). In patients with atrial fibrillation, HDW group had a higher incidence of ischemic stroke than the NDW group (2.2 versus 0.4 events per 100 patient years; p = 0.024). CONCLUSIONS: This study confirms the higher bleeding risk associated with HD/ESRD but suggests that warfarin use in these patients may not add significantly to this risk. We also demonstrated high rates of ischemic stroke in HD patients despite warfarin use. SUMMARY: Our study compares the frequency of major hemorrhage and secondarily, ischemic stroke in HD patients receiving or not receiving warfarin, with non-HD patients receiving warfarin. The major finding was that frequency of hemorrhage was higher in HD patients receiving warfarin than in non-HD patients receiving warfarin, but not different in HD patients with or without warfarin. A secondary finding was that INR variability was significantly higher in HD patients than non-HD patients on warfarin.

Induction of tolerance to 1-fluoro-2,4-dinitrobenzene contact sensitivity with hapten-modified lymphoid cells. II. Selective tolerance in F1 mice of T cell subsets recognizing 1-fluoro-2,4-dinitrobenzene associated with parental major histocompatibility complex antigens.

F1 animals were tolerized to 1-fluoro-2,4-dinitrobenzene (DNFB) contact sensitivity with parentally derived, in vitro hapten-modified spleen cells. This tolerant state was found, upon adoptive transfer to naive parental strain recipients, to affect only that T cell subpopulation that recognized the parental haplotype of the cell used as the tolerogen, and did not inhibit the ability of the remaining T cell subset to confer immunity. This demonstrates that this tolerant state involves the inactivation of a cell required for the expression of contact sensitivity by recognizing DNFB in association with self major histocompatibility complex gene products.

Purification to homogeneity of B cell stimulating factor. A molecule that stimulates proliferation of multiple lymphokine-dependent cell lines.

Murine B cell stimulating factor 1 (BSF-1) was purified to homogeneity from supernatants of a stimulated thymoma cell line. A protein of 18.4 kD with a unique N-terminal amino acid sequence was identified. BSF-1 had a sp act of at least 3.28 X 10(8) U/mg. In addition to its B cell-stimulatory activity, BSF-1 also stimulated the proliferation of several IL-2- and IL-3-dependent cell lines. We conclude that BSF-1 is both a growth factor and a differentiation factor. Finally, these results also suggest additional biologic properties of BSF-1 on lineages besides B lymphocytes.

Human interleukin 1. Purification to homogeneity.

We have purified human interleukin 1 (IL-1) to homogeneity by a simplified procedure that results in excellent yields of pure material that retains a high level of biological activity. IL-1, secreted by human peripheral blood macrophages that have been stimulated with Staphylococcus aureus, was purified by ion exchange chromatography and affinity chromatography on Procion Red agarose. The pure protein has a specific activity of 3.2 X 10(8) U/mg in the thymocyte mitogenesis assay, and is pyrogenic. No molecular weight heterogeneity was observed, in contrast to findings for mouse IL-1 and earlier reports of human IL-1. Purified IL-1, as analyzed by two-dimensional electrophoresis/electrofocusing gels, exhibited a series of charged species with isoelectric points ranging from 6.0 to 4.9, all with a molecular weight of approximately 17,500. Amino acid analysis indicated an abundance of acidic residues, in agreement with the low isoelectric points. There is little or no cysteine in the molecule. No evidence was found for the presence of carbohydrate moieties. The overall yield for this procedure was approximately 31% of the activity contained in the initial culture supernatant.

Recombinant granulocyte/macrophage colony-stimulating factor activates macrophages to inhibit Trypanosoma cruzi and release hydrogen peroxide. Comparison with interferon gamma.

