Sustained tumour eradication after induced caspase-3 activation and synchronous tumour apoptosis requires an intact host immune response

Effective anticancer treatments often result in the induction of large amounts of tumour cell death. In vivo, such dying tumour cells are a potential source of antigens for T-cell stimulation. Although apoptosis is generally considered nonimmunogenic, recent evidence suggests that some anticancer therapies that induce apoptosis can elicit antitumour immune responses. Here, a doxycycline-inducible, constitutively active caspase-3 (‘death switch'') was constructed in a murine tumour model to explore the impact of the host immune response to rapid, synchronous and substantial tumour cell apoptosis. In vitro, up to 80% of tumour cells underwent apoptotic cell death within 24 h and death was accompanied by the release of potential ‘danger signal'' molecules HMGB1 and HSP90. In vivo, death switch induction provoked rapid, pronounced tumour regression in immune-competent and immune-deficient mice, but sustained tumour eradication was observed only in immune-competent mice. Moreover, the majority of mice that were tumour free after death switch induction were protected from further tumour rechallenge. In addition, long-term remission after induction of the death switch was completely abrogated following depletion of CD8 T cells. These data suggest that sustained tumour eradication after substantial tumour apoptosis requires an antitumour host immune response that prevents tumour relapse. In many patients, cancer therapies produce encouraging initial responses that are only short lived. These results provide new insights that may have important implications for further development of strategies that result in long-term tumour clearance after initially effective anticancer treatment.     

Glomerular Immune Deposits Are Predictive of Poor Long-Term Outcome in Patients with Adult Biopsy-Proven Minimal Change Disease: A Cohort Study in Korea

Background and Objectives

There has been little published information on risk factors for poor long-term outcome in adult biopsy-proven minimal change disease (MCD).

Methods

Data from sixty-three adult, biopsy-proven primary MCD patients treated at a tertiary university hospital between 2003 and 2013 were analyzed. Baseline clinical and pathologic factors were assessed for the associations with composite outcome of creatinine doubling, end stage renal disease, or all-cause mortality.

Results

During a median (interquartile) 5.0 (2.8–5.0) years, the composite outcome occurred in 11.1% (7/63) of patients. The rate of glomerular immune deposits was 23.8% (15/63). Patients with glomerular immune deposits showed a significantly lower urine protein creatinine ratio than those without deposits (P = 0.033). The rate of non-responders was significantly higher in patients with glomerular immune deposits than in those without deposits (P = 0.033). In patients with deposits, 26.7% (4/15) developed the composite outcome, while only 6.3% (3/48) developed the composite outcome among those without deposits (P = 0.049). In multivariate Cox proportional hazards regression analysis, the presence of glomerular immune deposits was the only factor associated with development of the composite outcome (hazard ratio: 2.310, 95% confidence interval: 1.031–98.579, P = 0.047).

Conclusion

Glomerular immune deposits were associated with increased risk of a composite outcome in adult MCD patients. The higher rate of non-responders in patients with deposits might be related to the poor outcome. Future study is needed.     

Prevalence,incidence and characteristics of the metabolic syndrome (MetS) in a cohort of Mexican Mestizo early rheumatoid arthritis patients treated with conventional disease modifying anti-rheumatic drugs: the complex relationship between MetS and disease activity

