Immunorestorative properties of thymostimulin (TS) in patients with Hodgkin's disease in clinical remission

Summary Fifteen Hodgkin's disease patients (8 male, 7 female) aged 19–72 years, who had been in complete unmaintained remission for 1 year or more when the study was initiated, were given 50 mg thymostimulin (TS) IM daily for 60 consecutive days. When compared with 26–30 age- and sex-matched controls, as a group the patients' circulating E_NR⁺, OKT ₃ ⁺ , and OKT ₄ ⁺ cells were depressed (0.001P .06), whereas their OKT ₈ ⁺ cell population was not. Low (>1 SD or >2 SD below mean in controls) or borderline (mean value of two subsequent tests >1 SD below mean in controls) values of E_NR⁺, OKT ₃ ⁺ , and OKT ₄ ⁺ cells were seen in nine (group I) of the 15 patients tested, while the remaining six patients (group II) had normal T-cell proportions. Following TS treatment, the proportions of E_NR⁺, OKT ₃ ⁺ , and OKT ₄ ⁺ cells increased to normal in all group I patients. The T-cell levels, however, decreased to pretreatment values 60–70 days after completion of TS therapy. TS had no effect on the group II patients whose T-cell percentages had initially been normal. Spontaneous cell-mediated cytotoxicity (SCMC) was assessed in 11 patients, and irrespective of the baseline values, there was a significant enhancement (P<0.005) by day 15 of TS administration, which was maintained during treatment. SCMC, however, returned to pretreatment levels 60–70 days after TS was discontinued. The delayed skin test reactivity to DNCB was significantly depressed in all cases. Although TS restored the T-cell proportions, it failed to reverse DNCB reactivity from negative to positive in any of the patients tested. TS can thus restore defective T-cell frequencies and can enhance cytolytic functions that are potentially important in host immunosurveillance, but it apparently failed to improve the skin reactivity to neoantigen.     

Transfer factor in vitro abrogation of blocking factors and amplification of immunoreactivity directed to soluble tumor associated antigens in the leukocyte adherence inhibition assay

Summary 26 breast cancer patients with recurrent disease were studied in order to evaluate whether transfer factor (TF) could abrogate serum blocking factors (SBF) and transfer or amplify immune reactivity directed to tumor-associated antigens (TAA) by using the leukocyte adherence inhibition assay (LAI). When 11 leukocyte samples obtained from patients reactive to cancer extract were preincubated with autologous blocking sera in the presence of PBS, nonspecific TF, or specific TF, leukocytes from these patients gained reactivity against breast cancer extract in none out of 11, one out of 11, and nine out of 11 experiments, respectively. Specific TF was obtained from donors with positive LAI breast cancer extract. Mean percentage LAI differences between control extract and breast cancer extract were –4.5±2.6, 0.5±1.7, and 15.5±2.4. The results of specific TF were significantly different from those of nonspecific TF (P<0.005).When nine leukocyte samples obtained from patients unreactive to cancer extract were preincubated with autologous blocking sera in the presence of PBS, nonspecific TF, or specific TF, leukocytes from these patients gained reactivity against breast cancer extract in none out of nine, one out of nine, and seven out of nine experiments, respectively. Mean percentage LAI differences between control extract and breast cancer extract were –4.6±2.0, 0.6±2.3, and 11.8±3.5. The results of specific TF were again significantly different from those of nonspecific TF (P<0.005).When allogeneic blocking sera were utilized, similar results were obtained. When specific TF was administered in two doses, 1 week apart subcutaneously to six breast cancer patients who were unreactive to breast cancer extracts, four patients gained reactivity against breast cancer extracts.Our in vitro experiments support the presence of a specific component in transfer factor extract which can transfer or amplify cell-mediated reactivity with abrogation of SBF directed at TAA.Abbreviations BSS = Balanced Salt Solution - LAI = Leukocyte Adherence Inhibition - PBS = Phosphate Buffered Saline - RPMI = Roswell Park Memorial Institute - SBF = Serum Blocking Factors - TAA = Tumor Associated Antigen - TF = Transfer Factor     