Recombinant granulocyte/macrophage colony-stimulating factors (rGM-CSF) of mouse and human origins activated macrophages of the homologous species to inhibit the replication of the protozoan parasite T. cruzi. Activation could be induced with 10-100 ng/ml of rMu-GM-CSF, whether it was added before or after uptake of the parasite, in either adherent or suspension cultures. However, the degree of inhibition of parasite replication after exposure to rMu-GM-CSF was not as great as after treatment with rMu-IFN-gamma, and much more rMu-GM-CSF than rMu-IFN-gamma was required to achieve an equivalent antimicrobial effect. These results were mirrored by effects of the cytokines on enhancement of H2O2-releasing capacity in resident mouse peritoneal macrophages. In the latter tests, rMu-IFN-gamma and rHu-TNF-alpha afforded a 44-51-fold enhancement over the untreated control, with a 50% effective concentration (EC50) for rMu-IFN-gamma of approximately 0.05 ng/ml. Using rMu-GM-CSF or rM-CSF, enhancement of H2O2-releasing capacity was 14-15-fold over control, with EC50s of 1 and 14 ng/ml, respectively. However, peak enhancement of macrophage H2O2-releasing capacity was seen at least 24 h earlier with rMu-GM-CSF or rHu-M-CSF than with r-Mu-IFN-gamma or rHu-TNF-alpha. Thus, rMu-GM-CSF and rHu-GM-CSF displayed clear-cut macrophage-activating activity in vitro, but rMu-GM-CSF was less potent and less effective than rMu-IFN-gamma in the tests used.

Detection and characterization of high affinity plasma membrane receptors for human interleukin 1.

Interleukin 1 (IL-1) is a polypeptide hormone that acts as a central mediator of inflammation. Since IL-1 action is presumably mediated by specific cell surface receptor(s), we have characterized the binding of this hormone to cells. Purified human IL-1 was labeled to high specific activity with 125I, using Bolton-Hunter reagent. The labeled protein binds specifically to LBRM-33-1A5 (a murine T lymphoma line previously shown to produce IL-2 in response to phytohemagglutinin and IL-1) with an affinity of approximately 0.2-2 X 10(10)/M and, at saturation, to approximately 500 receptors per cell, on intact cells at 8 degrees C in the presence of sodium azide. The affinity of unmodified IL-1 for the murine plasma membrane receptor is 0.9-2 X 10(10)/M, as measured by the inhibition of 125I-IL-1 binding. The murine receptor specificity has been confirmed by demonstrating that, among a series of 12 polypeptide hormones, only IL-1 inhibits 125I-IL-1 binding to LBRM-33-1A5 cells. Treatment of surface-bound 125I-IL-1 with bivalent water-soluble crosslinkers identified a membrane polypeptide of Mr 79,500 to which IL-1 is crosslinked. A variety of cell types have been surveyed for the capacity to bind 125I-IL-1 specifically. The presence of specific binding correlates with the capacity of the cells tested to respond to IL-1. Our results indicate that the biological effects of the polypeptide hormone IL-1 are mediated by high affinity plasma membrane receptors. The identification of these receptors should provide valuable insight into the apparently diverse biological activities of IL-1.

Microbial epitopes act as altered peptide ligands to prevent experimental autoimmune encephalomyelitis.

Molecular mimicry refers to structural homologies between a self-protein and a microbial protein. A major epitope of myelin basic protein (MBP), p87-99 (VHFFKNIVTPRTP), induces experimental autoimmune encephalomyelitis (EAE). VHFFK contains the major residues for binding of this self-molecule to T cell receptor (TCR) and to the major histocompatibility complex. Peptides from papilloma virus strains containing the motif VHFFK induce EAE. A peptide from human papilloma virus type 40 (HPV 40) containing VHFFR, and one from HPV 32 containing VHFFH, prevented EAE. A sequence from Bacillus subtilis (RKVVTDFFKNIPQRI) also prevented EAE. T cell lines, producing IL-4 and specific for these microbial peptides, suppressed EAE. Thus, microbial peptides, differing from the core motif of the self-antigen, MBPp87-99, function as altered peptide ligands, and behave as TCR antagonists, in the modulation of autoimmune disease.

