Chromatin proteins from human lymphocytes: a gel electrophoretic comparison between normal, mitogen-stimulated, cell-line, and chronic lymphocytic leukemia lymphocytes.

Cell-line lymphocytes contained considerably more nonhistone chromatin protein than normal lymphocytes. The overall electrophoretic pattern of the nonhistone chromatin proteins from nonreplicating cell types with predominantly condensed chromatin (normal lymphocytes and chronic lymphocytic leukemia lymphocytes) was similar, as it was between replicating cell types with predominantly decondensed chromatin (mitogen-stimulated normal lymphocytes and cell-line lymphocytes), although all four cell types differed qualitatively. A "heavy" chromatin fraction from cell-line lymphocytes could be distinguished from a "light" fraction and had a pattern similar to that from cells of the condensed chromatin type. Normal lymphocytes contained two acid-soluble chromatin polypeptieds not found in cell-line lymphocytes.

Preparation of plasma membranes from line 10 and line 1 guinea pig hepatomas.

Gentle homogenization followed by differential and density gradient centrifugation was used to purify line 10 and line 1 guinea pig hepatoma plasma membranes in the form of ghosts. Yields of 15--25% allowed enough membranes to be obtained from a single ascites tumor-bearing animal for immunologic and biochemical studies. Although the plasma membrane marker enzyme (Na+ + k+)atpase was present in normal concentrations in both line 10 and line 1 hepatomas, 5'-nucleotidase was reduced over 100-fold in both tumors and phosphodiesterase I was increased 210-fold in the line 10 hepatomas.

Immunotherapy in infectious disease, autoimmunity and cancer.

The clinical and immunologic effects of transfer factor and of levamisole were evaluated in over 200 patients with a variety of diseases. With transfer factor, the most encouraging results were observed in patients with the Wiscott-Aldrich syndrome, chronic mucocutaneous candidiasis, coccidioidomycosis, Behcet's disease and malignant melanoma. With levamisole, the most promising results were observed in patients with recurrent aphthous stomatitis, rheumatoid arthritis and herpes simplex infections, especially ocular herpes. Immunologically, transfer factor usually caused conversion of skin test reactivity and conversion of in vitro tests of cellular immunity as well, whereas levamisole caused increases in skin test reactivity without a parallel change in in vitro para meters, suggesting that the two agents may have different mechanisms of action. In a limited number of patients with multiple sclerosis, reactivity to three viral antigens was found to be lower than that in normal subjects, as measured by lymphocyte stimulation. Following levamisole therapy, this reactivity increased to normal levels, but the patients did not show clinical benefit.

Plasmapheresis in idiopathic inflammatory myopathy. Experience with 35 patients.

Thirty-five patients with idiopathic inflammatory myopathy (IM) were treated with plasmapheresis. They had had inadequate clinical responses to prednisone or to prednisone and cytotoxic immunosuppressive drug therapy. All patients received cyclophosphamide or chlorambucil in addition to plasmapheresis; 33 also received prednisone. Strength increased in 32 patients during combined therapy; patients with the most active disease experienced substantial improvement that approached clinical remission in some cases. Clinical improvement was documented by muscle testing, forced vital capacity, electromyography, muscle biopsy, and serum creatine phosphokinase activity. The major side effect of treatment was herpes zoster. Primary biliary cirrhosis improved in one patient. Although the effects of plasmapheresis cannot be dissociated from those of the concomitantly administered immunosuppressive drugs, the improvement seen in patients with active disease suggests that plasmapheresis with immunosuppressive drug therapy may find a place in their treatment and implicates circulating factors in the pathogenesis of IM.

Patients with polymyositis and dermatomyositis who undergo plasmapheresis therapy. Pathologic findings.

Muscle biopsy specimens were studied in 20 of 26 patients who had polymyositis or dermatomyositis prior to treatment with plasmapheresis and immunosuppressive therapy. Morphologic findings from biopsy specimens were similar among patients with polymyositis and dermatomyositis, except that perifascicular atrophy was a prominent feature in patients with dermatomyositis and inconspicuous or absent in patients with polymyositis; immunofluorescent staining of muscle for immunoglobulins and/or complement was noted more frequently for patients with dermatomyositis than those with polymyositis. Patients judged clinically to have highly active disease had the most severe changes, which consisted of muscle necrosis, regeneration, phagocytosis, and inflammation; those with mild and moderately active disease were indistinguishable pathologically. No correlation between duration of disease and degree of pathologic alteration was found. Six of seven patients with both pretreatment and posttreatment biopsies showed marked improvement in the extent of pathologic alteration, and a statistically significant reduction in the degree of muscle atrophy following treatment.

Antibody to acetylcholine receptor in canine and human myasthenia gravis: differential cross-reactivity with human and rabbit receptor.