IntroductionA higher prevalence of metabolic syndrome (MetS) has been described in rheumatoid arthritis (RA), along with an association with disease activity. Objectives were to describe prevalence of MetS at RA diagnosis in a cohort of Mexican Mestizo early RA patients, and to define a causal association between MetS and disease activity.MethodsThe study population was a prospective cohort. At baseline and at fixed 6-months-intervals, patients had medical evaluations, fasting serum glucose, triglycerides, high-density lipoprotein cholesterol and acute reactant-phase determinations. MetS was defined according to international criteria and body mass index (BMI) ≥30 kg/m² was used as a surrogate of the waist circumference. The study was approved by the internal review board. Appropriated statistics and Cox regression analysis were used. All statistical tests were two-sided and evaluated at the 0.05 significance level.ResultsUp to March 2014, data from 160 patients were analyzed. At baseline, they were more frequently middle-aged females and had moderate to high disease activity. Prevalence of MetS varied from 11.3% to 17.5% in patients and was lower to that from matched controls (versus 26.3% to 30%, P ≤0.01).Up to last follow-up, 39 patients (34.5%) developed incidental MetS. In the Cox regression analysis, cumulative disease activity score (DAS) 28 (odds ratio (OR): 1.81, 95% confidence interval (CI): 1.346 to 2.433, P = 0.000) and baseline BMI (OR: 1.13, 96% CI: 1.035 to 1.236, P = 0.007) were the only predictors for incidental MetS.RA patients with incidental MetS accumulated more disease activity and had less frequent remission than their counterparts. Logistic regression analysis showed that incidental MetS (OR: 0.2, 95% CI: 0.01 to 0.99, P = 0.052) and baseline DAS28 (OR: 0.4, 95% CI: 0.2 to 0.9, P = 0.02) were the only predictors for achieving or maintaining sustained (≥6 months) remission.ConclusionsMetS prevalence in a cohort of early RA patients was lower than that from matched controls. Cumulative disease activity and higher BMI were risk factors for incidental Mets; higher baseline disease activity and incidental MetS prevented sustained remission. In addition to disease activity, MetS needs to be controlled to impact disease outcomes.     

Osteoclasts are active in bone forming metastases of prostate cancer patients

Background

Bone forming metastases are a common and disabling consequence of prostate cancer (CaP). The potential role of osteoclast activity in CaP bone metastases is not completely explained. In this study, we investigated ex vivo whether the osteolytic activity is present and how it is ruled in CaP patients with bone forming metastases.

Methodology

Forty-six patients affected by newly diagnosed CaP and healthy controls were enrolled. At diagnosis, 37 patients had a primary tumour only, while 9 had primary tumour and concomitant bone forming metastases. In all patients there was no evidence of metastasis to other non-bone sites. For all patients and controls we collected blood and urinary samples. We evaluated patients'' bone homeostasis; we made peripheral blood mononuclear cell (PBMC) cultures to detect in vitro osteoclastogenesis; we dosed serum expression of molecules involved in cancer induced osteoclatogenesis, such as RANKL, OPG, TNF-alpha, DKK-1 and IL-7. By Real-Time PCR, we quantified DKK-1 and IL-7 gene expression on micro-dissected tumour and healthy tissue sections.

Principal Findings

CaP bone metastatic patients showed bone metabolism disruption with increased bone resorption and formation compared to non-bone metastatic patients and healthy controls. The CaP PBMC cultures showed an enhanced osteoclastogenesis in bone metastatic patients, due to an increase of RANKL/OPG ratio. We detected increased DKK-1 serum levels and tissue gene expression in patients compared to controls. IL-7 resulted high in patients'' sera, but its tissue gene expression was comparable in patients and controls.

Conclusions

We demonstrated ex vivo that osteoclastogenesis is an active mechanism in tumour nesting of bone forming metastatic cancer and that serum DKK-1 levels are increased in CaP patients, suggesting to deeply investigate its role as tumour marker.     

Pharmacological removal of serum amyloid P component from intracerebral plaques and cerebrovascular Aβ amyloid deposits in vivo

Human amyloid deposits always contain the normal plasma protein serum amyloid P component (SAP), owing to its avid but reversible binding to all amyloid fibrils, including the amyloid β (Aβ) fibrils in the cerebral parenchyma plaques and cerebrovascular amyloid deposits of Alzheimer''s disease (AD) and cerebral amyloid angiopathy (CAA). SAP promotes amyloid fibril formation in vitro, contributes to persistence of amyloid in vivo and is also itself directly toxic to cerebral neurons. We therefore developed (R)-1-[6-[(R)-2-carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylic acid (CPHPC), a drug that removes SAP from the blood, and thereby also from the cerebrospinal fluid (CSF), in patients with AD. Here we report that, after introduction of transgenic human SAP expression in the TASTPM double transgenic mouse model of AD, all the amyloid deposits contained human SAP. Depletion of circulating human SAP by CPHPC administration in these mice removed all detectable human SAP from both the intracerebral and cerebrovascular amyloid. The demonstration that removal of SAP from the blood and CSF also removes it from these amyloid deposits crucially validates the strategy of the forthcoming ‘Depletion of serum amyloid P component in Alzheimer''s disease (DESPIAD)’ clinical trial of CPHPC. The results also strongly support clinical testing of CPHPC in patients with CAA.     

