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Umbilical cord blood (UCB) hematopoietic stem cells (HSC-CD34+) are valuable for treating malignant or nonmalignant disease. Processing UCB by HESPAN-6% and anti-CD34-Miltenyi particles provides insufficient cells for treating adults. Physicochemical-electrokinetic studies on UCB-mononuclear cells (MNCs) under conditions of delayed processing, ice or very low temperatures, and some cell separation media identified artifacts introduced by procedures. Adsorption of biomaterials from cell damage by temperature, degradation products after using enzymes, harsh reagents, dithiothreitol, and HESPAN affect cell properties and distribution. Miltenyi particles internalized by cells could release iron that accumulating in liver or spleen would then risk toxicity. Summary topics included the effects of temperature, HESPAN (fast sedimenting agent), glycoproteases, DNase, and dithiothreitol risk affecting cell receptors in recognition, "homing," leading to possible unintended iatrogenic bioeffects should such cells be transfused into humans. The loss of undetectable and uncaptured low CD34 antigen-bearing cells by Miltenyi particles seems to occur when the current methods of isolation of CD34+ cells and other cells are critically assessed. The purpose here is to highlight and suggest avoiding the procedural flaws involved. Preventing ice temperatures avoids ice-damaged platelets releasing biomaterials that are adsorbed on cells altering UBC-MNCs/HSC properties and cell loss. Omitting the positive selection with antibody-linked Miltenyi particles obviates the use of harsh reagents to release the cells. Internalized Miltenyi particles are a toxicity hazard that needs investigations. Achieving approximately 5% yields of CD34+ cells (153 × 10(5) /110 mL cord-placenta blood) is a major advance holding great promise, for the first time increasing the prospect of stem cell therapy of 70-kg adults, using a single UCB donation (with dose of 1.5 × 10(5) cells/kg) and considerably cheaper cultured red blood cells manufacture (multiple packs/2 × 10(12) ).

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BACKGROUND: Umbilical cord blood (UCB) is rich in the heavily glycosylated CD34 antigen-bearing hematopoietic stem cells that are valuable for transplantation therapy of malignant and nonmalignant disease. CD34+ cell yields (0.13%-0.25%-0.3%) of mononuclear cells (UCMCs) isolated by anti-CD34 monoclonal antibody (MoAb) on immunomagnetic particles (e.g., Miltenyi particles) are insufficient to treat adults. STUDY DESIGN AND METHODS: We fractionated UCMCs by physicochemical charge-based methods. Avoiding Miltenyi particles and HESPAN yielded 30 x 10(6) to 54 x 10(6) UCMCs/20 mL UCB by Ficoll-Isopaque for serial depletion fractionation, using nylon wool column (NWC) or direct rosetting with sheep red blood cells (SRBCs) without incubation in the cold. RESULTS: CD34+ cell yields (approx. 5.12%) were 39 times greater than 0.13% (Korean study, 11,098 UCB units) and 10 to 20 times greater than 0.25% to 0.3% harvested by anti-CD34 Miltenyi particles. SRBC depletion of most high-specific-gravity T cells achieved considerable enrichment of CD34+ and BY55+ cells. Using NWC achieved 2.5-fold enrichment of CD34+ cells and twofold enrichment of BY55+ cells. Direct SRBC rosetting provided better or higher enrichment of CD34+ cells. Overall CD34+ cell yield in low-density fraction was more than twice after direct rosetting (38% vs. 16%) in contrast to separation by NWC followed by SRBC rosetting. CD3+ cell yields (by three CD markers) were approximately 8.83%, far below approximately 30 x 10(7) /kg considered acceptable to avoid graft-versus-host disease. Natural killer cell yields (CD16+/CD56+ and BY55+/ CD160+) are in perfect agreement. CONCLUSIONS: Achieving approximately 5% CD34+ cell yields from single UCB donations, a major advance, holds great promise for CD34+ cell therapy of adults and larger children, and cheaper cultured RBC manufacture.

