NLRP3 inflammasome activation drives bystander cone photoreceptor cell death in a P23H rhodopsin model of retinal degeneration.

The molecular signaling leading to cell death in hereditary neurological diseases such as retinal degeneration is incompletely understood. Previous neuroprotective studies have focused on apoptotic pathways; however, incomplete suppression of cell death with apoptosis inhibitors suggests that other mechanisms are at play. Here, we report that different signaling pathways are activated in rod and cone photoreceptors in the P23H rhodopsin mutant rat, a model representing one of the commonest forms of retinal degeneration. Up-regulation of the RIP1/RIP3/DRP1 axis and markedly improved survival with necrostatin-1 treatment highlighted necroptosis as a major cell-death pathway in degenerating rod photoreceptors. Conversely, up-regulation of NLRP3 and caspase-1, expression of mature IL-1ß and IL-18 and improved cell survival with N-acetylcysteine treatment suggested that inflammasome activation and pyroptosis was the major cause of cone cell death. This was confirmed by generation of the P23H mutation on an Nlrp3-deficient background, which preserved cone viability. Furthermore, Brilliant Blue G treatment inhibited inflammasome activation, indicating that the 'bystander cell death' phenomenon was mediated through the P2RX7 cell-surface receptor. Here, we identify a new pathway in cones for bystander cell death, a phenomenon important in development and disease in many biological systems. In other retinal degeneration models different cell-death pathways are activated, which suggests that the particular pathways that are triggered are to some extent genotype-specific. This also implies that neuroprotective strategies to limit retinal degeneration need to be customized; thus, different combinations of inhibitors will be needed to target the specific pathways in any given disease.

An ex vivo gene therapy approach in X-linked retinoschisis.

PURPOSE: X-linked retinoschisis (XLRS) is juvenile-onset macular degeneration caused by haploinsufficiency of the extracellular cell adhesion protein retinoschisin (RS1). RS1 mutations can lead to either a non-functional protein or the absence of protein secretion, and it has been established that extracellular deficiency of RS1 is the underlying cause of the phenotype. Therefore, we hypothesized that an ex vivo gene therapy strategy could be used to deliver sufficient extracellular RS1 to reverse the phenotype seen in XLRS. Here, we used adipose-derived, syngeneic mesenchymal stem cells (MSCs) that were genetically modified to secrete human RS1 and then delivered these cells by intravitreal injection to the retina of the Rs1h knockout mouse model of XLRS. METHODS: MSCs were electroporated with two transgene expression systems (cytomegalovirus (CMV)-controlled constitutive and doxycycline-induced Tet-On controlled inducible), both driving expression of human RS1 cDNA. The stably transfected cells, using either constitutive mesenchymal stem cell (MSC) or inducible MSC cassettes, were assayed for their RS1 secretion profile. For single injection studies, 100,000 genetically modified MSCs were injected into the vitreous cavity of the Rs1h knockout mouse eye at P21, and data were recorded at 2, 4, and 8 weeks post-injection. The control groups received either unmodified MSCs or vehicle injection. For the multiple injection studies, the mice received intravitreal MSC injections at P21, P60, and P90 with data collection at P120. For the single- and multiple-injection studies, the outcomes were measured with electroretinography, optokinetic tracking responses (OKT), histology, and immunohistochemistry. RESULTS: Two lines of genetically modified MSCs were established and found to secrete RS1 at a rate of 8 ng/million cells/day. Following intravitreal injection, RS1-expressing MSCs were found mainly in the inner retinal layers. Two weeks after a single injection of MSCs, the area of the schisis cavities was reduced by 65% with constitutive MSCs and by 83% with inducible MSCs, demonstrating improved inner nuclear layer architecture. This benefit was maintained up to 8 weeks post-injection and corresponded to a significant improvement in the electroretinogram (ERG) b-/a-wave ratio at 8 weeks (2.6 inducible MSCs; 1.4 untreated eyes, p<0.05). At 4 months after multiple injections, the schisis cavity areas were reduced by 78% for inducible MSCs and constitutive MSCs, more photoreceptor nuclei were present (700/µm constitutive MSC; 750/µm inducible MSC; 383/µm untreated), and the ERG b-wave was significantly improved (threefold higher with constitutive MSCs and twofold higher with inducible MSCs) compared to the untreated control group. CONCLUSIONS: These results establish that extracellular delivery of RS1 rescues the structural and functional deficits in the Rs1h knockout mouse model and that this ex vivo gene therapy approach can inhibit progression of disease. This proof-of-principle work suggests that other inherited retinal degenerations caused by a deficiency of extracellular matrix proteins could be targeted by this strategy.

