Thyroxine feed-back on the regulation of thyrotrophin secretion

1. The role of thyroid hormone as a feed-back at the hypothalamic level in the control of thyrotrophin (TSH) secretion and release has been investigated by estimating the plasma and pituitary TSH levels following intrahypophysial and intrahypothalamic thyroid autotransplants.2. Thyroidectomized rats bearing thyroid autotransplants in the pituitary had a significantly lower (P < 0.001) plasma TSH than that of controls at 26 degrees C but not at 4 degrees C.3. Thyroidectomized rats bearing thyroid autotransplants in the supraoptic area showed a significantly lower (P < 0.001) level of plasma TSH and higher pituitary TSH at 4 degrees C but not at 26 degrees C.4. Study with both unilaterally and bilaterally thyroidectomized rats bearing thyroid autotransplants either in the pituitary or in the hypothalamus revealed that thyroxine feed-back operates at pituitary level in normal situations (26 degrees C) and there exists a feed-back through higher centres, specifically the TSR secreting area of the hypothalamus, in situations demanding higher thyroid function, as in cold exposure.     

Quantitation of the fourth complement component by electrophoresis in agarose gel containing antibodies

Electrophoresis in agarose gel containing antibodies can be used for quantitation of C4 in serum and plasma.

The normal range of the C4 concentration in serum and plasma varied between 40 and 200% of the standard pools. Low C4 values were found in systemic lupus erythematosus, in acquired haemolytic anaemia and in hereditary angioneurotic oedema.

Differences in C4 values were found between normal sera and the corresponding EDTA plasma, when tested after dilution in calcium-free and EDTA buffers, respectively. After storage of the samples for 1 day at 20°C or 37°C the C4 values decreased, most markedly in plasma; no further decrease was found on prolongation of storage, and the difference between serum and plasma C4 values disappeared. The plasma C4 levels gradually fell on storage at 4°C, but a clear difference between serum and plasma was still demonstrable after 5 days.

The method described is simple and quick and can be used routinely in large scale investigations.

     

The metabolism of autologous IgM and 19S rheumatoid factor in rheumatoid arthritis

The turnover of autologous preparations of radio-iodine labelled IgM and 19S rheumatoid factor was studied and compared in patients with severe rheumatoid disease. The IgM was isolated by block electrophoresis and column chromatography. 19S rheumatoid factor was isolated by a combination of euglobulin precipitation, column chromatography, and absorption onto and acid-elution from solid aggregated IgG.

Ten studies were made in seven patients, five with IgM, and five with 19S rheumatoid factor. In two patients the turnovers of 19S rheumatoid factor and IgM were studied simultaneously.

The turnover of IgM was similar to that reported for normal subjects and patients with other diseases: fractional catabolic rate 0·14–0·18, plasma and whole body T_½ 3·7–6·5 days, with 65–77% intravascular localization. The absolute catabolic rate for IgM was elevated (8–60 mg/kg/day).

The turnover of 19S rheumatoid factor isolated from serum was comparable in fractional catabolic rate (0·15–0·19) plasma and whole body T_½ (3·9–6·0 days) and intravascular localization (62–88%). No evidence of rapid catabolism of the `immune' elimination type was obtained.

     

Distribution of chylomicrons and albumin in dog kidney

1. Under specified experimental conditions the distribution space of labelled chylomicrons in the kidney was 13·8 ± 0·9 ml./100 g. tissue. The assumption is supported that this provides a measure for the quantity of intravascular plasma constituents.

2. Values for red blood cells and albumin distribution spaces were 5·2 ± 0·6 and 20·2 ± 1·0 ml./100 g tissue, respectively, in the whole kidney. The ratio of tissue haematocrit over simultaneous arterial haematocrit averaged 0·56. The extravascular albumin fraction amounted to about 31·0% of the total albumin in the whole kidney.

3. A statistically significant correlation was demonstrated between osmotic urine/plasma (U/P) ratios (within the approximate limits of 0·6-1·8) and quantities of extravascular albumin in the medulla.