Ferritin-bearing lymphocytes and T-cell levels in peripheral blood of patients with breast cancer

Summary Peripheral blood lymphocytes bearing surface ferritin and thymus-dependent lymphocyte (T cell) levels were determined in 15 breast cancer patients in stage I–II, 5 in stage III, 10 with benign breast disease, 4 with Thalassaemia, and 25 normal controls. The results of this study demonstrate that a subpopulation of lymphocytes (16.6%) bearing surface ferritin was found in patients with breast cancer in stage I–II. None were demonstrated in patients with either benign breast disease, or with Thalassaemia, the latter known to have high serum ferritin levels, and almost none (1.7%) in normal individuals. A significant decrease in the percentage of ERFC as compared with the percentage of T cells, determined with anti-T cell antiserum (P<0.01), was observed in patients with breast cancer in stage I–II. Yet, the mean T-cell percentage in this group of patients was significantly higher than the mean percentage of T cells in normal controls (P<0.01). In patients with benign breast disease, the percentage of T cells corresponded to the percentage of ERFC and did not significantly differ from those in normals. Stage III breast cancer patients seem to constitute a biologically distinct group, since the ferritin-positive lymphocyte subpopulation disappeared and the percentage of ERFC and T cells returned to the values of normal controls.Overnight incubation of lymphocytes from patients exhibiting a ferritin-positive lymphocyte subpopulation in culture media containing 20% FCS resulted in the removal of ferritin from the surface of the cells and in restoration of the percentage of ERFC.     

In vivo modulation of natural killer cell activity by tamoxifen in patients with bilateral primary breast cancer

Natural killer (NK) cell activity, the autologous mixed lymphocyte reaction (AMLR) and proportions of T cell subpopulations (CD3⁺/CD4⁺ and CD3⁺/CD8⁺) and NK cells (CD16⁺) were studied in 21 patients with bilateral primary breast cancer (BBC), 10 patients with single-breast cancer (SBC) and 20 healthy controls. All patients studied had no evidence of disease and had been off radiotherapy and/or chemotherapy for at least 1 year. Ten patients with BBC were also treated with tamoxifen. Patients with SBC had NK cell activity, AMLR responses and T cell subpopulations that were comparable to those of normal controls. In patients with BBC, a significant (P<0.01) increase in NK activity compared to that in normal controls (42±13% versus 21±10%, effector-to-target cell ratio, 251) and a significant (P<0.05) decrease in CD4⁺ T cell proportions (30±15% versus 49±13%) and absolute numbers (472±82/mm³ versus 953±131/mm³) were found. However, the proliferative response of BBC patients' T lymphocytes in AMLR was in the range of the normal controls. Lymphocytes derived from 10 BBC patients treated with tamoxifen exhibited NK cell activity that was comparable to that of normal controls and patients with SBC, and was significantly (P<0.01) reduced compared to the pretreatment period. BBC patients who received tamoxifen also show a reduction in the proportion of CD4⁺ T cells and in AMLR proliferative responses, which decreased compared to levels in normal controls. Taken together, these results indicate that long-term tamoxifen treatment modulates immune responses in BBC patients.     

Effects of mating system in Japanese quail

Summary A 17-generation selection experiment was conducted to study direct and correlated responses to mass selection under a mating system with alternating generations of full-sib inbreeding and wide outbreeding (population I) as compared with a mass selected, randomly mated population (population II). The selection criterion was an index of total egg mass to 78 days divided by adult female body weight. Estimates of heritabilities and genetic correlations are reported. Estimated heritabilities for the index were 0.38±0.04 and 0.29±0.05 in population I and II, respectively. Realized heritabilites were 0.10±0.05 and 0.12±0.03. For most traits studied the mean phenotypic values in the cyclic mated population decreased for inbred generations. Increased inbreeding levels also caused outbred generation means of population I to decrease through the first six or seven generations. After this period of adaptation to inbreeding selection response was positive for the index and positively correlated traits. Total response to selection under the cyclic inbred-outbred mating system did not exceed selection response made under random mating. However, the rate of response in the cyclically mated population exceeded that in the randomly mated population in later generations when the cyclically mated population had apparently adapted to inbreeding.Published with approval of the Director of the Montana Agricultural Experiment Station, Journal Series No. 1221     