Anti-acetylcholine receptor (AChR) antibody (ab) was found in the serum of a dog with acute myasthenia gravis (MG) by the use of Cowan 1 strain Staphylococcus aureus to bind radiolabeled anti-AChR ab-AChR immune complexes. Fifteen months later, when the dog was in remission, there was only a very low level of the anti-AChR ab. These observations strengthen the contention that anti-AChR ab is important in the pathophysiology of myasthenia gravis. Higher titers of the canine ab were measured with rabbit than with human AChR, whereas 17 human MG sera, selected to represent a wide range of anti-AChR ab titers, were all more reactive with human AChR. The degree of cross-reactivity of human anti-AChR ab with rabbit AChR varied widely, indicating a heterogeneous population of anti-AChR ab molecules in human myasthenia gravis sera.

Plasmapheresis in multiple sclerosis: preliminary findings.

In seven of eight patients with progressive multiple sclerosis subjected to long-term plasmapheresis in combination with azathioprine and pulsed prednisone therapy, we found modest improvement of neurologic function. There was no change in auditory and visual evoked responses or serum demyelinating activity. In six of seven patients, cerebrospinal fluid IgG content decreased. Three additional patients in acute, severe exacerbation refractory to prednisone therapy made a substantial recovery, which commenced with plasmapheresis therapy. In two of them, the onset of clinical improvement after plasmapheresis was corroborated by decreased latency or increased amplitude of somatosensory evoked potentials. These results suggest that blood-borne factors, possibly autoantibodies, may play a role in the pathogenesis of the disease. The lesions may be at least partially reversible with plasmapheresis therapy, but a controlled trial is necessary to confirm these preliminary findings.

Double-blind study of true vs. sham plasma exchange in patients treated with immunosuppression for acute attacks of multiple sclerosis.

We enrolled 116 patients in a multicenter, randomized, double-blind controlled trial of an 8-week course of 11 plasma exchange (PE) treatments in exacerbations of MS. The control group received sham PE, and both groups received identical treatment with IM ACTH and oral cyclophosphamide. Serum IgG decreased in the PE and sham treatment groups by 76% versus 22% by treatment 5, and by 64% versus 14% by treatment 11. PE also produced significant reductions in IgA, IgM, C3, and fibrinogen. PE patients had moderately enhanced improvement at 2 weeks relative to the sham group. PE patients with relapsing/remitting disease had significantly enhanced improvement at 4 weeks and there was also an increased improvement at 12 months, although this latter effect disappeared when we analyzed relapsing/remitting patients as a separate subgroup. Life table analysis showed the median time to recover preattack disability status was shorter in PE- than in sham-treated relapsing/remitting patients (4 vs. 13 weeks), a result confirmed by raw disability status scores in which there was recovery to their average preattack disability score by 3 months. PE given with ACTH plus cyclophosphamide enhances recovery from an exacerbation of disease in relapsing/remitting patients, although we observed no clear long-term benefits.

Intrafamily fragment analysis of the T cell receptor beta chain CDR3 region.

The length distributions of the third complementarity determining region (CDR3) of the T cell receptor (TCR) beta chain were determined quantitatively from peripheral blood lymphocytes of healthy donors. RT-PCR products of 26 V beta families and subfamilies were analyzed by an A.L.F. DNA sequencer and Fragment Manager software. We established a normal reference of CDR3 lengths for most of the V beta families and subfamilies. The known range of CDR3 lengths for the V beta chain was expanded to 24 amino acids, and quantitative measurements were made for each fragment length allowing intrafamily CDR3 fragment length comparisons. Importantly, we were able to analyze intrafamily CDR3 fragment distribution without optimizing PCR conditions, thereby circumventing a major obstacle found in intrafamily TCR beta chain comparisons.

Increased proliferation of blood mononuclear cells after plasmapheresis treatment of patients with demyelinating disease.

Induction of lymphocytic proliferation has been postulated to be a mechanism whereby plasmapheresis may enhance the action of cytotoxic immunosuppressive drugs. This study found increased spontaneous proliferation of peripheral blood mononuclear cells following intensive plasmapheresis treatment of patients with multiple sclerosis (MS) or Guillain-Barré syndrome (GBS). The increased proliferative response was reduced below baseline in four of six MS patients who received subsequent immunoglobulin intravenous (IGIV) and pulsed cyclophosphamide therapy, but not in three MS patients receiving IGIV alone. In five GBS patients with low baseline proliferation, proliferation also increased after plasmapheresis. High baseline proliferation found in three GBS patients may have reflected antecedent infection, since it fell during plasmapheresis in the two patients in whom it was measured. Plasmapheresis could possibly augment the effectiveness of cytotoxic drugs in controlling autoimmunity by inducing lymphocytes to proliferate, thereby making them more susceptible to drug action.