     

Transport of potassium at the blood—brain barrier

1. The pial surface of different regions of the central nervous system of the rabbit have been bathed with artificial cerebrospinal fluid (c.s.f.), containing different concentrations of potassium. The object has been to change the composition of the interstitial fluid with respect to this ion, where it is adjacent to subarachnoid c.s.f.

2. Two techniques, subarachnoid perfusion from the supracallosal space between the hemispheres to the cisterna magna and barbotage from the cisterna magna, have been used. If the artificial c.s.f. contains Evans Blue, the former procedure results in maximum staining of the pia and underlying nervous tissue of the pons-medulla and spinal cord. The latter procedure results in maximum staining of the medial and supero-lateral surfaces of the cerebral hemispheres, particularly anteriorly.

3. During subarachnoid perfusion at 0·06 ml./min with the potassium-free fluid, most regions of the brain took up significantly greater amounts of ⁴²K than was the case during perfusion with the fluids containing 3 and 10 m-equiv/l. For blue cerebral cortex, the tissue subjected directly to the inflowing fluid and showing the biggest differences, the ratios, c.p.m. per g brain/c.p.m. per ml. plasma, were 1·71 ± 0·12 (+44%), 1·19 ± 0·05 and 1·07 ± 0·08 (-10%) during perfusion with the fluids containing, 0, 3 and 10 m-equiv/l. respectively.

4. During barbotage, the uptake of ⁴²K into pons-medulla and spinal cord from blood plasma, the concentration in the latter being effectively kept near constant, was, at the end of 2 hr, greater when the fluid contained potassium, 0 m=-equiv/l. rather than 10 m-equiv/l. Thus the ratio, c.p.m per g brain/c.p.m. per ml. plasma was (0·99 ± 0·04 (+36%) as against 0·73 ± 0·05 for pons-medulla where the difference was greatest.

5. Simultaneous measurements of the entry of [¹⁴C]urea from blood to different regions of the central nervous system revealed no significant differences due to the differing concentrations of potassium imposed by either barbotage or subarachnoid perfusion. This appears to exclude a non-specific cause for the changes in ⁴²K uptake, an example of which might be a changing blood flow.

6. Reasons are given for supposing that the big increase in ⁴²K uptake due to the potassium-free fluid must be due to events occurring at the blood—brain barrier. This might be some form of interaction, possibly the single file effect, such that a low potassium concentration in the interstitial fluid potentiates ⁴²K influx across the blood—brain barrier. Alternatively it might be due to a low potassium concentration in this fluid greatly reducing active potassium movement from interstitial fluid to blood. The former explanation would conform neatly with the present results; but the latter would additionally be compatible with other evidence concerning the homoeostasis of potassium concentration in c.s.f. and the interstitial fluid of brain.

     

Hepatitis B virus DNA stability in plasma samples under short-term storage at 42°C

We evaluated the stability of hepatitis B virus (HBV) DNA in plasma samples stored at 42°C for external quality assessment (EQA) panels of viral load. To assess the stability of plasma samples containing different concentrations of HBV DNA, serial dilutions of HBV-infected samples with a viral load of 6.40 log₍₁₀₎ IU/mL were made to yield viral loads of 5, 4, and 3 log₍₁₀₎ IU/mL. These were incubated at 42°C for up to 7 days and then frozen at -70°C. Viral load testing for HBV DNA was performed for all samples using COBAS^¯ AmpliPrep/COBAS^¯ TaqMan^¯ HBV Test (v.2.0, Roche, Switzerland). Results were compared with fresh frozen plasma samples as a benchmark to establish acceptable measurements on the days following sample collection. Although the results of this study demonstrated a decrease in HBV DNA viral load ranging from 0.005 to 0.30 log₍₁₀₎ IU/mL after storage at 42°C for up to 7 days, these values did not exceed 0.5 log₍₁₀₎, which is the estimated intra-assay variation for molecular tests. Thus, the insignificant decrease in viral load suggests that shipment of HBV in plasma samples at temperatures of up to 42°C is permissible if they are frozen within 7 days.     

