Positive outcomes following Autologous Matrix-Induced Chondrogenesis (AMIC) in the treatment of retropatellar chondral lesions: a retrospective analysis of a patient registry.

BACKGROUND: The patellofemoral joint is a challenging environment for treating chondral defects. Among the surgical options for the treatment of chondral defects, the single-stage Autologous Matrix-Induced Chondrogenesis (AMIC) procedure uses a porcine collagen I/III membrane to enhance bone-marrow stimulation. However, longer term outcomes data are rare for this specific indication. In order to provide real-world information, an ongoing registry has been established to record patient data and outcomes when AMIC is used to treat chondral and osteochondral lesions. METHODS: Patient data were retrieved from an ongoing, prospective, multisite registry of patients who had undergone AMIC treatment of chondral defects. We identified 64 patients who had undergone AMIC for patellofemoral chondral defects and for whom pre-operative and at least 1 post-operative score were available were included in this retrospective data analysis. Outcomes were assessed via the KOOS, VAS pain, and the Lysholm scores. Outcomes at the post-operative time-points were analysed using a factorial ANOVA with post-hoc testing while linear regression was used to assess associations between the change in the Lysholm score and lesion size. RESULTS: There was a significant improvement in Lysholm, VAS pain, and KOOS scores from pre-operative to the 1st year post-operative (p < 0.001), and this was maintained during the follow-up. CONCLUSIONS: The forces exerted on the patellofemoral joint make this a challenging scenario for chondral repair. Our data demonstrates that the AMIC procedure with a collagen I/III membrane is an effective treatment for retropatellar cartilage lesions, and provides reliable results, with decreased pain and improved function. Importantly, these improvements were maintained through the follow-up period.

Outcome of Autologous Matrix Induced Chondrogenesis (AMIC) in cartilage knee surgery: data of the AMIC Registry.

INTRODUCTION: Autologous Matrix-Induced Chondrogenesis (AMIC) is an innovative treatment for localized full-thickness cartilage defects combining the well-known microfracturing with collagen I/III scaffold. The purpose of this analysis was to evaluate the medium-term results of this enhanced microfracture technique for the treatment of chondral lesions of the knee. METHODS AND MATERIALS: Patients treated with AMIC (Chondro-Gide, Geistlich Pharma, Switzerland) were followed using the AMIC Registry, an internet-based tool to longitudinally track changes in function and symptoms by the Lysholm score and VAS. RESULTS: A series of 57 patients was enrolled. The average age of patients (19 females, 38 males) was 37.3 years (range 17-61 years). The mean defect size of the chondral lesions was 3.4 cm(2) (range 1.0-12.0 cm(2)). All defects were classified as grade III (n = 20) or IV (n = 37) according to the Outerbridge classification. Defects were localized at the medial (n = 32) or lateral (n = 6) condyle, at the trochlea (n = 4) and at the patella (n = 15). The follow-up period was 2 years. The majority of patients were satisfied with the postoperative outcome, reporting a significant decrease of pain (mean VAS preop = 7.0; 1 year postop = 2.7; 2 years postop = 2.0). Significant improvement of the mean Lysholm score was observed as early as 1 year after AMIC and further increased values were noted up to 2 years postoperatively (preop. 50.1, 1 year postop. 79.9, 2 year postop. 85.2). CONCLUSIONS: AMIC is an effective and safe method of treating symptomatic chondral defects of the knee. However, further studies with long-term follow-up are needed to determine if the grafted area will maintain structural and functional integrity over time. LEVEL OF EVIDENCE: Prognostic study, Level IV.

Fine-mapping the immunodominant antibody epitopes on consensus sequence-based HIV-1 envelope trimer vaccine candidates.

