Application of a new ultra-microculture system. II. Stimulation of human B lymphocytes

An ultra-microtechnique for culturing human B-lymphocytes in glass capillary tubes using a volume of 2 microliter is described. The advantage of this ultra-microculture system is that only a small number of lymphocytes and minute amounts of culture medium (or test factors) are required. Optimal culture conditions for the formation of Ig-secreting plaque-forming cells (PFC) after stimulation of mononuclear cells with pokeweed mitogen are given. Furthermore it is shown that immunoglobulin secreted into culture supernatants by purified B cells in the presence of T cell subsets can be measured in a microELISA.     

Daily versus thrice-weekly interferon alfa-2b plus ribavirin for the treatment of chronic hepatitis C in HIV-infected persons: a multicenter randomized controlled trial

Among HIV-infected persons, chronic hepatitis C virus (HCV) infection causes substantial morbidity and mortality. However, few studies have evaluated the safety and efficacy of interferon alfa (IFN) and ribavirin (RBV) therapy in co-infected persons. Accordingly, a randomized, controlled, open-label, multicenter trial was conducted to establish the safety, tolerability, and efficacy of IFN alfa-2b 3 mIU daily plus RBV 800 mg/d compared with IFN alfa-2b 3 mIU thrice weekly (TIW) plus RBV 800 mg/d in HCV treatment-naive, HIV-infected subjects with compensated liver disease and stable HIV disease. The primary endpoint was sustained virologic response (SVR), defined as an undetectable HCV RNA level 24 weeks after discontinuation of HCV therapy. At study entry, subjects in both groups were similar with respect to age, gender, HCV genotype, and HIV disease status. Of 180 randomized subjects, 162 received at least 1 dose of study medication, constituting the modified intention-to-treat population. After 12 weeks of therapy, 122 (75%) had serum HCV RNA levels assessed; of these subjects, early virologic response (undetectable HCV RNA or >2 log10 decrease from baseline) was observed in 33 (42%) and 13 (16%) of subjects taking daily and TIW IFN, respectively (P < 0.001). SVR was observed in 15 (19.0%) and 7 (8.4%) of subjects taking daily and TIW IFN, respectively (P = 0.05). Adverse events were similar in both groups. However, while no deaths or opportunistic infections were observed, nearly 30% of subjects stopped treatment due to adverse events and 7 subjects experienced a serious adverse event. In conclusion, SVR was achieved in 19% of HIV/HCV coinfected subjects treated with daily IFN plus RBV, but the effectiveness of therapy was substantially diminished by relatively high rates of treatment-related toxicity.     

Preparation of the domains and domain-containing fragments of the human gamma-chain

Under certain conditions trypsin will split only a small number of peptide bonds in intact IgG, or the isolated γ-chain, and fragments thereof, viz. the bonds between the domains. The rates of splitting differ for different bonds. Using repeated tyrptic digestion and repeated gel chromatography all domains and all possible fragments consisting of sequential domains of the human γ-chain could be isolated, with the exception of V_H, of which the cyanogen bromide cleavage products were obtained. All fragments could be unambigously identified by their apparent molecular weights, as determined by SDS-PAA gel electrophoresis, by their origin and their further split-products, and by their amino acid compositions. Overall yields ranged from 10 to 45% on a molar basis.     

Theta coupling in the human electroencephalogram during a working memory task

The role of coupling between prefrontal and temporo-parietal brain areas within the theta frequency range of the human electroencephalogram was explored in a working memory task. During encoding of visual information higher theta amplitudes were observed in the right compared to the left hemisphere. Retrieval of visuospatial and verbal information elicited a more bilateral activation pattern. These effects were accompanied by theta coupling between dorsolateral prefrontal and right posterior temporal electrode sites during encoding. During retrieval prefrontal and bilateral temporo-parietal brain areas were coupled. These results support the idea of working memory functions being dependent on distributed prefrontal-temporal networks.     

Lymphokine-activated killer (LAK) cells can be focused at sites of tumor growth by products of macrophage activation

Successful adoptive cancer immunotherapy presumably depends on the accumulation of tumoricidal leukocytes at the sites of tumor growth. Large numbers of lymphokine-activated killer (LAK) cells can be generated in vitro by growth in high concentrations of interleukin-2 (IL-2), but relatively few arrive at the tumor site after intravenous injection. We hypothesize that the delivery of LAK cells to tumor sites may be augmented by previously demonstrated lymphocyte-recruiting factors, including activated macrophage products such as interleukin-1 (IL-1) and tumor necrosis factor. 111Indium-labeled LAK cells were injected intravenously into syngeneic mice bearing the macrophage activator endotoxin (LPS) in one hind footpad, and saline solution was injected into the contralateral footpad. Significantly more activity was recovered from the LPS-bearing footpad at all times during a 96-hour period. Recombinant IL-1 also attracted more LAK cells after injection into tumor-free hind footpads. Furthermore, LAK cells preferentially homed to hind footpads that were bearing 3-day established sarcomas after intralesional injections of LPS, IL-1, or tumor necrosis factor when compared with contralateral tumor-bearing footpads injected with saline solution alone. In preliminary experiments, mice with hind-footpad tumors appeared to survive longer after combined systemic IL-2 and LAK therapy if intralesional LPS was administered. These studies demonstrate that macrophage activation factors that have been shown capable of attracting circulating normal lymphocytes can also effectively attract LAK cells from the circulation. By the stimulation of macrophages at the sites of tumor growth, more LAK cells can be attracted. It is hoped that by "focusing" the migration of LAK cells to tumors, LAK cells and IL-2 would effect tumor regression more efficiently and with less toxicity.     