Analysis of waiting times on Irish renal transplant list.

INTRODUCTION: A number of recipient variables have been identified which influence waiting list times for a renal allograft. The aim of this study was to evaluate these factors in the Irish population. METHODS: We examined patients accepted onto the transplant list from January 1, 2000 until December 31, 2005. Inclusion criteria were adults listed for kidney only, deceased donor transplants. We included patients previously transplanted. Patients were censored, but still included in the analysis, if they died while on the list, permanently withdrew from the list or if they were not transplanted at the time of the study. RESULTS: There were a total of 984 patients accepted onto the waiting list during the study period, of which 745 of these were transplanted. Factors significantly associated with longer waiting times included age above 50 yr, blood group O and high peak panel reactive antibodies level. Gender and patient body mass index were not associated with longer waiting times. CONCLUSION: We have identified factors associated with a longer waiting time on the Irish cadaveric renal transplant list. This information can help our patients make informed decisions regarding likely waiting times and the merits of living related transplantation.

Encapsulating peritoneal sclerosis: experience of a tertiary referral center.

AIM: Encapsulating peritoneal sclerosis (EPS) is arguably the most serious complication of chronic peritoneal dialysis (PD) therapy with extremely high mortality rates. We aimed to establish the rates of EPS and factors associated with its development in a single center. METHODS: We retrospectively reviewed the records of all our PD patients from 1 January 1989 until 31 December 2008. All suspected cases were confirmed at laparotomy. Multifactorial models adjusted for potentially confounding variables such as age and sex. RESULTS: Eleven cases of EPS were identified giving a prevalence rate of 1.98%. Median duration on PD was substantially longer in affected versus unaffected patients (42.5 months versus 13.8 months; p = 0.0002). EPS patients had experienced a mean of 3.54 previous cases of peritonitis (1 infection per year versus 0.71 per year in unaffected patients; p = 0.075). Six patients died (54.5%) due to intra-abdominal sepsis including all five who presented with small bowel obstruction. Three patients had an omentectomy and adhesiolysis performed with a successful outcome. CONCLUSION: Our study reinforces the link between duration on PD and EPS. While mortality was high in our cohort, emerging surgical techniques demonstrate a favorable outcome that can be achieved even in severely affected cases.

Nephrogenic systemic fibrosis.

Nephroaenic systemic fibrosis (NSF) is a potentiallv fatal dermatiological condition found exclusively in patients with advanced renal I failure. There is minimal literature regarding the epidemiology and outcomes of patients with NSF in Ireland. A retrospective chart review was performed for all patients with NSF in Ireland. Ireland's experience with the disease was examined in light of international reports. There have been three cases of NSF in Ireland; an area which serves 1915 dialysis patients--giving a point prevalence among Irish end-stage kidney disease patients of 0.002. There was a large variation in disease severity between the three patients. All three patients had significant exposure to gadolinium chelate. Caution with gadolinium administration must be exercised in patients with advanced renal failure.

Serological visualization of interleukin 2.

Interleukin 2, a lymphokine that acts as a second signal of cellular immune response by way of its action as a T-cell growth factor, was morphologically identified by immunoperoxidase staining. With the use of a monoclonal antibody to interleukin 2 and several complex-forming antisera, the lymphokine was readily distinguished in cytocentrifuge preparations of peripheral blood leukocytes stimulated with a T-cell mitogen. When preparations of cloned interleukin 2 producer and responder cells were stained by the same procedures, discrete patterns of both responder and producer cell phenotypes were revealed. Interleukin 2 producer T cells exhibited a characteristic intense, ringlike cytoplasmic staining, whereas the responder cells (as exemplified by interleukin 2-dependent cell lines) exhibited a less intensive, spotlike membrane staining. In addition, intense membrane localization of interleukin 2, reminiscent of potential capping phenomena, could be observed in stained preparations of cloned responder cells.