The HIV-1 envelope glycoprotein (Env) trimer is the key target for vaccines aimed at inducing neutralizing antibodies (NAbs) against HIV-1. The clinical candidate immunogen ConM SOSIP.v7 is a stabilized native-like HIV-1 Env trimer based on an artificial consensus sequence of all HIV-1 isolates in group M. In preclinical studies ConM SOSIP.v7 trimers induced strong autologous NAb responses in non-human primates (NHPs). To fine-map these responses, we isolated monoclonal antibodies (mAbs) from six cynomolgus macaques that were immunized three times with ConM SOSIP.v7 protein and boosted twice with the closely related ConSOSL.UFO.664 immunogen. A total of 40 ConM and/or ConS-specific mAbs were isolated, of which 18 were retrieved after the three ConM SOSIP.v7 immunizations and 22 after the two immunizations with ConSOSL.UFO.664. 22 mAbs (55%) neutralized the ConM and/or ConS virus. Cross-neutralization of ConS virus by approximately one-third of the mAbs was seen prior to ConSOSL.UFO.664 immunization, albeit with modest potency. Neutralizing antibodies predominantly targeted the V1 and V2 regions of the immunogens, with an apparent extension towards the V3 region. Thus, the V1V2V3 region is immunodominant in the potent NAb response elicited by two consensus sequence native-like HIV-1 Env immunogens. Immunization with these soluble consensus Env proteins also elicited non-neutralizing mAbs targeting the trimer base. These results inform the use and improvement of consensus-based trimer immunogens in combinatorial vaccine strategies.

Rod-cone interaction in human scotopic vision.

Thresholds of a test flash were measured at various time intervals from onset of a conditioning flash under parafoveal scotopic conditions; rods or cones were selectively stimulated by utilizing either 420- or 680-nanometer light. Rod-cone interaction was indicated because conditioning flash presentation increased test threshold above control level for heterochromatic as well as for homochromatic stimulus pairs. The time course of these t.. reshold changes indicates that the rod system has a longer latency than the cone system.

Activation of renal cortical adenylate cyclase by circulating immunoreactive parathyroid hormone fragments.

Three distinct immunoreactive species of parathyroid hormone (PTH) are present in human serum. One has an estimated mol wt of 9,500 and probably represents glandular hormone, the second 7,000-7,500 mol wt, and the third 4,500-5,000 mol wt. In order to assess the biological activity of these circulating forms of PTH, we determined their ability to activate renal cortical adenylate cyclase. The 9,500 mol wt and 4,500-5,000 mol wt fractions produced four- to sixfold increases in cyclic 3',5'-AMP accumulation above control; the 7,000-7,500 mol wt fraction was inactive. None of the fragments had any effects on phosphodiesterase activity. Antiserum to bovine PTH did not block the activation of adenylate cyclase by either the gragments or bovine PTH. The data suggest that a large proportion of circulating immunoreactive human PTH is biologically active and that the biologically and immunologically active sites of the hormone are distinct.

The role of phosphate in the secretion of parathyroid hormone in man.

In man, oral administration of 1 g of phosphorus resulted in a 60-125% increase in serum immunoassayable parathyroid hormone (PTH) concentration. Peak PTH levels were attained in 1 hr, and PTH returned to base line levels in 2 hr. This increase in PTH appeared to be initiated by a very small decrease of total and ionized calcium and was abolished by a calcium infusion. There was no correlation between serum phosphorus and PTH. The experiments show that oral phosphorus administration initiates a calcium-mediated control system for PTH secretion and that this system operates very sensitively in man.

Evidence for secondary hyperparathyroidism in idiopathic hypercalciuria.

Circulating levels of immunoreactive parathyroid hormone (PTH) were measured in 40 patients with idiopathic hypercalciuria (IH) before and during reversal of hypercalciuria with thiazide, and in four normal subjects before and during induction of hypercalciuria with furosemide. 26 patients with IH had elevated serum PTH levels. The remaining patients had normal levels. Although the correlation was not complete, high PTH levels were generally found in patients who had more severe average urinary calcium losses. When initially elevated. PTH levels fell to normal or nearly normal values during periods of thiazide administration lasting up to 22 months. When initially normal, PTH levels were not altered by thiazide. Reversal of hyperparathyroidism by thiazide could not be ascribed to the induction of hypercalcemia, since serum calcium concentration failed to rise in a majority of patients. Renal hypercalciuria produced by furosemide administration elevated serum PTH to levels equivalent to those observed in patients with IH. The findings in this study help to distinguish between several current alternative views of IH and its relationship to hyperparathyroidism. Alimentary calcium hyperabsorption cannot be the major cause of IH with high PTH levels, because this mechanism could not elevate PTH. Idiopathic hypercalciuria cannot be a variety of primary hyperparathyroidism, as this disease is usually defined, because PTH levels are not elevated in all patients and, when high, are lowered by reversal of hypercalciuria. Primary renal loss of calcium could explain the variable occurrence of reversible hyperparathyroidism in IH, since renal hypercalciuria from furosemide elevates serum PTH in normal subjects. Consequently, a reasonable working hypothesis is that IH is often due to a primary renal defect of calcium handling that leads, by unknown pathways, to secondary hyperparathyroidism.