Increased antibody production in peripheral blood mononuclear cells after plasma exchange therapy in multiple sclerosis.

It has been difficult to study antibody synthesis in vivo following plasma exchange (PE) because of the intra- and extravascular compartmentation of immunoglobulins, and their altered lymphatic flow and catabolism after the procedure. There is fragmentary experimental and clinical evidence that antibody synthesis may increase due to removal of autoregulatory factors by PE. The present study showed increased in vitro spontaneous immunoglobulin (Ig) G and IgM production, as well as increased spontaneous lymphocytic proliferation, after intensive PE in 10 multiple sclerosis (MS) patients. Cytofluorometric control of cultured mononuclear cell populations revealed no change with treatment except for a small increase in B lymphocytes inadequate to account for the increased immunoglobulin production. These findings indicate an activation of the immune system through PE, and suggest the possible need for adjunctive immunosuppression to control antibody production in autoimmune diseases with subacute, relapsing, or chronic courses when PE is employed as a therapeutic modality.

Treatment of refractory familial hypercholesterolemia by low-density lipoprotein apheresis using an automated dextran sulfate cellulose adsorption system. The Liposorber Study Group.

A subgroup of patients with familial hypercholesterolemia (FH) respond inadequately to standard diet and drug therapy, and are therefore at high risk for the premature development or progression of coronary artery disease. This study evaluated low-density lipoprotein (LDL) cholesterol and lipoprotein (a) removal in a multicenter, controlled trial with a new LDL apheresis procedure (Liposorber LA-15 System). The study comprised patients with FH who had not responded adequately to diet and maximal drug therapy. There were 54 patients with heterozygous FH (45 randomized to treatment and 9 control subjects) and 10 with homozygous FH (all of whom received LDL apheresis). The study included three 6-week treatment phases and a 4-week rebound phase. Treatments were administered at 7- to 14-day intervals. Mean acute reductions in LDL cholesterol were 76% in heterozygous FH patients and 81% in homozygous ones. Time-averaged levels of LDL cholesterol were reduced 41% (243 to 143 mg/dl) in heterozygous FH patients and 53% (447 to 210 mg/dl) in homozygous ones. The substantial acute reduction of lipoprotein (a) (means: 65%, heterozygous FH; 68%, homozygous FH) has not been reported with other therapies. The Liposorber LA-15 System represents an important therapeutic option in FH patients who respond inadequately to diet and drug therapy.

Long-term effects of low-density lipoprotein apheresis using an automated dextran sulfate cellulose adsorption system. Liposorber Study Group.

The short-term effectiveness of low-density lipoprotein (LDL) apheresis using a dextran sulfate cellulose adsorption column technique was previously examined in a 9-center, 22-week controlled trial in 64 patients with familial hypercholesterolemia (FH) who did not adequately respond to diet and drug therapy. Forty-nine patients (40 treatment, 9 controls) subsequently received LDL apheresis procedures as part of an optional follow-up phase. This study reports on the long-term safety, lipid lowering, and clinical efficacy of LDL apheresis for the 5-year period that includes both the initial controlled study and follow-up phase. During this time, patients received a total of 3,902 treatments of which 3,314 treatments were given during the follow-up phase. Adverse events were infrequent, occurring in 142 procedures (3.6%). Immediate reduction in LDL cholesterol was 76% both in homozygotes and in heterozygotes. Patients with homozygous FH had a progressive decrease in pretreatment LDL cholesterol level along with an increase in high-density lipoprotein (HDL) cholesterol level. There was no appreciable change in pretreatment lipoprotein level over time in heterozygotes. The rate of cardiovascular events during therapy with LDL apheresis and lipid-lowering drugs was 3.5 events per 1,000 patient-months of treatment compared with 6.3 events per 1,000 patient-months for the 5 years before LDL apheresis therapy. These findings support the long-term safety and clinical efficacy of LDL apheresis in patients with heterozygous and homozygous FH who are inadequately controlled with drug therapy.

Chronic arsenic poisoning masquerading as Landry-Guillain-Barré syndrome.

Acute arsenic intoxication may present as Landry-Guillain-Barré syndrome because of similarities in clinical symptoms involving the gastrointestinal tract, weakness, and sensory symptoms. Electrodiagnostic findings may be similar with demyelinating changes predominating early in both diseases. A case is presented of repeated arsenic poisoning over two years misdiagnosed as Landry-Guillain-Barré syndrome. Proximal F-loop latency (M-wave latency at wrist + F-wave latency at wrist - 2 M-wave latency at axilla) helped to establish the correct diagnosis. Serial electrodiagnostic studies were done documenting the evolution of chronic repeated arsenic poisoning from a picture showing demyelination to one with severe axonal loss.