Impact of MIF Gene Promoter Polymorphism on F508del Cystic Fibrosis Patients

Macrophage migration Inhibitory Factor (MIF) is a pro-inflammatory cytokine sustaining the acute response to gram–negative bacteria and a regulatory role for MIF in Cystic Fibrosis has been suggested by the presence of a functional, polymorphic, four-nucleotide repeat in this gene''s promoter at position −794, with the 5-repeat allele displaying lower promoter activity. We aimed at assessing the association of this polymorphism with disease severity in a group of Cystic Fibrosis patients homozygous for F508del CFTR gene mutation. Genotype frequencies were determined in 189 Cystic Fibrosis and 134 control subjects; key clinical features of patients were recorded and compared among homozygous 5-allele patients and the other MIF genotypes. Patients homozygous for the 5-repeat allele of MIF promoter displayed a slower rate of lung function decline (p = 0.027) at multivariate survival analysis. Multiple regression analysis on age-normalized respiratory volume showed no association of the homozygous 5-repeat genotype with lung function under stable conditions and no correlation with P.aeruginosa chronic colonization. Therefore, only the Homozygous 5-repeat genotype at MIF −794 is associated with milder disease in F508del Cystic Fibrosis patients.     

Measurement of tumour volume by MRI to evaluate risk of pelvic nodal metastases in early cervical carcinoma patients

BackgroundApart from the FIGO staging system there are several other factors, including tumour volume and lymph node status, which considerably influence local tumour control and survival of cervical carcinoma patients.AimThe study aimed to determine the prognostic value of cervical tumour volume measured on the basis of MRI in terms of pelvic nodal metastases prediction in early cervical carcinoma patients.MethodsThe records of 49 early stage cervical carcinoma patients treated with preoperative brachytherapy and radical hysterectomy were analyzed. All patients underwent diagnostic MRI, which was the basis for tumour volume calculations as well as the evaluation of pelvic lymph nodes status and parametrial invasion. In each case the postoperative pathological diagnosis was obtained. The correlation between the occurrence of nodal metastases and such variables as tumour histology, grade and tumour volume, FIGOMRI stage IIB, and patients' age was evaluated. Logistic regression analysis was employed to determine correlations between tumour volume and histological pelvic nodal involvement.ResultsA statistically significant correlation between pelvic lymph node involvement and such parameters as tumour volume and parametrial invasion was proven. The probability of lymph node metastasis is 20% for tumour volume of 17 cm³ and increases up to 50% for tumour volume of 40 cm³. An increase of tumour volume by 1 cm³ increased the risk of lymph node disease by 6.2%.ConclusionsThe study demonstrates that tumour volume may be considered a predicting factor in early cervical carcinoma patients, since it strongly correlates with pelvic lymph node histological status.     

Lymphocyte transformation in vitro to RII mouse milk antigen among woman with breast disease.

The cell-mediated immune responses of 110 women with benign or malignant breast disease were tested in in vitro lymphocyte transformation assay with an antigen preparation made from RIII mouse milk containing mammary tumor virus. About 50% of patients responded positively to the milk preparation. In contrast, 25% of normal women or women with other gynecological malignancies responded positively to the antigen (P = 0.015). The data demonstrate a similar response pattern among women with malignant or benign breast disease. In addition, a subpopulation of normal women with positive response to this antigen is clearly defined.     