Long-Term Survival of Streptococcus pneumoniae at Room Temperature on Dorset Egg Medium

Forty-five isolates of Streptococcus pneumoniae were inoculated on Dorset egg and supplemented Columbia agar base media, incubated overnight at 37°C, and then kept at room temperature (RT; 21°C) or 4°C. Long-term viability was best at RT for both media, with all isolates remaining viable on Dorset egg medium for 44 days; viability was 90 and 57% on Columbia agar base medium after 7 and 30 days. We recommend the use of Dorset egg medium for the maintenance of pneumococci at RT.     

Increased immunoglobulin production in silver catfish (Rhamdia quelen) exposed to agrichemicals

Fish vaccination has been increasingly exploited as a tool to control pathogen infection. The production of immunoglobulin following vaccination might be affected by several factors such as management procedures, water temperature, and the presence of xenobiotics. In the present study, we aimed to investigate the kinetics of immunoglobulin production in silver catfish (Rhamdia quelen) inoculated with inactivated Aeromonas hydrophila and kept at two different water temperatures (17.4±0.4° or 21.3±0.3°C). The effect of a second antigen inoculation and exposure of fish to sublethal concentrations of the herbicides atrazine and glyphosate at 10% of the lethal concentration (LC_50-96h) on specific serum antibodies were also investigated. Antibodies to A. hydrophila were detected as early as 7 days post-inoculation and increased steadily up to 35 days. The kinetics of antibody production were similar in fish kept at 17.4±0.4° and 21.3±0.3°C, and reinoculation of antigen at 21 days after priming failed to increase specific antibody levels. Intriguingly, we found that, in fish exposed to atrazine and glyphosate, the secretion of specific antibodies was higher than in non-exposed inoculated fish. These findings are important for the design of vaccines and vaccination strategies in Neotropical fish species. However, because atrazine and glyphosate are widespread contaminants of soil and water, their immune-stimulating effect could be harmful, in that fish living in herbicide-contaminated water might have increased concentrations of nonspecific antibodies that could mediate tissue injury.     

Thermal regulation of the immune response in South American toads (Bufo marinus)

Thermal regulation of the immune response was studied in toads following single injections of bacteriophage f2. The immune response was markedly inhibited in animals kept at 15° as compared to the controls (25°). The appearance of serum antibodies was delayed in animals kept at 15° for the first post-immunization week but their peak antibody levels were similar to those in toads maintained at 25° throughout. Transfer of animals from 25° to 15° 2 weeks after immunization only temporarily depressed the serum antibody levels but caused a marked delay in conversion from heavy to light antibodies.

Our results are in keeping with the hypothesis that lowered environmental temperatures inhibit a variety of metabolic processes which may be concerned with the utilization of antigen and/or the synthesis of antibodies.

     

Serum associated leucocyte locomotion inhibition and leucocyte motility in malignant melanoma: effect of BCG treatment

Serum-associated leucocyte locomotion inhibition (SALLI) and leucocyte motility were investigated in patients with malignant melanoma. Ten days after tumour excision twelve out of eighteen patients' sera exhibited a SALLI exceeding the normal range of 15%. The mean SALLI thus reached was 59·2±5·2%. No correlation was observed between SALLI and the level of invasion or the stage of the disease.

Six patients selected at random who had a mean SALLI of 71·5±5·8% after tumour excision were further treated by BCG immunotherapy and presented after 8·2±2·9 months of therapy with a significantly (P<0·01) lower SALLI of 32·6±8·1%. In eight patients treated exclusively by surgical excision, SALLI remained basically unchanged in the course of 10±2·8 months (29·0±8·0% vs 30·4±12·9%).

The mean index of leucocyte locomotion (LL) of eight melanoma patients who had received BCG for 11·2±2·3 months was 5·9±0·9 cells/field and thus significantly (P<0·01) higher in comparison with the mean index of LL (2·8±0·5) found in eight patients treated by surgical excision only 12·4±2·1 months before testing.

In addition, patients receiving BCG had a significantly higher (P<0·05) mean value of LL than fifteen healthy controls who presented with a mean index of LL of 3·4±0·3 cells/field. Our results permit the suggestion that BCG decreases SALLI and increases LL in melanoma patients.