Glycaemic control and lipid concentrations in a cohort of people with diabetes over 7 years of follow-up: a regional audit of diabetes care in the UK.

AIM: To determine changes in glycaemic control and lipids over time since the introduction of the Quality and Outcomes Framework. METHODS: In adults with diabetes (Hampshire, UK), HbA1c and lipid measurements were retrieved from a regional National Health Service biochemical database in 2006 and 2013 and analysed using anova and logistic regression modelling. RESULTS: In 2006, 8568 people with diabetes were identified. In 2013, 5815 had follow-up data, 1207 people were lost to follow-up and 1546 had died. At baseline, HbA1c concentrations were 62.1 ± 16.1, 64.7 ± 16.7 and 64.5 ± 17.6 mmol/mol for those with follow-up data, those lost to follow-up and those who died, respectively. The mean age was 60.2 ± 14.5, 57.6 ± 18.0 and 73.9 ± 10.5 years, respectively, for the three groups. Total cholesterol, HDL cholesterol and triglyceride concentrations were similar between groups. The mean HbA1c concentration for those with complete follow-up data was 62.1 ± 16.1 mmol/mol in 2006 and 61.7 ± 17.3 mmol/mol in 2013. Quality and Outcomes Framework targets for cholesterol (< 5 mmol/l) were achieved by 79% of people in 2006 and 83% in 2013 (P < 0.001). Baseline age and HbA1c were associated with death at follow-up: the odds ratio per year increase in baseline age was 1.10 (95% CI 1.09-1.10; P < 0.001) and per unit increase in HbA1c it was 1.02 (95% CI 1.02-1.03; P < 0.001). CONCLUSIONS: Glycaemic control showed remarkable stability over 7 years of follow-up, despite increasing patient age and duration of diabetes. More patients achieved lipid targets in 2013 than in 2006. Although baseline HbA1c was a predictor of death at follow-up, baseline HbA1c differed little between survivors, non-survivors and those lost to follow-up.

Infection risk factors associated with seropositivity for Toxoplasma gondii in a population-based study in the Central Region, Ghana.

About 20-90% of the world's population has had contact with Toxoplasma gondii parasites. The aim of this study was to determine the seroprevalence and risk factors associated with T. gondii infection in the Central Region, Ghana. A community-based cross-sectional study was conducted in three selected communities. Serum samples were tested for the presence of anti-T. gondii IgG and IgM antibodies by ELISA. A serological criterion for seropositivity was a positive test result for any of the two anti-Toxoplasma IgG or IgM antibodies or a combination of both. In all, 390 participants of mean age 47.0 years consisting of 118 (30.%) males and 272 (69.7%) females were tested. The overall seroprevalence of T. gondii was 85% (333/390) where fishermen, farmers and fishmongers, respectively, had the highest seropositivity. IgG and IgM antibodies were detected in 329 (84%) and 25 (6%), respectively, while both IgG and IgM antibodies were detected in 21 (5%) of the participants. Respectively, 1% (4/390) and 79% (308/390) of participants tested positive for IgM-only and IgG-only antibodies. There was a significant relationship between Toxoplasma seropositivity and contact with soil, presence of a cat in the surrounding area, age, sources of drinking water, level of formal education, and socioeconomic status. The results suggest that the seashore may serve as a good ground for sporulation and survival of Toxoplasma oocysts.

Late effects of childhood cancer treatment: severe hypertriglyceridaemia, central obesity, non alcoholic fatty liver disease and diabetes as complications of childhood total body irradiation.

BACKGROUND: Childhood cancer survivors may develop a number of endocrine complications linked to organ failure, such as hypogonadism, diabetes and growth hormone deficiency. However, increasing evidence now suggests that total body irradiation treatment, specifically, is linked with future risk of insulin resistance, hepatic steatosis and dyslipidaemia, possibly because total body irradiation affects adipocyte differentiation and impairs subcutaneous adipose tissue depot expansion during times of positive energy balance. CASE REPORT: We describe a 20-year-old woman who developed pancreatitis with severe hypertriglyceridaemia (serum triglycerides > 300 mmol/l) that required plasmapheresis. She had received total body irradiation prior to her bone marrow transplant at age 6 years for relapsed acute lymphoblastic leukaemia. She developed ovarian failure at age 12 years. At age 15 years she was noted to have hyperglycaemia, increased blood pressure, hepatic steatosis and mild hypertriglyceridaemia. She presented with severe hypertriglyceridaemia and eruptive xanthoma, and developed pancreatitis 12 h after admission. She was treated with plasmapheresis and intravenous insulin and made an excellent recovery. We implicate and discuss total body irradiation as the major contributing factor to her severe hypertriglyceridaemia, compounded by worsening glycaemic control, oestrogen deficiency and a changing adult lifestyle. CONCLUSION: Children who have received total body irradiation are at risk of diabetes and an exaggerated form of the metabolic syndrome with hypertriglyceridaemia, which can be life-threatening. We suggest that survivors of total body irradiation treatment require careful lifelong monitoring of their metabolic status.