7-Ketocholesterol-Induced Inflammation Signals Mostly through the TLR4 Receptor Both In Vitro and In Vivo

The cholesterol oxide 7-ketocholesterol (7KCh) has been implicated in numerous age-related diseases such as atherosclerosis, Alzheimer''s disease, Parkinson''s disease, cancer and age-related macular degeneration. It is formed by the autooxidation of cholesterol and especially cholesterol-fatty acid esters found in lipoprotein deposits. This molecule causes complex and potent inflammatory responses in vitro and in vivo. It is suspected of causing chronic inflammation in tissues exposed to oxidized lipoprotein deposits. In this study we have examined the inflammatory pathways activated by 7KCh both in cultured ARPE19 cells and in vivo using 7KCh-containing implants inserted into the anterior chamber of the rat eye. Our results indicate that 7KCh-induced inflammation is mediated mostly though the TLR4 receptor with some cross-activation of EGFR-related pathways. The majority of the cytokine inductions seem to signal via the TRIF/TRAM side of the TLR4 receptor. The MyD88/TIRAP side only significantly effects IL-1β inductions. The 7KCh-induced inflammation also seems to involve a robust ER stress response. However, this response does not seem to involve a calcium efflux-mediated UPR. Instead the ER stress response seems to be mediated by yet identified kinases activated through the TLR4 receptor. Some of the kinases identified are the RSKs which seem to mediate the cytokine inductions and the cell death pathway but do not seem to be involved in the ER stress response.     

Production of migration inhibitory factor in response to bacterial and fungal antigens in patients with untreated Graves' disease

Tests for the production of migration inhibitory factor by peripheral blood leukocytes in response to ubiquitous bacterial and fungal antigens were carried out in patients with untreated Graves'' disease and in healthy control subjects. Dose-response studies, tests for the production of this factor after 72 hours'' stimulation with phytohemagglutinin as a test for reserve, and tests before and after 24 hours'' preculture to deplete suppressor cells were also performed in some patients. The antigens used were Candida, Trichophyton-Oidiomyces-Epidermophyton, mumps live attenuated virus and purified protein derivative of tuberculin. The production of migration inhibitory factor was measured by the agarose microdroplet method.The production of migration inhibitory factor in response to all the antigens except mumps virus was slightly greater in the patients than in the control subjects, although the differences were not significant. The dose-response characteristics and the production of migration inhibitory factor after stimulation with phytohemagglutinin were similar in the two groups. The production of migration inhibitory factor in response to suboptimal concentrations of Candida, Trichophyton-Oidiomyces-Epidermophyton and mumps virus was not enhanced in either group after 24 hours'' preculture apart from a slight increase in response to mumps virus in the patients.These results fail to support the suggestion that patients with Graves'' disease have a deficiency of suppressor cells.     

Explaining the effects of socio-economic deprivation on survival in a national prospective cohort study of 1909 patients with head and neck cancers

Background: Socio-economic differences in survival from head and neck cancers are among the largest of any malignancies. Population-based data have been unable to explain these differences. Aims: To describe survival from head and neck cancers in a large cohort of patients for whom a range of socio-economic, demographic, behavioural and casemix data was available. Methods: Prospective cohort study using data from the Scottish Head and Neck Audit on all patients diagnosed with a head and neck cancer in Scotland between 1st September 1999 and 31st August 2001 linked to General Register Office for Scotland death records to 30th June 2006. Cox proportional hazards models were produced to describe adjusted hazards of death according to socio-economic circumstances, using validated area-based DEPCAT scores. Results: Data on 1909 patients were analysed. 71.0% were male and mean age was 64.3 (SD 12.2) years. Overall 5-year survival was 45.6% (95% CI: 43.4–47.8%). In order of strength of association in univariate regression, World Health Organisation Performance Status, disease stage, patient age, tumour site, smoking status, alcohol use, tumour differentiation, and deprivation were significant predictors of all-cause mortality but after multiple adjustment, deprivation was no longer an independent predictor of survival. Conclusions: Socio-economic differentials in survival from head and neck cancers are determined by a mixture of risk factors, some of which may be amenable to targeted earlier detection methods and lifestyle interventions. However, further research is needed to understand the impacts of performance status in more deprived patients.     

Wilms' tumour: adjuvant treatment with actinomycin D and vincristine.