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OBJECTIVE: This case report discusses the treatment of 2 patients with cervicogenic headache (CHA) attending the Outpatient Clinic of the Hungarian National Institute for Rheumatology and Physiotherapy (Budapest, Hungary) and reviews the pathophysiology, therapeutic strategy, and problems associated with the treatment of CHA. CLINICAL FEATURES: Patient 1 was a 27-year-old female who sustained a whiplash injury. A sharp, shooting headache developed, readily induced, and aggravated by just bending the neck backward or by turning her head. Magnetic resonance imaging revealed a disk protrusion at C4-C5 pressing the anterior cerebrospinal space. Patient 2 was a 62-year-old female who sustained a whiplash injury; her cervical movements became restricted, which precipitated headaches. Magnetic resonance imaging revealed a paramedian disk hernia between the C4 and C5 vertebrae that intruded into the right ventral cerebrospinal space. INTERVENTION AND OUTCOME: After 4 weeks of manipulative therapy for patient 1, both active and passive range of motion returned to normal, and the high tumor necrosis factor-alpha (TNF-alpha) level (63 pg/mL) was substantially reduced (28 pg/mL). Patient 2 was started on manipulative therapy twice a week for 4 weeks; after 2 months, the patient became symptom-free, and high TNF-alpha level (72 pg/mL) was reduced greatly (35 pg/mL). CONCLUSION: Two patients with whiplash injury and disk herniation developed CHA associated with very high TNF-alpha levels. After manipulative therapy, these patients became symptom-free, and their TNF-alpha levels decreased substantially.

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Hepatitis virus infection persistent worldwide (approximately 600 m people) results in chronic hepatitis progressing to hepatocellular carcinoma (HCC) in many (approximately 1 m deaths/year). The review examines the usefulness of treating chronic viral hepatitis, including decompensated patients, by intentional coinfection with an attenuated infectious bursal disease virus (IBDV; apathogenic in man, stable at pH 2, orally administered). Learning lessons from the IBDV studies, the case is made to treat human immunodeficiency virus (HIV) infected patients (worldwide prevalence approximately 50 m people) by coinfecting with apathogenic hepatitis G virus (GBV-C). These ideas are reinforced by (i) eight out of ten studies reporting a beneficial effect of GBV-C viremia on HIV-related mortality or response to therapy and (ii) the recent reports of improved or delayed survival of HIV patients, naturally coinfected with an apathogenic virus.

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The changes induced in the distribution of the electrophoretic mobility (EPM) of human peripheral blood lymphocytes (HPBL), by various methods used to prepare the lymphocyte suspensions and eliminate platelets from them, were investigated on blood samples collected from healthy individuals and thrombopenic patients. Data showed that the distribution of the lymphocyte EPMs, i.e., the "lymphocyte electrophoregram," was dependent on the method chosen to enrich the suspension in the cell type of interest. The relative percentages of the low and high mobility cells, the two main subpopulations defined by lymphocyte electrophoresis, were different. The most striking artifactual differences in the lymphocyte electrophoregram were induced by the method of elimination of platelets; the distribution was unimodal and asymmetric when thrombin was used and bimodal when the blood sample, or the lymphocyte suspension, was placed on ice for 30 min (as is the practice in some laboratories). The "split" of the lymphocyte electrophoregram was found to be reversible within 90 min. Similar changes were observed on lymphocyte suspensions and blood samples of thrombopenic patients when the step for the elimination of platelets was not involved.

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Receptors for opiates, opiate-like substances, and their antagonists, such as naloxone (a close chemical and conformational congener of morphine), on brain cell homogenates and neuroblastoma X glioma hybrid cells in tissue culture have been reported. The present study on the binding of [3H]naloxone to lymphocytes and platelets freshly isolated from the peripheral blood of 39 healthy adult human volunteers showed that (1) [3H]naloxone bound to lymphocytes and platelets at 4 degrees C, reaching equilibrium in 30 min, and was not removed by washing (three times) with the suspending medium; (2) the binding of [3H]naloxone to cells decreased in the presence of increasing amounts of unlabeled naloxone, approaching a plateau; (3) significant amounts of the radioligand remained bound in the presence of micromolar quantities of the unlabeled ligand; and (4) in the absence of Na+ ions, 1 to 10 nmol of morphine hydrochloride for 10(6) lymphocytes, and 1 to 25 nmol of morphine hydrochloride for 10(8) platelets, decreased the binding of [3H]naloxone by 43 to 57%. It is concluded that at least some of the [3H]naloxone binding sites on human lymphocytes and platelets are specific opioid receptor sites of the mu type (Enkephalins define the delta sites.) The observations on the binding of naloxone to cells do not appear to be artifacts. Opioid receptor sites on lymphocyte and platelet membranes may have properties similar to those on nerve cell membranes.