     

Water uptake by Bufo melanostictus, as affected by osmotic gradients, vasopressin and temperature

1. The rate of water uptake across the skin was studied in the live toad, Bufo melanostictus. When toads were kept in distilled water at 29° C the uptake of water amounted to 16·9 ± 1·3 μl./cm²/hr; when bathed in sucrose or urea solutions, the water uptake diminished with increasing osmotic pressure. There was no water uptake observed when toads were kept in 200 m-osmolar sucrose or urea.

2. Intramuscular injections of vasopressin increased the rate of water uptake from distilled water. There was a good relation between doses and responses over various time intervals. A dose of 4 m-u. vasopressin/g body wt. doubled the rate of water uptake over a period of 1 hr. The same dose of vasopressin doubled the rate of water uptake when the toads were kept in solutions of sucrose or urea of different osmolarity.

3. The rate of water uptake when the toads were bathed in sodium chloride solutions was consistently 8 μl./cm²/hr greater than when bathed in sucrose or urea solutions of equal osmolarity. There was no water uptake when the sodium chloride solution was 285 m-osmolar.

4. Vasopressin (4 m-u./g) injected intramuscularly doubled the rate of water uptake from sodium chloride solutions of different osmolarity.

5. With solutions of potassium chloride, sodium nitrate, and potassium nitrate, in concentrations up to 150 m-osmoles/l., the rate of water uptake was found to be the same as with solutions of sodium chloride of the same osmolarity. Similarly, it was doubled by injection of vasopressin (4m-u./g).

6. The effect of temperature on the rate of water uptake before and after injection of vasopressin was investigated in toads kept in distilled water, sucrose, or sodium chloride solutions. For temperatures between 20 and 37° C, vasopressin (4 m-u./g) reduced the activation energy involved in the process of water uptake by 4000 cal.

7. The results agree with the view that water uptake follows a diffusion process which is facilitated by vasopressin, possibly as a result of increasing the size or number of available pores.

     

Autoradiographic demonstration of inhomogeneous distribution of sodium in single oocytes of Bufo bufo

1. Autoradiography of frozen cells labelled with ²²Na has been used to locate a sequestered fraction of internal Na in the oocyte which exchanges very slowly or not at all with external Li.

2. Relative grain density in nucleus and cytoplasm, measured photometrically, was used as an indication of ²²Na distribution within the oocyte. In test experiments grain density fell to 50% within 19 μm of the edge of the section. Owing to the large diameter of the oocyte (> 600 μm) and its nucleus (> 200 μm), this resolution was adequate to determine cytoplasmic/nuclear (C/N) ratios of grain density.

3. In oocytes fully loaded with ²²Na, the mean C/N ratio was 0·92 ± 0·03 (n = 11). After 5 hr exchange in Li-substituted Na-free Ringer solution, the mean C/N ratio was 2·18 ± 0·04 (n = 11). After 5 hr exchange in Ringer solution as a control, the mean C/N ratio was 1·39 ± 0·18 (n = 7). The cytoplasm thus contained a fraction of ²²Na inexchangeable with Li, and more slowly exchangeable with Na than that in the nucleus.

4. The non-Li-exchangeable fraction of internal Na thus revealed appeared to be quantitatively similar to that already demonstrated by studies of ²²Na fluxes and of internal Na activity by means of Na-sensitive micro-electrodes.

     

Cutaneous thermoreceptors in primates and sub-primates

1. Cutaneous thermoreceptors were examined electrophysiologically in primates (monkey, baboon) and in sub-primates (dog and rat) by recording from single units dissected from peripheral nerves.

2. Thermal stimuli were delivered from thermodes in contact with the skin.

3. Primate `cold' receptors had spot-like receptive fields and were found in both hairy and glabrous skin. The conduction velocities of the axons ranged from 0·6 to 15·3 m/sec.

4. The discharge from the primate receptors characteristically appeared in bursts with intervals of silence within the range temperatures of 18-40° C. Static and dynamic sensitivity curves were established, with maxima about 30° C.

5. Cold receptors in the lip of the dog had maximal sensitivity at 31-37° C. The axons were myelinated with conduction velocities less than 20 m/sec.