In an unselected series of 49 children with Wilms'' tumour treated in 1969-74 the 5-year relapse-free survival and survival rates were 78% and 81%, respectively, whereas in the series of children treated in 1963-68 the corresponding rates were 49% and 70%. The significant improvement in the relapse-free survival rate was a result of adjuvant treatment with actinomycin D and vincristine (AMD + VCR), which, in some patients, eradicated occult metastatic disease. In the treatment of lung metastases the combination of whole-lung irradiation and maintained chemotherapy with AMD + VCR proved excessively toxic: in 5 of 11 patients acute diffuse pneumonitis developed, and it was fatal in 3. Adjuvant AMD + VCR therapy is advocated in all patients with Wilms'' tumour except children less than 12 months old with a tumour of moderate size, limited to the kidney and completely resectable.     

Deposition of IgM and complement at the dermoepidermal junction in acute and chronic cutaneous graft-vs-host disease in man.

The presence of cutaneous immunoglobulin and complement was investigated in 88 patients with and without graft-vs-host disease (GVHD) after transplantation of bone marrow from HLA identical siblings for the treatment of acute leukemia or aplastic anemia. For comparison, skin biopsies from the patients obtained before transplantation, from 58 healthy individuals (mostly marrow donors) and from four syngeneic marrow recipients were studied. A direct immunfluorescent staining technique was used. Dermo-epidermal IgM deposits were found in 11% of healthy individuals and patients before grafting but were present in 86% of patients with chronic and 39% of patients with acute GVHD. Patients with allogeneic grafts who never had GVHD or who had recovered from it and patients with syngeneic grafts showed findings not different from those in healthy individuals. Findings similar to those with IgM, although less striking, were made for C3, i.e., patients who had chronic or acute GVHD had a high incidence and intensity of C3 deposits at the dermo-epidermal junction. This observation raises the possibility that humoral immunity is involved in the development of GVHD.     

Immunotherapy of bovine ocular squamous cell carcinoma by repeated intralesional injections of live bacillus Calmette-Guérin (BCG) or BCG cell walls

Summary Thirty cows of the Dutch Friesian and the Maas-Rijn-Ijssel breed with histologically confirmed ocular squamous cell carcinoma were treated by repeated intralesional injection of live bacillus Calmette-Guérin (BCG) (n = 14) or a BCG cell-wall vaccine (n = 16). Complete regression of the primary tumour was observed in 64% and 57% of the animals respectively. In the 2-year follow-up period there was no recurrence of primary tumours. This sharply contrasts with the recurrence frequency (40%–50%) after complete remission induced by a single intralesional injection with BCG, observed in an earlier study. In 1 animal a new primary tumour developed. At necropsy metastases were present in 33% of the treated animals: in 3 of 17 animals that showed complete regression of the primary tumour and in 7 of 13 animals with partial regression or progressive disease. This did not differ significantly from results obtained after a single treatment (27%). Delayed-type hypersensitivity toM. bovis purified protein derivative (PPD) was more persistent in animals showing regression of the primary tumour than in non-responding animals. Of the animals with a positive PPD response 6 months after treatment, 79% showed tumour regression. Regression was observed in only 28% of the animals not responding to PPD after the same period of time. In conclusion: (a) recurrence of the primary tumour was not observed after repeated BCG treatment; (b) the frequency of metastases was not decreased compared to results obtained with a single treatment; (c) regression was correlated with a positive delayed-type hypersensitivity reaction to PPD (P <0.05) 6 months after treatment; (d) no significant differences were observed when the clinical results of treatment with live BCG and the BCG cell wall vaccine were compared.     

Oxidant/antioxidant status in blood of patients with malignant breast tumour and benign breast disease

The aim of this study was to investigate the alterations in lipid peroxidation and antioxidant enzyme defences in the blood of patients with malignant breast tumour and benign breast disease. Forty patients with malignant breast tumour, 20 patients with benign breast disease and also 20 healthy control subjects were recruited for the study. Malondialdehyde levels in plasma and erythrocytes, and the activities of erythrocyte CuZn-superoxide dismutase, catalase, glutathione peroxidase and glucose-6-phosphate dehydrogenase were measured. Malondialdehyde levels were higher in patients with both benign breast disease and malignant breast tumour compared with control subjects. The activities of all antioxidant enzymes were higher in patients with malignant breast tumour, while only glutathione peroxidase and CuZn-superoxide dismutase activities were higher in patients with benign breast disease. Except for glucose-6-phosphate dehydrogenase, the antioxidant enzymes studied correlated positively with the malondialdehyde levels in patients with malignant breast tumour. On the other hand, only glucose-6-phosphate dehydrogenase activity was increased by the level of malignancy. The activity increases in erythrocyte antioxidant enzymes may be a compensatory upregulation in response to increased oxidative stress especially in patients with malignant breast tumour.     