Circadian rhythm in serum parathyroid hormone concentration in human subjects: correlation with serum calcium, phosphate, albumin, and growth hormone levels.

A circadian variation in serum calcium, albumin and PTH concentration in normal subjects has been demonstrated. The levels of the three blood constituents were remarkably constant during the day, but striking night and early morning changes occurred. Serum calcium levels were highest at 8:00 p.m. and reached a nadir between 2:00 and 4:00 a.m. Serum albumin levels were parallel to those of serum calcium. PTH levels began to rise after 8:00 p.m., reached the highest levels between 2:00 and 4:00 a.m., and fell to baseline values by 8:00 a.m. The nocturnal fall in serum calcium levels appears to be secondary to dilution of serum proteins by increasing blood volume. The nocturnal rise in PTH levels appears to be independent of serum calcium levels within the normal range but it can be abolished by induced hypercalcemia. Serum phosphate levels were lowest between 8:00 a.m. and 10:00 a.m. and highest between 2:00 a.m. and 4:00 a.m. The data presented suggest that circadian changes in serum phosphate levels are not mediated in toto by parathyroid hormone but they are exaggerated when the secretion of this hormone is inhibited. They are independent of growth hormone levels and activity but they are greatly modified during a prolonged fast.

Metabolic consequences of oral administration of 24,25-dihydroxycholecalciferol to uremic dogs.

24,25-dihydroxycholecalciferol [24,25-(OH)(2)D(3)], once considered a relatively inert metabolite of vitamin D(3), has been recently recognized as a metabolically active product in some species. In previous studies, we have shown that infusion of 24,25(OH)(2)D(3) into the thyroid artery of normal dogs results in prompt and complete suppression of parathyroid hormone (PTH) secretion. In this study, we have examined the metabolic consequences of oral administration of this metabolite in dogs with experimentally induced renal hyperparathyroidism. Dogs with comparable degrees of renal insufficiency (glomerular filtration rate, 10-15 ml/min) were treated for 3 wk with daily doses of either 2 mug of 24,25(OH)(2)D(3) or 50% ethanol, the vehicle in which the metabolite was suspended. After a 6-wk recovery period, treatments were reversed: dogs who had previously served as controls received the metabolite while dogs previously treated with metabolite received the vehicle. Administration of 24,25(OH)(2)D(3) resulted in a 40-60% decrease of immunoreactive PTH. This was associated with a small (0.1-0.2 mg/dl) but unequivocal decrease of serum ionized calcium. Calcium balance, which was slightly negative under control conditions, became slightly but definitively positive on treatment with 24,25(OH)(2)D(3). All other parameters measured, including total serum calcium, magnesium, phosphorus, creatinine, electrolytes, phosphorus excretion, and phosphorus balance, remained unchanged. The data support the hypothesis that 24,25(OH)(2)D(3) not only decreases PTH secretion but also functions as an anabolic hormone in bone under the conditions of this experiment.

Inhibition of parathyroid hormone secretion by 25-hydroxycholecalciferol and 24,25-dihydroxycholecalciferol in the dog.

We studied the effects of vitamin D metabolites on parathyroid hormone (PTH) secretion. Test materials were injected into the cranial thyroid artery of the dog, and immunoreactive PTH was measured frequently in serum samples from the inferior thyroid vein and the femoral vein. This model for the study of secretion had previously been validated with the use of known modulators on PTH secretion. In control experiments, injection of 100% ethanol, the vehicle in which cholecalciferol (D(3)) metabolites were suspended, resulted in no change in PTH secretion. Likewise, native vitamin D(3), in doses ranging from 250 to 1,250 ng had no effect on PTH secretion. 25-Hydroxycholecalciferol, 25-(OH)D(3), in doses of 125-240 ng, caused complete suppression of PTH secretion. When 24,25-dihydroxycholecalciferol, 24,25-(OH)(2)D(3), was injected in doses of 50-250 ng, suppression of PTH secretion was again complete; in doses of 5 ng, injection of this metabolite resulted in significant but incomplete suppression of secretion. In doses of 50-250 ng, 1,25-(OH)(2)D(3) strongly stimulated PTH secretion, but in a dose of 5 ng this metabolite had no effects. Injection of equal doses of 1,25-(OH)(2)D(3) and 24,25-(OH)(2)D(3) resulted in significant suppression of PTH secretion. Hypocalcemia-induced stimulation of PTH secretion was suppressed by 24,25-(OH)(2)D(3) while hypercalcemia-induced suppression of PTH secretion was stimulated by 1,25-(OH)(2)D(3). In all experiments showing suppression of PTH secretion, peripheral PTH decreased. Arguments are presented for considering the suppressive effects of D(3) metabolites as physiologic modulators. However, this stimulating effect of 1,25-(OH)(2)D(3) occurred only in pharmacologic doses and hence probably has no physiologic relevance.