Selection of 3LL tumor subline resistant to natural effector cells concomitantly selected for increased metastatic potency

Summary The numbers of strongly adherent monocytes in the peripheral blood of normal subjects and cancer patients were determined. The method used was to place peripheral blood mononuclear cells in microwells and culture them for 1 week. At the end of that period, adherent macrophages were counted in the Coulter counter after release. Adherent cells per milliliter of blood, per total cells, and per mononuclear cells or monocytes plated were markedly diminished in the peripheral blood mononuclear cells of 44 melanoma, 23 breast cancer, 18 lung cancer, nine colon cancer, and 27 leukemia patients. Median values were 14.8×10⁴ adherent cells per ml peripheral blood for 86 normal subjects, as against 2.5×10⁴ per ml in the peripheral blood of the 125 patients (P<0.001). There was a poor correlation between the adherent cell numbers and the peripheral blood leukocyte counts, but an excellent correlation of the different adherent cell counts with each other. The number of adherent cells in the peripheral blood varied inversely with age in the cancer patients, but not in the normal subjects (r=0.29, P<0.005). When patients under age 50 were compared to the controls, the deficiency of adherent cells was slightly more severe in patients with stage IV lung cancer than in those with stage III lung cancer. In contrast, there was no difference in the degree of deficiency between patients with stage III melanoma and no evident disease and patients with stage IV disseminated metastatic disease. The implications of these results are discussed.     

Presymptomatic direct detection of adenomatous polyposis coli (APC) gene mutations in familial adenomatous polyposis

The recent identification of the familial adenomatous polyposis (FAP) gene (designated as APC) enables conclusive genetic testing of at-risk family members for the specific mutation in families in which the germline gene mutation has been characterized. Presymptomatic molecular diagnosis of FAP was performed by direct direction of mutations in lymphocyte DNA in four families. Each of the families has a different mutation of the APC gene. Twenty-seven offspring of affected individuals (a priori risk of 50%) were tested. Ten of the 27 had already developed clinical features of FAP. Of the remaining seventeen, two had had a negative colon exam at an early age, and nine had never had colon exams (mean age, 12.1±3.1 SD years). Six children from this group (54%) were found to carry their affected parent's mutation. No change in the conventional FAP colon screening regimen is recommended for these children. In contrast, when direct tests indicate that an individual does not have the FAP mutation, we recommended that screening be decreased. Reduction of uncertainty for at-risk FAP family members is an important benefit of genetic testing.     

Effect of co-culturing of half-destroyed and intact 4-cell mouse embryos in varying ratios on subsequent in vitro development

We studied the co-culturing effect of intact and half-destroyed 4-cell mouse embryos on blastocyst formation rate and cell counts. A laser beam was used to produce a hole and destroy an adjacent blastomere in two opposite areas of the zona in the experimental group (n = 342), and to open two opposite zonal holes in the controls (n = 318). Control and half-destroyed embryos were cultured together in varying ratios of 10:0, 7:3, 5:5, 3:7, and 0:10 (group 1-5, respectively) for 48 h in 10 μl drops of cleavage medium. They were then separated and cultured in blastocyst medium for 24 h. The results showed that half-destroyed embryos had no effect on the blastulation rates of controls (97-100%, P = 0.28). Neither was there a difference in the number of ICM (27.3 ± 6.7, 29.4 ± 9.9, 27.7 ± 9.3, 26.5 ± 6.4, in group 1-4, respectively; P = 0.491), TE (47.7 ± 18.6, 52.3 ± 13.9, 48.4 ± 19.2, 57.3 ± 12.9, in group 1-4, respectively; P = 0.101), nor total cells (75.0 ± 19.5, 81.3 ± 17.1, 76.1 ± 19.6, 83.7 ± 16.2, in group 1-4, respectively; P = 0.188) in the resulting blastocysts. However, among half-destroyed embryos, cleavage arrest decreased (58.3%, 39.6%, 17.9%, and 8.3%, in group 5 to 2, respectively; P < 0.001) and blastocyst development increased (38.3%, 58.2%, 72.6%, and 88.9%, in group 5 to 2, respectively; P < 0.001) following co-culturing with intact controls. These embryos had a higher number of ICM cells (P = 0.035), but no significant changes in TE (P = 0.262) and total cell counts (P = 0.065). The findings indicate that the co-culturing of half-destroyed with intact embryos increased the blastulation rate of the first but had no effect on the latter.     