Improvements in glycaemic control and cholesterol concentrations associated with the Quality and Outcomes Framework: a regional 2-year audit of diabetes care in the UK.

AIMS: To determine whether there have been changes in glycaemic control and lipids in a cohort of people with repeated glycated haemoglobin (HbA(1c)) measurements since the implementation of the Quality and Outcomes Framework (QOF) for diabetes care. METHODS: Retrospective retrieval of computer-held biochemical measurements of glycaemic control (HbA(1c)) and lipid profiles in adults in Hampshire, UK between 2006 and 2008. Routine data on age, sex, HbA(1c) and plasma lipids were available on an NHS database on 8997 adults with data available for HbA(1c) in both 2006 and 2008. RESULTS: In 2006, 39.7% of adults had glycaemic control within the QOF threshold (HbA(1c) < 7.5%); by 2008, this proportion had risen to 52.1% (P < 0.001). In 2006, 11.8% of subjects had poor glycaemic control (HbA(1c) > 10.0%); by 2008, this proportion had decreased to 10.1% (P < 0.001). The proportion of subjects achieving HbA(1c) and cholesterol targets (both HbA(1c) < 7.5% and total cholesterol < or = 5.0 mmol/l) was 30.2% in 2006; in 2008, this proportion had increased to 43.7% (P < 0.001). Individuals with poorer glycaemic control (HbA(1c) > 10.0%) were younger and had higher cholesterol concentrations than people with good (HbA(1c) < 7.5%) or moderate (HbA(1c) 7.5-10.0%) glycaemic control (P value for trend, both P < 0.001). CONCLUSION: Since the introduction of performance indicators for primary care and the incorporation of pay for performance in 2004, there has been marked improvement in the management of hyperglycaemia and hypercholesterolaemia among people with diabetes with data available in 2006 and 2008. It remains to be seen whether the new HbA(1c) audit target (HbA(1c) < 7.0%) introduced in 2009 will result in a further improvement in glycaemic control.

Intralesional application of medical grade honey improves healing of surgically treated lacerations in horses.

BACKGROUND: Infection and dehiscence of simple lacerations is common in horses, and consistently effective methods of prevention are yet to be found. Honey has been shown to promote wound healing when applied topically; however, intralesional application prior to wound closure has not been reported. OBJECTIVES: To examine whether intralesional application of medical grade honey (MGH) would reduce the incidence of infection and dehiscence following wound closure. STUDY DESIGN: Prospective, open-label randomised block design clinical study. METHODS: Lacerations, treated by field practitioners, were divided into treatment and control groups using block randomisation. Horses in the treatment group received a single intralesional treatment with l-mesitran gel (MGH). Data were collected at the time of wound closure and at suture removal. RESULTS: Data from 127 horses were included, 69 MGH-treated and 58 control cases. No adverse effects of the MGH were recorded. MGH-treated horses were more likely to completely heal (P = 0.006, odds ratio [OR] 3.40 95% confidence interval [CI] 1.41-8.20), to have no signs of infection (P = 0.007, OR 3.64, CI 1.42-9.26) and for the veterinarians to report some degree of satisfaction (P = 0.04, OR 2.72, CI 1.05-7.09) compared to control cases. Numbers needed to treat for complete healing was 5.1 (CI 2.8-40). MAIN LIMITATIONS: Clinical studies have inherent flaws compared to wound healing models, because of variability between wounds. There were more horses with limb injuries in the control group, although not statistically significant, this may have biased the results. Clinical satisfaction and signs of infection were subjective evaluations and evaluators were not blinded to the treatment group. CONCLUSIONS: Intralesional application of MGH to lacerations prior to wound closure may be beneficial in preventing infection and dehiscence. Larger, blinded studies focusing on wounds at a specific location with more objective assessment should be pursued.