AUR Memorial Award. Two computer models for selection of optimal magnetic resonance imaging (MRI) pulse sequence timing

Two computer modeling techniques have been developed that aid in the selection of optimal magnetic resonance imaging (MRI) pulse sequences and timing intervals for specific clinical situations. The "parameter sensitivity" technique provides a means of selecting three separate MRI scans which are individually sensitive to changes in each of the three NMR tissue parameters N, T1, and T2. The "contrast" technique allows selection of a single optimal MRI sequence using the expected changes in all three tissue parameters simultaneously. Excellent correlation is demonstrated between the models and images obtained in a normal volunteer and in a patient with multiple sclerosis. The two methods compliment each other; the parameter sensitivity method is most useful in situations where subtle changes in tissue parameters are expected, whereas the contrast method is suited to circumstances where large differences in tissue parameters are anticipated and the magnitude and direction of these changes are known.     

Correction to: Factor Structure and Equivalence of Maternal Resources for Care in Bangladesh,Vietnam, and Ethiopia

Maternal and Child Health Journal - The article “Factor Structure and Equivalence of Maternal Resources for Care in Bangladesh, Vietnam, and Ethiopia”, written by Sulochana Basnet,...     

Increased cation transport inmdr1-gene-expressing K562 cells

Cation-transport properties were compared in a human leukemic cell line (K562) and its vincristineselected,mdr1-gene-expressing sublines (K562/Vcr30 and K562/Vcr150) by the capacity of the cells to accumulate the potassium analogue thallium (²⁰¹Tl). Determination of the time course of thallium accumulation in the absence and presence of ouabain, an inhibitor of sodium-potassium adenosine triphosphatase (ATPase), showed that the initial (at 20 min) rate of ouabain-resistant uptake was about 70% higher in the K562/Vcr30 cells than in the parental line. The maximal rate (V_max) of ouabain-resistant uptake was 78 mmol/h for K562 cells and 115 mmol/h for K562/Vcr30 cells, and the Michaelis constant (K _m) was 0.37 and 0.18 mmol, respectively. Bumetanide (50 M), a specific inhibitor of ouabain-resistant Na–K–Cl cotransport, inhibited the elevated²⁰¹TI uptake in K562/Vcr150 cells but had no effect on cellular vincristine accumulation. Incubation with different multidrug resistance (MDR)-reversing agents (verapamil as well as cyclosporin A and its analogue PSC833) had no significant effect on²⁰¹Tl uptake. Membrane depolarization by an elevation of the potassium concentration in the incubation medium did not affect vincristine accumulation in any cell line, which indicated that the changed drug-transport properties inmdr1-gene-expressing cells were not due to membrane hyperpolarization. It was concluded that P-glycoprotein-positive cells have a more efficient ouabain-resistant cation-transport mechanism than to cells without P-glycoprotein. A functional relationship between this phenomenon and MDR was not identified.     

Brain-specific protein C activation during carotid artery occlusion in humans

BACKGROUND AND PURPOSE: Activation of plasma protein C (PC) zymogen by thrombin-thrombomodulin at the endothelial surface is an important endogenous antithrombotic mechanism. It is unknown whether activated protein C (APC) is generated in vivo in the cerebrovasculature, because there is only limited thrombomodulin expression in human brain vascular endothelium. Therefore, we tested the hypothesis that carotid occlusion produces brain-specific PC activation. METHODS: Blood samples were simultaneously collected from the ipsilateral internal jugular vein and radial artery before and during carotid cross-clamping and on "de-occlusion" in 8 awake patients undergoing routine carotid endarterectomy. Plasma PC zymogen and circulating APC levels were measured using enzyme immunocapture assay and expressed as percent of pooled plasma controls. RESULTS: Internal jugular vein APC levels increased 28% exclusively during carotid occlusion and then decreased 32% with de-occlusion (F=8.1, P<0.005). PC zymogen increased only 5.9% with occlusion (F=6.3, P<0.02), consistent with hemoconcentration. There were no changes in radial artery PC or APC levels. CONCLUSIONS: These findings demonstrate brain-specific protein C activation in humans during carotid occlusion and suggest a protective role for endogenous APC generation during cerebrovascular occlusion.