Sister chromatid exchange in lymphocytes from patients with malignant lymphoma

Summary Sister chromatid exchange (SCE) frequencies were studied in differentially stained lymphocytes from 47 patients with malignant lymphoma. Thirteen patients were untreated when studied. The mean SCE frequency [±standard error (SE)] for these patients was 12.7±0.9 per mitosis. The mean score for 40 controls was 6.1±0.3. SCE mean scores were significantly higher in the untreated patients than in the controls (P<0.001). Seven patients were treated with radiotherapy alone. They demonstrated a mean SCE frequency (8.8±0.8) significantly lower (P<0.01) than that found in untreated patients. Eleven patients received cyclophosphamide within 4 weeks prior to study. They demonstrated a mean SCE frequency (14.3±1.3) significantly higher (P<0.05) than that found in patients who had received regimens that did not contain cyclophosphamide in the prior 4 weeks (11.1±1.3) or who had been off drugs for at least 8 weeks (10.1±0.8). Our data suggest that untreated patients with malignant lymphoma have elevated SCE frequencies, which may be further increased by certain chemotherapeutic agents.     

Tartrate-resistant acid phosphatase 5b as a serum marker of bone metastasis in breast cancer patients

Diagnosis and follow-up of bone metastases in breast cancer patients usually rely on symptoms and imaging studies. Tartrate-resistant acid phosphatase 5b (TRACP 5b) is a specific marker of osteoclasts and is herein proposed as a marker of bone metastasis in breast cancer patients. An immunoassay using a monoclonal antibody, 14G6, was used to measure the activity of serum TRACP 5b at pH 6.1 in 30 early breast cancer patients without bone metastasis and in 30 aged-matched breast cancer patients with bone metastasis. Another 60 normal volunteers were recruited as controls. Bone alkaline phosphatase (BAP), a traditional marker of bone turnover, was also measured in selected cases. The overall mean TRACP 5b activity in normal women was 2.83 ± 1.1 U/I, and it increased with age. The mean TRACP 5b activity in early breast cancer patients did not differ from that of the normal group (2.93 ± 0.64 vs. 2.83 ± 1.1 U/I; p=0.66), whereas it was significantly higher in breast cancer patients with bone metastasis (5.42 ± 2.5 vs. 2.83 ± 1.1 U/I; p<0.0001). BAP activity was significantly higher in breast cancer patients with bone metastasis than in early breast cancer patients (p=0.004). Serum TRACP 5b activity correlated well with BAP activity in breast cancer patients with bone metastasis (p<0.0001), but not in normal individuals or in patients without bone metastasis. TRACP 5b activity can be considered a surrogate indicator of bone metastasis in breast cancer patients.     