Drastic cutaneous multi-focal orf infection in goats, causing severe dysfunctioning.

A flock of 76 goats was struck by orf infection at the Al-Ahsa oasis in eastern Saudi Arabia. The morbidity rate was 89%, while the case fatality rate was 3%. The clinical signs were most severe in three adult goats, which survived, and in two kids, which died of the infection. The lesions were multi-focal and so serious that they prevented the infected goats from seeing, eating, lactating or walking. The virus was isolated in Vero cell culture and a scab suspension was used to experimentally infect susceptible goats. These newly infected animals suffered only mild disease but the orf virus was re-isolated and the goats seroconverted. To the best knowledge of the authors, these were the most drastic orf lesions seen during the last 17 years in goats in Saudi Arabia.

The Construction of the Concepts "Democracy" and "Republic" in Arabic in the Eastern and Southern Mediterranean, 1798-1878.

This article illuminates the construction of the concepts "democracy" and "republic" in the Arabic-speaking regions of the eastern and southern Mediterranean between 1798 and 1878. Examining these ideas through conceptual analysis on two levels, language construction and political discourse, the article reveals the layers these concepts acquired and their reception in the context of state reforms in the Ottoman Empire. While both "democracy" and "republic" evolved in Arabic after the French Revolution and acquired their modern morphological forms and content primarily between the 1820s and 1876, "republic" came into use and was perceived as relevant to local circumstances earlier than "democracy."

The localization of androgen receptors in human bone.

Androgens have important effects on the human skeleton, and deficiency has been associated with bone loss in both males and females. The skeletal actions of androgens may be mediated directly via the androgen receptor (AR) or indirectly via the estrogen receptor after aromatization to estrogens. The presence of androgen receptors has been demonstrated in bone cells and chondrocytes in vitro, but their presence in human bone in situ has not been reported. In order to provide further evidence for a direct action of androgens on bone via androgen receptors, we have used specific monoclonal antibodies to investigate the expression of human AR in normal developing and osteophytic bone of both sexes. In the growth plates from the developing bone, androgen receptors were predominantly expressed in hypertrophic chondrocytes and in osteoblasts at sites of bone formation. They were also observed in osteocytes in the bone, and in mononuclear cells and endothelial cells of blood vessels within the bone marrow. In the osteophytes, androgen receptors were widely distributed at sites of endochondral ossification in proliferating, mature, and hypertrophic chondrocytes and at sites of bone remodeling in osteoblasts. They were also expressed in osteocytes and mononuclear cells within the bone marrow. The pattern and number of cells expressing the receptor was similar in both sexes. Our results show for the first time the presence and distribution of androgen receptors in normal developing human and osteophytic bone in situ and further provide evidence for a direct action of androgens on bone and cartilage cells.

The localization of the functional glucocorticoid receptor alpha in human bone.

Glucocorticoids have well-documented effects on the skeleton, although their mechanism of action is still poorly understood. The actions of glucocorticoids on bone cells are mediated, in part, directly via specific receptors. The presence of these receptors has been demonstrated in both rodent and human osteoblastic cells in vitro, but their presence in human bone in vivo has not been reported. In this study, we have used specific affinity purified polyclonal antibodies to the functional glucocorticoid receptor alpha (GRalpha) to investigate its expression in both developing and adult human bone using sections of neonatal rib, calvarial, and vertebral bones, tibial growth plates from adolescents, and iliac crest biopsies from adults who were to undergo liver transplantation. In the tibial growth plates, GRalpha was predominantly expressed in the hypertrophic chondrocytes within the cartilage. In the primary spongiosa, the receptor was highly expressed by osteoblasts at sites of bone modeling. Within the bone marrow, receptors were also detected in mononuclear cells and in endothelial cells of blood vessels. In the neonatal rib and vertebrae, GRalpha was widely distributed at sites of endochondral bone formation in resting, proliferating, mature, and hypertrophic chondrocytes. They were also highly expressed in osteoblasts at sites of bone modeling. At sites of intramembranous ossification in neonatal calvarial bone and rib periosteum, GRa was widely expressed in cells within the fibrous tissue and in osteoblasts at both the bone-forming surface and at modeling sites. In the iliac crests from adults, GRalpha was predominantly expressed in osteocytes. The receptors were not detected in osteoclasts. Our results show for the first time the presence of the functional GRalpha in human bone in situ and suggest that the actions of glucocorticoids on bone may be mediated, in part, directly via the GR at different stages of life. The absence of receptor expression in osteoclasts also suggests that the effects of glucocorticoids on bone resorption may be mediated indirectly.