On the pathogenesis of hyperparathyroidism in chronic experimental renal insufficiency in the dog.

Healthy adult dogs were subjected to stepwise reduction of nephron population so as to create the transition from normal renal function to advanced renal insufficiency. Studies were performed at each level of renal function. Glomerular filtration rate (GFR), renal phosphate clearance, and serum radioimmunoassayable parathyroid hormone (PTH) levels were measured. Two groups of animals were studied. In one, phosphorous intake was maintained at 1200 mg/day. As GFR declined, fractional phosphate excretion rose reciprocally, and PTH levels increased over 20-fold. In the second group, phosphorous intake was maintained at less than 100 mg/day. As GFR fell, fractional phosphate excretion changed little, and no increment in PTH levels occurred. The data suggest that the control system regulating phosphate excretion contributes importantly to the pathogenesis of secondary hyperparathyroidism in advancing renal insufficiency.

Metabolism of bovine parathyroid hormone. Immunological and biological characteristics of fragments generated by liver perfusion.

The metabolism of bovine parathyroid hormone (PTH) by the perfused rat liver was studied. Labeled hormone, with or without cold hormone, was infused into the circulating perfusion medium containing various calcium concentrations. Pefusate samples at various time periods after the introduction of PTH into the system were chromatographed on Bio-gel P-10; radioactivity and/or immunoreactivity were measured in eluted fractions. Before the perfusion, all immuno- and radioactivity eluted in a single peak, with an apparent mol wt of 9,500 (peak I). After perfusion for 15 min, two other peaks with approximate mol wt of 7,000 (peak II) and 3,500 (peak III) were discernible. Peak I contained both NH2-terminal and COOH-terminal immunoreactivity and was biologically active at all time periods tested. The relative contribution of NH2-terminal and COOH-terminal immunoreactivity to the total immunoreactivity remained constant in this peak throughout the perfusion. In every respect, peak I had the characteristics of intact hormone. At all times, peak II consisted of only COOH-terminal immunoreactivity and was biologically inactive. At early time periods, peak III contained predominantly NH2-terminal immunoreactivity and was biologically active. With time, the relative contribution of NH2-terminal immunoreactivity decreased strikingly while that of COOH-terminal immunoreactivity increased. The three peaks identified in these experiments were analogous in size, biological activity, and immunological characteristics to those we have previously described for fractionated human hyperparathyroid serum. The rate of metabolism of PTH appeared to be regulated by the calcium concentration in the medium. At a high concentration of calcium (greater than 11 mg/100 ml), PTH metabolism was greatly retarded. At a low concentration of calcium (smaller than 5 mg/100 ml), the rate of metabolism was greatly increased. The physiological significance of our observations on the metabolism of PTH by isolated perfused rat liver is not known. However, since such metabolism results in a biologically active fragment, it is suggested that metabolism of intact hormone may be required before full biological expression is possible.

Parathyroid hormone and the hypercalcemia of immobilization.

Serial measurements of serum calcium and immunoreactive parathyroid hormone (PTH) were performed in two young patients with hypercalcemia of immobilization. Serum PTH was elevated in both patients. With mobilization, both serum PTH and serum calcium returned to normal levels and remained so during six months of follow-up. The hyperparathyroidism of immobilization is an unexplained, reversible disorder that should be treated by medical measures and aggresive attempts at early mobilization.

Acute actions of 1,25-dihydroxy-vitamin D3 in normal man: effect on calcium and parathyroid status.