Anti-tyrosinase antibodies in malignant melanoma

 Anti-tyrosinase antibodies were measured by enzyme-linked immunosorbent assay in sera of patients with malignant melanoma with either metastatic disease or no evidence of disease, in patients with melanoma and associated hypopigmentation (MAH), in patients with vitiligo and in healthy volunteers. The mean relative absorbance (A _rel) was calculated by dividing the absorbance of each sample by the mean value for the control group. Using this method, the A _rel of the control group was 1.000(SE 0.083). A _rel of patients with metastatic disease (1.516; SE 0.225) was significantly higher (P = 0.03) than the value for the controls, but insignificantly higher than that for patients with no evidence of disease (1.216; SE 0.148). Patients with no evidence of disease, in whom the primary lesion originated in the lower limb, had a significantly higher (P = 0.01) A _rel than the healthy volunteers. Patients with metastatic disease showed higher A _rel if their primary lesions were confined to the area of the head and neck or to the lower limb. Patients with vitiligo had higher A _rel values for their anti-tyrosinase antibody than any of the other groups. However, those with melanoma and MAH (vitiligo-like) had the same A _rel of anti-tyrosinase antibodies as the controls or the patients with metastatic melanoma. This observation reflected the possible absorption of anti-tyrosinase antibodies to melanoma antigens, and pointed to the participation of anti-tyrosinase antibodies in the destruction of normal melanocytes in patients with melanoma, as part of the immune reaction towards this disease. Received: 4 January 1996 / Accepted: 11 April 1996     

Validity of a model of cultured myocardial cells for assessment of cardioplegia

Myocardial protection is usually studied in vitro on perfused heart preparations, but never directly on cultured cardiomyocytes. We evaluated a model of cultured newborn rat cardiomyocytes to study both the cytotoxicity and the protective effect against chemical hypoxia of three cardioplegic solutions (St Thomas' I, Bretschneider, St Thomas' II) under normothermic (37°C) and hypothermic (4°C) conditions. Cytotoxicity was evaluated in 50% and 100% concentrations of the cardioplegic solutions with incubation times from 90 to 360 min. Myocardial protection was studied in 50% cardioplegic solution with metabolic inhibitors. Immediate and late viabilities, after 24 h of recovery in the medium, were evaluated by simultaneous staining with fluorescein diacetate and propidium iodide.At 37°C, the 50% concentration of the three cardioplegic solutions did not modify cell viability. At 37°C, with 360 min of incubation, the 100% concentration of the St Thomas' I and Bretschneider solutions diminished immediate viability (mean ± SD: medium 87% ± 2%; St Thomas' I 58% ± 5%; Bretschneider 37% ± 8%; St Thomas' II 89% ± 3%) as well as late viability (medium 69% ± 2%; St Thomas' I 32% ± 3%; Bretschneider 24% ± 7%; St Thomas' II 65% ± 4%). At 4°C, immediate and late viabilities were unaffected by cardioplegic solutions.At 37°C, after 360 min incubation time, metabolic inhibitors diminished immediate viability to 29% ± 1% and late viability to zero. None of the three cardioplegic solutions used at 50% concentration prevented this effect.At 4°C, immediate viability was not significantly affected by metabolic inhibitors (73% ± 10%), but the use of Bretschneider cardioplegic solution seemed to be detrimental (53% ± 9%). On the other hand, recovery phase after pretreatment with metabolic inhibitors with or without cardioplegic solutions for 360 min significantly diminished late viability (medium 63% ± 7%; metabolic inhibitors 17% ± 8%; St Thomas' I 17% ± 6%; Bretschneider 8% ± 6%; St Thomas' II 15% ± 3%) and again cardioplegia was inefficient. In conclusion, in this in vitro model for the study of cardioplegic solutions, only pure concentrations of the St Thomas' I and Bretschneider solutions under normothermic conditions were cytotoxic. The well-known protective effects of hypothermia against ischemia and reperfusion injury were both reproduced. Therefore, and even though cardioplegia failed to have any protective effect, probably owing to a severe metabolic inhibition, this model may be useful for studying myocardial protection.     