6. `Warm' receptors, with maximal sensitivity at 40° C and non-myelinated axons, were abundant in the scrotal nerve of the rat. The `cold' receptors had maximal responses at 23-28° C.

7. The `spurious' thermoreceptor behaviour of slowly adapting mechanoreceptors is described and the way in which they may distort integrated potential records from whole nerves is analysed.

     

IgA/IgC cryoglobulinaemia with vasculitis

A mixed IgA/IgG cryoglobulin complex was found in the serum of a 61-year-old man suffering from rheumatoid arthritis, Raynaud's phenomenon and vascular purpura. The purified complex was progressively insoluble at temperatures below 37°C. Reversible loss of cryoprecipitability was seen in conditions of extreme pH, in concentrated urea solutions and in 2-mercaptoethanol. Inactivation of complement at 56°C did not affect cryoprecipitability. The complex contained no complement and showed no anticomplementary activity. Analytical ultracentrifugation of the cryoglobulin at 37°C showed 7S and 11S components present in equal concentration.

The component immunoglobulins of the complex were separated by anion exchange chromatography at 37°C. Crossmixing studies with purified normal immunoglobulins indicated that the patient's IgA component was essential for cryoprecipitability of the complex. RA latex tests for anti-IgG activity at 37°C showed strong agglutination with both cryoglobulin complex and the isolated IgA component; this protein was found to be monoclonal with type K light chains.

Vasculitis, induced by skin testing the patient with his own plasma and isolated cryoglobulin was found to be histologically indistinguishable from that occuring spontaneously; reduction of symptoms paralleled reduction of cryoglobulin on treatment. These observations strongly support the hypothesis that the cryoglobulin complex is responsible for the patient's vasculitis.

     

The clearance and antidiuretic potency of neurohypophysial hormones in man,and their plasma binding and stability

1. Bio-assay techniques have been used to measure plasma levels of neurohypophysial hormones in man, following either a single injection or a continuous infusion.2. The median half-life of oxytocin after a single injection of 2 u. was 3·2 min (2·0-5·7, 95% confidence limits); this increased significantly (P < 0·01) to 4·8 min (4·4-6·1) when the hormone was infused at a rate of 500 m-u./min. The vasopressins had appreciably longer half-lives. After a single injection of 1 or 1·5 u. 8-lysine vasopressin (LVP), the half-life was 5·7 min (3·6-6·0). Continuous infusions of the hormones at a rate of 120 m-u./min yielded half-lives of 5·5 min (5·0-7·1) for LVP, and 5·6 min (3·9-9·5) for 8-arginine vasopressin (AVP).3. The apparent volumes of distribution of the hormones were all of the order of two thirds the extracellular volume.4. In accordance with its shorter half-life, the clearance of oxytocin was greater than that of the vasopressins (1·5 l./min, compared with 1·0 l./min).5. The antidiuretic potencies of the hormones were studied in over-hydrated subjects, by measuring the rate of urine excretion following an I.V. injection. Duration of antidiuretic action increased in the order: oxytocin, LVP, AVP. A 5:1 mixture of oxytocin and AVP was not as long-lasting as AVP alone. 8·5% (4-22) of an administered dose of AVP was excreted in the urine, and this amount was significantly correlated with urine volume (r = +0·67, P < 0·05).6. Ultrafiltration of human plasma containing exogenous hormones showed that 30% (13-50) of AVP was bound, the degree of binding being independent of concentration over the range used (50-400 μ-u./ml.) In contrast, oxytocin was completely unbound.7. Exogenous oxytocin was more stable than exogenous AVP in human plasma. At 4° C there was no significant loss of oxytocin until 7 days, whereas 20% of AVP was inactivated in 2 days. At 37° C a 20% loss of AVP occurred within 4 hr, and a 50% loss within 24 hr; corresponding times for oxytocin were 24 and 48 hr.     