Remission in psoriatic arthritis: is it possible and how can it be predicted?

Introduction

Since remission is now possible in psoriatic arthritis (PsA) we wished to examine remission rates in PsA patients following anti tumour necrosis factor alpha (TNFα) therapy and to examine possible predictors of response.

Methods

Analysis of a prospective patient cohort attending a biologic clinic, between November 2004 and March 2008, was performed prior to commencing therapy and at regular intervals. Baseline clinical characteristics including demographics, previous disease-modifying antirheumatic drug (DMARD) response, tender and swollen joint counts, early morning stiffness, pain visual analogue score, patient global assessment, C reactive protein (CRP) and health assessment questionnaire (HAQ) were collected.

Results

A total of 473 patients (152 PsA; 321 rheumatoid arthritis (RA)) were analyzed. At 12 months remission, defined according to the disease activity score using 28 joint count and CRP (DAS28-CRP), was achieved in 58% of PsA patients compared to 44% of RA patients, significant improvement in outcome measures were noted in both groups (P < 0.05). Analysis of a subgroup of PsA and RA patients matched for DAS28-CRP at baseline also showed higher numbers of PsA patients achieving remission. Linear regression analysis identified the HAQ at baseline as the best predictor of remission in PsA patients (P < 0.001).

Conclusions

DAS28 remission is possible in PsA patients at one year following anti-TNF therapy, at higher rates than in RA patients and is predicted by baseline HAQ.     

La forme scléronodulaire de la maladie de Hodgkin: expérience du CHU de Sherbrooke

Scleronodular type of Hodgkin''s disease: experience at the Centre hospitalier universitaire de SherbrookeThe nodular sclerosis type of Hodgkin''s disease appears to be a distinct clinical entity. However, the incidence, the initial localization of the tumour and the survival of the patients are variable. The present study was carried out on a group of 17 patients, all French Canadians living in the province of Quebec, from a total of 31 with Hodgkin''s disease, an incidence of 55%. There were more males (10) than females (7). The mean age of the group was 37 years, but that of the females was lower than that of the males. The mediastinum was involved at the onset in 47% of the patients. The initial staging (according to the classification of Rye) in 76% of the patients was I or II.Four patients showed disease below the diaphragm. The lungs were infiltrated three times, the spleen six times, and the liver five times. The duration of survival of the 17 patients was twice that of the patients with the three other types of the disease.     

Phase III, randomised, multicentre trial of maintenance immunotherapy with low-dose interleukin-2 and interferon-α for metastatic renal cell cancer

This is the first phase III randomised trial to evaluate maintenance immunotherapy in metastatic renal cell cancer (mRCC). Patients were randomised to receive treatment with a 4-week cycle of subcutaneous low doses IL-2 + IFN in months 1, 3 and 5, and then every 3 months until the first documented disease progression (arm A, suspension), or the same regimen, with chronic maintenance of immunotherapy, regardless of tumour response, until death or intolerable toxicity (arm B, maintenance). The primary endpoint was overall survival (OS); secondary endpoints were time from first progression to death (TFPTD) and tolerability. One hundred and eighty-three patients were enrolled between January 1998 and November 2003. After a median follow-up of 53.9 months, response rate, median OS and median TFPTD were 14.7% (6.3% CR) versus 11.3% (5.5% CR), 14 versus 14 months, 6 versus 5 months, in arms A and B, respectively with no significant differences between the groups. Cox regression analysis showed that the use of chemotherapy after first progression (HR 0.54; 95% CI 0.35–0.86; p = 0.008), PS = 0 (HR 0.53; 95% CI 0.35–0.81; p = 0.001) and female gender (HR 0.63; 95% CI 0.41–0.98; p = 0.038) were significantly associated with a longer TFPTD; treatment arm was not significant (HR 0.88; 95% CI 0.60–1.31; p = 0.54). Toxicity was mainly limited to WHO grades 1 or 2. Chronic maintenance immunotherapy after disease progression is feasible, but does not significantly increase OS or the TFPTD.     