The Influence of Temperature on Metabolic and Cellular Protection of the Heart during Long-Term Ischemia: A Study Using P-31 Magnetic Resonance Spectroscopy and Biochemical Analyses

We have compared the influence of two different cold temperatures (below 10°C) for cardiac ischemia by measuring a large variety of hemodynamic and metabolic parameters during ischemia and reflow. Isolated isovolumic rat hearts were arrested with a preservation solution which was developed in our laboratory and then submitted to 5 h of cold storage (4°C, group I; and 7.5°C, group II) in the same solution. After an additional period of 50 min of ischemia at 15°C with intermittent cardioplegic infusion, hearts were reperfused for 60 min at 37°C. Function was assessed during the control period and reflow. High-energy phosphates and intracellular pH were followed by³¹P magnetic resonance spectroscopy. Analyses of metabolites and enzymes were performed by biochemical assays and HPLC in coronary effluents and in freeze-clamped hearts to assess cellular integrity. The energetic pool was better preserved at 4°C during ischemia (ATP at the end of 4°C ischemia, 59 ± 7% in group I vs 31 ± 5% in group II,P< 0.01) and reflow (P< 0.05) but membrane protection was higher when increasing the temperature to 7.5°C (reduction of creatine kinase leakage, 89 ± 16 IU/min in group I vs 51 ± 5 IU/min in group II,P< 0.05). As a result, functional recovery, represented by the rate pressure product, was higher in hearts preserved at 7.5°C (52 ± 6% recovery in group I vs 77 ± 7% in group II at the end of reflow,P< 0.05). Altogether, cold storage at 7.5°C provides a better protection than storage at 4°C.     

Does vinblastine add to the potency of alpha interferon in the treatment of renal cell carcinoma?

The combination of alpha interferon and vinblastine has been reported to yield a response rate of 30-40% in previously untreated patients with metastatic renal cell carcinoma. This combination was given to nine patients with advanced metastatic renal carcinoma after they failed or relapsed on alpha-interferon alone, to attempt to evaluate the role of vinblastine in this combination. Neither complete nor partial response was observed. Two patients had disease stabilization for two and seven months. Our preliminary results suggest that vinblastine did not add to the efficacy of interferon in this group of patients.     

Immunization with a tumor-cell-lysate-loaded autologous-antigen-presenting-cell-based vaccine in melanoma

The discoveries of human melanoma-associated antigens in molecular terms have renewed interest in peptide- or peptide- and antigen-presenting-cell (APC)-based cancer vaccines. Considering the limited scope of immunization using defined peptides, we have studied an alternative approach of specific immunization with tumor-lysate-loaded autologous APC (adherent peripheral mononuclear cells cultured in 1000 U granulocyte/macrophage-colony-stimulating factor for 14 days) as a surrogate vaccine. Seventeen patients (11 with active metastatic disease) were intradermally immunized with the vaccine in a phased dose escalation (10⁵–10⁷ cells/injection) monthly for 4 months. Thirteen patients completed all four immunizations showing no toxicity (3 patients had to be taken off study because of progressive disease and 1 patient went off study as a result of myocardial infarction due to multi-vessel coronary artery disease). None has shown any immediate or delayed toxicity attributable to the immunization and none has shown any evidence of autoimmunity. One patient showed a partial regression of a subcutaneous nodule. Thirteen patients are alive after 4+ months to 30+ months (17-month median survival for the group). Nine patients showed evidence of delayed-type hypersensitivity at the vaccine sites. Monitoring of biological response in conventional natural killer or cytolytic T lymphocyte assays with pre- and post-immune peripheral blood lymphocytes revealed no consistent differences. The vaccine-infiltrating lymphocytes (VIL) from nine specimens were adequately expanded following in vitro stimulation with the respective autologous-lysate-loaded APC for phenotypic and functional analyses. Five of the nine ex vivo expanded VIL were predominantly CD8⁺. Evidence of an antigen-specific CD8⁺ T cell response (cytotoxicity and/or tumor necrosis factor production) was detected in three of the five CD8⁺ VIL. These observations suggest that this type of vaccine is feasible, that it has biological activity, and that the approach may be improved through additional strategic manipulations. Received: 27 March 1998 / Accepted: 14 May 1998     

Alterations in isoforms of glutathione S-transferase in liver and kidney of cadmium exposed rhesus monkeys: Purification and kinetic characterization