Effect of incubation temperature on mitogen responses of lymphocytes from adult peripheral blood and from cord blood

Lymphocytes from normal adults, when cultured with phytohaemagglutinin (PHA) or Concanavalin A (Con A) at 39°C, showed an enhancement and earlier onset of ³H-thymidine incorporation compared with cultures incubated at 37°C. In contrast, the peak responses of cord blood lymphocytes incubated at either 35°C or 39°C did not differ significantly from those incubated at 37°C. Cultures of adult lymphocytes showed an exponential rise and fall in ³H-thymidine incorporation, which was much more rapid at 39°C than at 37°C. However, the kinetics of thymidine incorporation into mitogen-stimulated cord blood lymphocytes incubated at 39°C were similar to those at 37°C. The following results suggested that temperature acted predominantly on the proliferative phase of the transformation response. Firstly, by inhibiting binding of Con A using methyl-α-D-mannopyranoside, it was found that activation of adult lymphocytes took place within the same time period at both 39°C and 37°C. Secondly, cultures incubated at 37°C for 3 days, and labelled for 4 hr at either 37°C or 39°C showed no significant difference in ³H-thymidine uptake, whereas cultures incubated at 39°C for 3 days, and labelled for 4 hr at 37°C showed significantly higher responses than those both incubated and labelled at 37°C. Thirdly, the major increase in thymidine uptake occurred after incubation at 39°C for the second and third days of culture. These findings were consistent with a shortening of the cell cycle at the higher temperature. Thus, the failure of cord blood lymphocytes to show increased thymidine uptake after incubation at 39°C apparently reflects an insensitivity to temperature of certain of the metabolic pathways involved in cell replication in the neonate.     

The influence of stimulus parameters on contractions of isolated frog muscle fibres

1. Reversible changes in twitch and tetanus contractions of isolated frog muscle fibres were produced by varying the duration and strength of a transversely applied d.c. stimulus.

2. When the stimulus strength was at least 1·1 times threshold the peak force of the contraction elicited was reduced by stimulus durations longer than 1 msec at 20 °C, and by durations longer than 3 msec at 2-5 °C.

3. As the strength of a prolonged stimulus was progressively increased to 3 times rheobase or more, peak twitch and tetanus force declined to 40-70% of the amplitude with short (0·2-0·5 msec) stimuli. The time to peak of the twitch was slightly decreased and the first phase of relaxation was accelerated.

4. Cine-micrography of fibres which were permitted to shorten under a light load revealed that the side of the fibre adjacent to the anode was unactivated under conditions which also reduced peak force output.

5. With stimulus durations longer than 20 msec, peak force, time to peak, and relaxation time increased as the stimulus strength increased from 2·5 to 3 times rheobase. This might have been produced by a cathodal contracture following the action potential.

6. The results are discussed in relation to previous studies of activation in striated muscle.

     

Thyroid-stimulating antibody (TSAb) detected in sera of Graves'' patients using human thyroid cell cultures

As a homologous system is required to evaluate the effect of thyroid-stimulating antibody (TSAb) present in the serum of Graves' patients, primary cultures obtained from normal human thyroid gland have been used and the stimulatory effect measured as an increase of cAMP intracellular levels.

Monolayer cell cultures were stimulated by IgG purified from sera of Graves' patients or control subjects and compared to the effect of bovine TSH. Bovine TSH produced a dose-dependent increase in cAMP intracellular levels between 0·05 mU and 2·5 mU/ml, reaching a maximal value after 30 min with higher doses. While normal IgG had no effect, IgG prepared from untreated patients with frank Graves' disease elicited a significant increase in cAMP accumulation at a concentration between 0·05 and 0·5 mg/ml within 60 min in thirteen out of fourteen patients. A longer incubation period showed no further increase in cAMP values, even if in one case a higher concentration (5·0 mg/ml) of Graves' IgG had a delayed response. When the cAMP intracellular level modifications produced by Graves' IgG preparations in thyroid cell cultures were compared to those evoked in thyroid slices, an identical percentage (93%) of positive cases was obtained, without a coincidence of negative cases. Using thyroid slices the cAMP intracellular increase above basal levels was higher, if considered as a percentage, but in cultured cells a very low IgG concentration was sufficient to detect the presence of TSAb. No correlation between the two assays was found.

In conclusion, normal human cultured thyroid cells appeared to be a more suitable substrate when compared to human thyroid slices for detecting the presence of TSAb in Graves' disease and for studying its effect on thyroid cells. However, a 100% TSAb positivity was present in our Graves' patient series only when both assays were used.