Subcellular localization of the heat-stable glucocerebrosidase activator substance in Gaucher spleen

The spleen in Gaucher's disease contains relatively large quantities of a heat-stable activator of the glucocerebrosidase of normal human tissues (Ho, M. W., and O'Brien, J. S. (1971) Proc. Nat. Acad. Sci. USA68, 2810–2813) that has been shown to be an 11,000 molecular weight acidic glycoprotein (Peters, S. P., et al. (1977) J. Biol. Chem.252, 563–573). In an effort to determine the subcellular location of the activator, a mannitol-sucrose homogenate of fresh, unfrozen spleen obtained from a 26-year-old patient with adult, nonneuropathic (Type 1) form of Gaucher's disease was subjected to subcellular fractionation. The tissue used in these experiments exhibited a β-glucocerebrosidase deficiency (11% of control tissue characteristic of Gaucher's disease. Mitochondrial and lysosomal fractions obtained by centrifugation of the spleen homogenate at 6900 and and 20,000g, respectively, contained greater than 80% of the recovered acid phosphatase and heat-stable glucocerebrosidase activator activities. In addition, 60% of the residual glucocerebrosidase activity was recovered in the mitochondrial and lysosomal fractions. The lysosomal and mitochondrial fractions were subjected to equilibrium sucrose density gradient centrifugation. Analysis of the sucrose gradient of the crude mitochondrial fraction demonstrated the mitochondrial marker enzyme (cytochrome oxidase) banding with a specific gravity of 1.19 g/ml, whereas the heat-stable activating factor banded in an acid phosphatase-rich fraction having a specific gravity of 1.12 g/ml. Sucrose gradient analysis of the crude lysosomal fraction obtained from differential centrifugation indicated the activating factor banding with a specific gravity of 1.12 g/ml. Coincident with the activating factor was glucocerebrosidase and acid phosphatase activity. Electron microscopic examination of fractions from each of the sucrose density gradients demonstrated that the glucocerebrosidase activating factor was located in the same acid phosphatase-rich fractions that contained the characteristic Gaucher deposits. Furthermore, when Gaucher deposits were isolated and purified independently by a sucrose gradient procedure, they were found to contain high concentrations of the heat-stable glucocerebrosidase activator. The specific activity of the glucocerebrosidase activating factor was approximately 15-fold greater in the extensively purified Gaucher deposits than in the crude extract of Gaucher spleen from which the deposits were isolated. These observations indicate that the heat-stable activator is associated with the storage deposits contained in lysosomes of the Gaucher cell.     

Immunoresponsiveness in Ulcerative Colitis and Crohn's Disease—Effect of Colectomy and Suppression of Disease Activity

We evaluated the effect of medically induced symptomatic disease improvement on in vitro tests of cell-mediated immune responses in 33 patients with Crohn''s disease. When results obtained in 17 patients with ulcerative colitis were compared with those of 10 patients with ulcerative colitis who had undergone a colectomy, no significant correlation was detected between individual clinical and laboratory variables or the Crohn''s disease activity index and in vitro tests of cell-mediated immunity. A different pattern emerged from the longitudinal tests of cell-mediated immunity: when these test results were initially abnormal in patients with Crohn''s disease, clinical improvement as assessed by the Crohn''s disease activity index was associated with normalizing cell-mediated immunity. In contrast, when the test results were initially normal, clinical improvement was not associated with any change in the immune response. Following colectomy in patients with ulcerative colitis, some abnormalities of suppressed immune responses remained, although patients were cured of their disease. Factors other than clinical disease activity may be responsible for the suppressed immunoresponsiveness in some patients with inflammatory bowel disease, and variable changes in cell-mediated immunity occur after both surgical and medical treatment.