Exposure of animals to cadmium (Cd) (25 mg kg-1 body wt day-1) for 10 weeks resulted in preferential accumulation of the metal in liver and kidney. Cd accumulation concomitantly increased zinc (Zn) concentration in both the organs. However, significant decrease in copper level was observed in liver, whereas kidney showed increase in copper (Cu) level. Cd exposure resulted in decreased total GST activity in liver (63%) and kidney (41%) as compared to control group monkeys on normal diet (group I). On isoelectric focusing (IFP) control liver GST segregated into thirteen isoenzymes, while in Cd-treated experimental animals (group II) liver GST resolved into nine isoenzymes. Similarly kidney GST from control animals separated into seven isoenzymes as compared to four isoenzymes from Cd-treated animals. Kinetic analysis showed that Cd exposure did not alter the affinity constant (Km) of GST for GSH and CDNB whereas maximal velocity (Vmax) for these substrates decreased as compared to controls in both the organs, indicating inhibition in GST synthesis by Cd. Cd resulted in a noncompetitive type of inhibition with respect to GSH in vitro. On isoelectric focussing GST of liver and kidney in group II resolved into nine and four isoenzymes as compared to thirteen and seven in group I, showing loss of four basic isoenzymes in case of liver and three isoenzymes in case of kidney. Monkey liver and kidney expressed all the three classes of GST isoenzymes i.e. , µ and , which were serologically identical to human , µ and GSTs. (Mol Cell Biochem 166: 55-63, 1997)     

Decreased Brain Protein Levels of Cytochrome Oxidase Subunits in Alzheimer's Disease and in Hereditary Spinocerebella Ataxia Disorders

Abstract : Controversy exists as to the clinical importance, cause, and disease specificity of the cytochrome oxidase (CO) activity reduction observed in some patients with Alzheimer's disease (AD). Although it is assumed that the enzyme is present in normal amount in AD, no direct measurements of specific CO protein subunits have been conducted. We measured protein levels of CO subunits encoded by mitochondrial (COX I, COX II) and nuclear (COX IV, COX VIc) DNA in autopsied brain of patients with AD whom we previously reported had decreased cerebral cortical CO activity. To assess disease specificity, groups of patients with spinocerebellar ataxia type I and Friedreich's ataxia were also included. As compared with the controls, mean protein concentrations of all four CO subunits were significantly decreased (-19 to -47%) in temporal and parietal cortices in the AD group but were not significantly reduced (-12 to -17%) in occipital cortex. The magnitude of the reduction in protein levels of the CO subunits encoded by mitochondrial DNA (-42 to -47%) generally exceeded that encoded by nuclear DNA (-19 to -43%). In the spinocerebellar ataxia disorders, COX I and COX II levels were significantly decreased in cerebellar cortex (-22 to -32%) but were normal or close to normal in cerebral cortex, an area relatively unaffected by neurodegeneration. We conclude that protein levels of mitochondrial- and nuclear-encoded CO subunits are moderately reduced in degenerating but not in relatively spared brain areas in AD and that the decrease is not specific to this disorder. The simplest explanation for our findings is that CO is decreased in human brain disorders as a secondary event in brain areas having reduced neuronal activity or neuronal/synaptic elements consequent to the primary neurodegenerative process.     

Successful treatment of metastatic melanoma by adoptive transfer of blood-derived polyclonal tumor-specific CD4+ and CD8+ T cells in combination with low-dose interferon-alpha