The expression of thyroid hormone receptors in human bone.

The mechanism of action of thyroid hormones on bone is poorly understood. Thyroid hormones may act on bone cells either indirectly by increasing secretion of growth hormone (GH) and insulin-like growth factor-1 (IGF-1), or directly by influencing target genes via specific nuclear receptors. The presence of thyroid hormone receptors (TRs) has been demonstrated in human and rodent osteoblast-like cells and cell lines and recently in osteoclasts derived from an osteoclastoma in vitro. However, their presence in human bone in situ has not been reported. We have used specific polyclonal antibodies to TR-alpha 1, -alpha 2, and -beta 1 to investigate the expression of these receptors in sections of human osteophytes and heterotopic bone. Osteoblasts and osteoclasts were identified by alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP), respectively, whereas chondrocytes were identified morphologically. At sites of endochondral and intramembranous bone formation, TR-beta 1 and the splice variant -alpha 2 were widely expressed by proliferating, mature, and hypertrophic chondrocytes and also in cells within the fibrous tissue and at the bone forming surfaces, respectively. They were also detected in osteoblasts, osteoclasts, and a few osteocytes at sites of bone remodeling. In contrast, TR-alpha 1 was the least expressed and was present mainly in osteoblasts at remodeling sites and in a few mature and undifferentiated chondrocytes. Our results show, for the first time, the presence and distribution of TRs in human bone in situ and suggest that the skeletal actions of thyroid hormones may be mediated via these receptors. Further studies are required to define the role of the individual receptor isoforms in bone metabolism.

The localization of thyroid hormone receptor mRNAs in human bone.

Thyroid hormones have well-documented effects on the skeleton although the mechanism of their action on bone is poorly understood. We have recently reported the presence of different thyroid hormone receptor isoforms in human bone. However, there is evidence to suggest that the expression of thyroid hormone receptor (TR) protein may not necessarily correlate with its mRNA. In this study, we used specific digoxigenin-labeled ribo probes to investigate the expression of TRalpha1, variant TRalpha2, TRbeta1, and in particular TRbeta2 mRNA in human osteophytic bone and osteoclastoma tissue in situ. The number of positive cells was expressed as the percentage of the total number of cells of the same phenotype. In osteophytes, at sites of endochondral ossification, TRalpha1, variant TRalpha2, TRbeta1, and TRbeta2 mRNA were widely distributed in undifferentiated, proliferating, mature and hypertrophic chondrocytes. At sites of bone remodeling, TRalpha1 mRNA was expressed in the majority (> 90%) of osteoblasts. TRbeta1 and the variant TR-alpha2 mRNA were moderately expressed in approximately 75% of cells with only a few osteoblasts (< 25%) expressing TRbeta2 mRNA. All the TR transcripts were highly expressed in multinucleated osteoclasts in osteoclastoma tissue. The distribution of TR mRNAs was similar to TR receptor protein expression (as we have previously reported) in both osteophytic bone and osteoclastoma tissue except TRalpha1 mRNA that was highly expressed in osteoclasts and in undifferentiated, proliferating, mature, and hypertrophic chondrocytes in contrast to its receptor protein expression. This study highlights the importance of studying both TR mRNA and receptor proteins in triiodothyronine (T3) responsive tissues. This is also the first demonstration of the presence of TRbeta2 mRNA in bone. The role of TRbeta2 in mediating the actions of thyroid hormones in bone is not known and requires further investigation.

Plasma urate in diabetes: relationship to glycaemia, glucose disposal, microvascular complications and the variations following oral glucose.

In order to provide further insights into the conflicting reports of associations between diabetes and uric acid metabolism, we studied 175 adult diabetic patients (56 IDDM, 119 NIDDM) and 114 matched control subjects. Plasma uric acid (PUA) concentrations were not significantly different between diabetic and control subjects, despite increased urinary urate in diabetic patients. There were no significant associations, in diabetic patients, between PUA and (i) type of diabetes, (ii) glycaemic control, (iii) retinopathy and (iv) proteinuria. Plasma urate did not correlate with the KG constant for glucose disposal rate during IVGTT, thus indicating that PUA may not be related to insulin action. In a separate study, PUA rose sharply, peaking at 30 min, and fell subsequently in both newly diagnosed NIDDM patients (n = 20) and subjects with impaired glucose tolerance (n = 15) in response to standard OGTT, in contrast to normal controls (n = 35) in whom PUA rose gradually to a peak at 120 min. Mean rise in PUA from baseline to peak was significant (P less than 0.05) in the diabetic group only. These differences in PUA response during an OGTT between subjects with abnormal glucose metabolism and normal controls may be a feature in the metabolic evolution of diabetes and need further investigation.