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Triiodothyronine (T3)-receptor characteristics of isolated circulating human mononuclear cells have been studied in a group of obese patients who claimed to be unable to lose weight on conventional 4.2 MJ (1000 kcal) diets. The cells of the obese patients exhibited a lower receptor capacity than those of a control group of non-obese subjects but the difference was not significant. There was a significant fall (P less than 0.01) in receptor capacity in the obese patients after 12 weeks in a 1.34 MJ (320 kcal) per d formula diet and this provides a further mechanism whereby a fall in metabolic rate takes place in response to severe dietary restriction. Some patients who also received T3 (60 micrograms/d) in addition to the formula diet showed a further fall in the receptor numbers. These findings may partly account for the previously reported resistance to thyroid hormones in obesity.

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Obese patients taking a 1.34 MJ/day formula diet showed a steady decline in the rate of weight loss after several weeks and this was associated with a fall in RMR, serum T3 levels and the number of T3 receptors on peripheral lymphocytes. The addition of T3 (60 micrograms/day) to the dietary regime produced a significantly greater weight loss by the 12th week. Analysis of individual thyroid hormone levels revealed those patients taking the T3 tablets who achieved the greatest weight loss had the lowest serum T3 levels but the greatest suppression of serum T4 values. The serum T3 levels in the T3 treated subjects were negatively correlated with the lymphocyte rece 12th week. Analysis of individual thyroid hormone levels revealed those patients taking the T3 tablets who achieved the greatest weight loss had the lowest serum T3 levels but the greatest suppression of serum T4 values. The serum T3 levels in the T3 treated subjects were negatively correlated with the lymphocyte rece 12th week. Analysis of individual thyroid hormone levels revealed those patients taking the T3 tablets who achieved the greatest weight loss had the lowest serum T3 levels but the greatest suppression of serum T4 values. The serum T3 levels in the T3 treated subjects were negatively correlated with the lymphocyte receptor capacity. It is postulated that the fall in the number of T3 receptors is the primary event leading to the observed serum hormone level changes in the T3-treated patients.

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The distribution of the anodic electrophoretic mobilities (EPM) of human peripheral blood lymphocytes was determined for lymphocytes isolated from umbilical cord blood and from blood of individuals 6 months to 93 years of age. The distribution was bimodal in infants up to 2 years of age and suggested a small percentage of cells with a mobility of 0.95 micrometer s-1 V-1 cm. this value was chosen to discriminate between low-mobility cells (LMCs) and high-mobility cells (HMCs). The relative percentage of LMCs increased from birth to 2 years and two types of LMCs could be distinguished. The distribution was unimodal and asymmetric in children and adults and nearly Gaussian in aged people. Substantial differences between the distributions of the lymphocyte EPMs were seen on comparison of the histograms for individuals of similar ages. The analysis of the distribution of the lymphocyte EPMs on cell suspensions enriched in, or depleted of T or B cells confirmed the mobility of most T cells to be higher than the mobility of most B cells, whatever the age of the individual. The distribution of lymphocyte EPMs determined in the same adult over a 6 year period showed minor variations.

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Spleen lymphocytes and erythrocytes from congenic mice of diffrent haplotypes were characterized on a precise biophysical basis (the anodic electrophoretic mobility, EPM) to correlate any subtle differences in the cell surface topochemistry with the H-2 specificity. Spleen T lymphocytes from A.CA (H-2f) and A.SW (H-2s) mice exhibited high values EPM, which were significantly different. In contrast, significant differences in the EPM of B cells and erythrocytes were not observed. Cell electrophoresis of spleen T lymphocytes (without " contaminating " B cells), before and after the chemical modification of the cell surface by treatment with small/non-toxic concentrations of maleic anhydride, showed the number of lysine side chain amino-groups in the periphery of cells with H-2f specificity to be about twice those on cells with H-2s haplotype. Such a difference was observed both in the case of premature T lymphocytes and mature T lymphocytes. The difference in the number of cationogenic amino-groups in the cell periphery contributing a positive charge, would explain the oberved difference in the EPM of H-2f and H-2s spleen T lymphocytes, and suggest that the macromolecules coded by the H-2 genes or other genes under H-2 control lead to delicate differences in the chemical composition of the surface membranes of cells of the two H-2 haplotypes, expressed only on high EPM lymphocytes of spleen (T or T-like cells).

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Lymphocytes can be separated by electrophoresis into cells of high and of low electrophoretic mobility. Cells of high mobility are T cells and those of low mobility are B cells, with distinct immunological functions. Biophysical and biochemical studies on physically isolated and immunologically defined T and B cells in CBA mice showed striking differences in the chemical composition of the surface membranes.

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