A phase I/II study was conducted to test the feasibility and safety of the adoptive transfer of tumor-reactive T cells and daily injections of interferon-alpha (IFNα) in metastatic melanoma patients with progressive disease. Autologous melanoma cell lines were established to generate tumor-specific T cells by autologous mixed lymphocyte tumor cell cultures using peripheral blood lymphocytes. Ten patients were treated with on average 259 (range 38–474) million T cells per infusion to a maximum of six infusions, and clinical response was evaluated according to the response evaluation criteria in solid tumors (RECIST). Five patients showed clinical benefit from this treatment, including one complete regression, one partial response, and three patients with stable disease. No treatment-related serious adverse events were observed, except for the appearance of necrotic-like fingertips in one patient. An IFNα-related transient leucopenia was detected in 6 patients, including all responders. One responding patient displayed vitiligo. The infused T-cell batches consisted of tumor-reactive polyclonal CD8+ and/or CD4+ T cells. Clinical reactivity correlated with the functional properties of the infused tumor-specific T cells, including their in vitro expansion rate and the secretion of mainly Th1 cytokines as opposed to Th2 cytokines. Our study shows that relatively low doses of T cells and low-dose IFNα can lead to successful treatment of metastatic melanoma and reveals a number of parameters potentially associated with this success.     

Cellular hypertrophy in cardiomyopathic patients is associated with lower creatine-stimulated mitochondrial respiration

The mitochondrial respiration rate and morphometric indices in endomyocardial biopsy samples were measured in 43 patients with dilated cardiomyopathy selected in accordance to WHO criteria by endomyocardial biopsy studies after excluding of various forms of myocarditis, alcoholic cardiomyopathy and other specific diseases of the heart. A group of 13 patients with unusually high mean myocyte diameter, 30±4 m, and nuclear size, 57±5 m, was selected. The remainder of patients (n=30) had significantly lower mean myocyte diameter and nuclear size, 23±3 and 42±6 m, respectively, (p<0.01). Creatine-stimulated elevation in mitochondrial respiration rate as measured in saponin-skinned was found in the former group to be much lower (36±4%) as compared with the remainder (90±12%). Also, the former group of patients had higher left ventricular enddiastolic pressure and volume index with concomitantly decreased ejection fraction. The results indicate that marked nuclear and cellular hypertrophy is associated with lower creatine-stimulated mitochondrial respiration rate and more severe cardiac failure. They suggest that disorders in energy supply to myofibrils may be related to disturbances in cellular genetic apparatus.     

High resolution respirometry of permeabilized skeletal muscle fibers in the diagnosis of neuromuscular disorders

High resolution respirometry in combination with the skinned fiber technique offers the possibility to study mitochondrial function routinely in small amounts of human muscle. During a period of 2 years, we investigated mitochondrial function in skeletal muscle tissue of 13 patients (average age = 5.8 years). In all of them, an open muscle biopsy was performed for diagnosis of their neuromuscular disorder. Mitochondrial oxidation rates were measured with a highly sensitive respirometer. Multiple substrate-inhibitor titration was applied for investigation of mitochondrial function. About 50 mg fibers were sufficient to obtain maximal respiratory rates for seven different substrates (pyruvate/malate, glutamate/malate, octanoylcarnitine/malate, palmitoylcarnitine /malate, succinate, durochinol and ascorbate/TMPD). Decreased respiration rates with reference to the wet weight of the permeabilized fiber could immediately be detected during the course of measurements.In 4 patients with mitochondrial encephalomyopathy (MEM) the respiration pattern indicated a specific mitochondrial enzyme defect, which was confirmed in every patient by measurements of the individual enzymes (one patient with PDHC deficiency, one with complex I deficiency and two patients with combined complex I and IV deficiency). In the 6 patients with spinal muscular atrophy (SMA) oxidation rates were found to be decreased to 23 ± 5% of controls. The normalized respiration pattern was comparable to that of the controls indicating a decreased content of mitochondria in SMA muscle with normal functional properties. Also in the 3 patients with Duchenne muscular dystrophy (DMD) decreased oxidation rates (42 ± 5%) were detected. In addition a low RCI (1.2) indicated a loose coupling of oxidative phosphorylation in the mitochondria of these patients.It is concluded that investigation of mitochondrial function in saponin skinned muscle fibers using high resolution respirometry in combination with multiple substrate titration offers a valuable tool for evaluation of mitochondrial alterations in muscle biopsies of children suffering from neuromuscular disorders. (Mol Cell Biochem 174: 71–78, 1997)