     

Motor innervation, motor unit organization and afferent innervation of m. extensor digitorum communis of the baboon's forearm

1. One hundred and fifty efferent axons innervating m. extensor digitorum communis (EDC) were isolated in filaments of C7 and C8 ventral roots of baboons. Conduction velocities were measured antidromically by stimulating the muscle nerve and recording from the filaments, and fell into two groups: a fast (49-84 m/sec) and a slow (22-41m/sec), presumably fusimotor group. The threshold for these latter axons exceeded the strength needed to elicit the maximal motor twitch.

2. Stimulation of ventral root filaments containing slow axons produced no contractile tension in EDC.

3. Stimulation of ventral root filaments containing fast-group axons elicited all-or-nothing twitches of motor units of EDC. The twitch tensions of 66·3% of the units were < 2·0 g wt.; only 8·7% were > 5·0 g wt. Tetanus-twitch ratios were 1·4-4·7 in a sample of 14 units. Contraction times were between 15 and 35 msec in 97% of the units. There was no correlation between contractile properties and axonal conduction velocity.

4. Afferent volleys from the stimulated EDC nerve were recorded from C6 or C7 dorsal roots. The threshold was below the threshold for a just-detectable motor twitch in ten out of eleven baboons. Conduction velocity of the earliest component of the muscle afferent volley was 67-83 m/sec.

5. The conduction velocities of twenty-eight spindle afferents, identified by their responses to linear stretches of EDC and by their unloading by maximal twitches, were all < 70 m/sec. Higher dynamic sensitivity tended to be associated with higher conduction velocity.

     

Correlation of disease activity and Clq-binding immune complexes with the neutrophil inclusions which form in the presence of SLE sera

Intracytoplasmic inclusions containing immunoglobulin (Ig) and complement (C3) are found in normal neutrophils (PMN) after incubation with sera from patients with SLE. These inclusions are believed to be immune complexes removed by phagocytosis from the SLE patients' sera in vitro. Similar inclusions were also noted in the circulating PMN from some patients with SLE. In the present study we have examined the relationship between the presence of intracytoplasmic inclusions and various clinical and laboratory features of SLE. Blood from forty-five patients with SLE was drawn and separated at 37°C. Fresh heparinized blood was also obtained from normal volunteers and allowed to stand for 90 min at 37°C. The buffy coat cells from both normal and SLE groups were removed, centrifuged, washed and examined (direct method) or incubated in the SLE sera for 90 min at 37°C (indirect method). Slides of washed cells were prepared in the cytocentrifuge, stained with fluorescein-conjugated goat anti-human IgG, IgM, IgA and C3 and examined under ultra-violet light.

By the direct method, 24% of patients had small intracytoplasmic inclusions in their neutrophils when stained for IgG suggesting that immune complexes were phagocytosed in vivo. None of twenty-one normal controls had similar inclusions. By the indirect method, 62% of SLE patients were positive for IgG, 15% for IgM, 8% for IgA and 31% for C3. None of the twelve normal controls were positive.

By the indirect method, PMN inclusions containing both IgG and IgM correlated with clinical activity (P<0·001), depressed serum complement (CH50, P=0·026; and C3, P<0·051), cryoglobulinaemia (P=0·014), anti-nDNA antibodies (P<0·001) and Clq-binding immune complexes (P=0·008). A suggestive correlation with granulocytopenia was also observed. The presence of inclusions containing IgG alone did not correlate with any of these parameters. C3 and IgM appeared to be mutually exclusive, i.e. neither was present simultaneously. These findings suggest (1) that normal PMN on exposure to SLE sera develop intracytoplasmic inclusions by phagocytosis of immune complexes, (2) the presence of such complexes correlates with a number of parameters of disease activity, particularly when IgG and IgM are both present and (3) such complexes may be phagocytosed in vivo as suggested by the presence of inclusions in vivo and contribute to a number of granulocyte disturbances seen in patients with SLE. These abnormalities in granulocyte function may be important, predisposing factors for infection in patients with active SLE.