Indigenous knowledge system for treatment of trypanosomiasis in Kaduna state of Nigeria.

A survey was carried out in Kaduna State of Nigeria to establish the indigenous knowledge system for treating trypanosomiasis in domestic animals. Questionnaire and interviews were, respectively, administered to, or conducted with about 200 livestock farmers and traders spread around the state. Data obtained revealed the use of several plants either alone or in combination, for the treatment and management of trypasonomiasis. The most common plants encountered were Adansonia digitata, Terminalia avicennoides, Khaya senegalensis, Cissus populnea, Tamarindus indica, Lawsonia inermis, Boswellia dalzielli, Pseudocedrela kotschi, Syzyium quinensis, Sterculia setigera, Afzelia africana, Prosopis africana, Lancea kerstingii. The method of preparation and mode of administration of some of these plants in the treatment of trypanosomiasis are reviewed and discussed.

Targeting inflammation in emerging therapies for genetic retinal disease.

Genetic retinal diseases such as age-related macular degeneration and monogenic diseases such as retinitis pigmentosa account for some of the commonest causes of blindness in the developed world. Diverse genetic abnormalities and environmental causes have been implicated in triggering multiple pathological mechanisms such as oxidative stress, lipofuscin deposits, neovascularisation, and programmed cell death. In recent years, inflammation has also been highlighted although whether inflammatory mediators play a central role in pathogenesis or a more minor secondary role has yet to be established. Despite this, numerous interventional studies, particularly targeting the complement system, are underway with the promise of novel therapeutic strategies for these important blinding conditions.

Simultaneous detection of the two main proliferation driving EBV encoded proteins, EBNA-2 and LMP-1 in single B cells.

Epstein Barr virus (EBV) is carried by almost all adults, mostly without clinical manifestations. Latent virus infection of B lymphocytes induces activation and proliferation that can be demonstrated in vitro. In healthy individuals, generation of EBV induced malignant proliferation is avoided by continuous immunological surveillance. The proliferation inducing set of the virally encoded genes is expressed exclusively in B cells in a defined differentiation window. It comprises nine EBV encoded nuclear proteins, EBNA 1-6, and three cell membrane associated proteins, LMP-1, 2A and 2B, designated as latency Type III. Outside this window the expression of the viral genes is limited. Healthy carriers harbor a low number of B lymphocytes in which the viral genome is either silent or expresses one virally encoded protein, EBNA-1, latency Type I. In addition, EBV genome carrying B cells can lack either EBNA-2 or LMP-1, latency Type IIa or Type IIb respectively. These cells have no inherent proliferation capacity. Detection of both EBNA-2 and LMP-1 can identify B cells with growth potential. We devised therefore a method for their simultaneous detection in cytospin deposited cell populations. Simultaneous detection of EBNA-2 and LMP-1 was reported earlier in tissues derived from infectious mononucleosis (IM), postransplantation lymphoproliferative disorders (PTLD) and from "humanized" mice infected with EBV. We show for the first time the occurrence of Type IIa and Type IIb cells in cord blood lymphocyte populations infected with EBV in vitro. Further, we confirm the variation of EBNA-2 and LMP-1 expression in several Type III lines and that they vary independently in individual cells. We visualize that in Type III LCL, induced for plasmacytoid differentiation by IL-21 treatment, EBV protein expression changes to Type IIa (EBNA-2 negative LMP-1 positive). We also show that when the proliferation of EBV infected cord blood lymphocyte culture is inhibited by the immunomodulator, PSK, the majority of the cells express latency Type IIa pattern. These results show that by modifying the differentiation state, the proliferating EBV positive B cells can be "curbed". Type IIa expression is a characteristic for EBV positive Reed-Sternberg (R/S) cells in EBV positive Hodgkin's lymphomas. For survival and proliferation, the R/S cells require the contribution of the in vivo microenvironment. Consequently, Type IIa lines could not be established from Hodgkin's lymphoma in vitro. We propose that these experimental cultures can be exploited for study of the Type IIa cells.