The present study was undertaken to evaluate the acute effect of 1,25-dihydroxy-vitamin D3 (1,25 (OH)2D3) on serum Ca, P and immunoreactive parathyroid (iPTH) and urinary Ca, P. and cyclic AMP. In 8 normal subjects, samples were collected over intervals of 30 to 60 min during a control day and on a treatment day following oral ingestion of 1,25(OH)2D3, 2.7 microgram. For the entire group there were no significant changes in serum Ca. P, iPTH or urinary P. Urinary Ca increased significantly 7 h after administration of 1,25(OH)2D3, and urinary cAMP decreased at 12 h. In 4 patients (group A). showing an increase in serum Ca by 0;2 to 0.4 mg/dl, serum iPTH decreased in 3, and the decrease in urinary cAMP appeared sooner. Among 4 patients showing no change in serum Ca after 1,25(HO)2D3 (group B), 3 showed an increase in iPTH. These data document the early onset of action of 1,25(OH)2D3 following its administration to normal man; increments in urinary Ca provide the most sensitive index of its action. The data provide no support for the view that 1,25(OH)2D3 exerts any direct inhibitory effect on the secretion of parathyroid hormone.

d-fenfluramine treatment of binge eating disorder.

OBJECTIVE: The purpose of this study was to assess the efficacy of the appetite suppressant d-fenfluramine in the treatment of binge eating disorder. METHOD: The authors conducted an 8-week double-blind, placebo-controlled clinical trial of the drug with 28 severely obese female patients meeting full criteria for binge eating disorder. The primary outcome measure was number of binges per week, as recorded in binge diaries and reviewed weekly with the principal investigators. RESULTS: Random effects linear regression analysis showed that the rate of binge eating in the d-fenfluramine group fell three times more rapidly than that in the placebo group, a result that was both clinically and statistically significant. At 4-month follow-up the binge frequency of the d-fenfluramine group had increased to pretreatment levels and no longer differed from that of the placebo group. CONCLUSIONS: d-Fenfluramine reduced the frequency of binge eating by obese women with binge eating disorder.

Oncogenous osteomalacia. Review of the world literature of 42 cases and report of two new cases.

The clinical and biochemical data in 42 reported cases of oncogenous osteomalacia are reviewed, and data in two previously unreported cases are recorded. It is likely that the syndrome is more common than suggested by the paucity of reports and may account for a substantial fraction of nonfamilial, adult-onset "idiopathic" osteomalacia. Tumors associated with the syndrome are characteristically benign, of mesenchymal origin, highly vascular, and composed principally of giant and spindle cells. Complete excision of tumors results in cure of the osteomalacia in the majority of patients. The syndrome presumably represents an instance of humor-induced phosphaturia but supporting experimental data are scanty. Plasma levels of 1,25-dihydroxycholecalciferol are uniformly low, and treatment with this metabolite is generally very effective; however, abnormal vitamin D metabolism cannot by itself account for the syndrome.

Activation of cellular oncogenes by chemical carcinogens in Syrian hamster embryo fibroblasts.

Carcinogen-induced point mutations resulting in activation of ras oncogenes have been demonstrated in various experimental systems such as skin carcinogenesis, mammary, and liver carcinogenesis. In many cases, the data support the conclusion that these point mutations are critical changes in the initiation of these tumors. The Syrian hamster embryo (SHE) cell transformation model system has been widely used to study the multistep process of chemically induced neoplastic transformation. Recent data suggest that activation of the Ha-ras gene via point mutation is one of the crucial events in the transformation of these cells. We have now cloned the c-Ha-ras proto-oncogene from SHE cDNA-libraries, and we have performed polymerase chain reaction and direct sequencing to analyze tumor cell lines induced by different chemical carcinogens for the presence of point mutations. No changes were detectable at codons 12, 13, 59, 61, and 117 or adjacent regions in tumor cell lines induced by diethylstilbestrol, asbestos, benzo(a)pyrene, trenbolone, or aflatoxin B1. Thus, it is not known whether point mutations in the Ha-ras proto-oncogene are essential for the acquisition of the neoplastic phenotype of SHE cells. Activation of other oncogenes or inactivation of tumor suppressor genes may be responsible for the neoplastic progression of these cells. However, in SHE cells neoplastically transformed by diethylstilbestrol or trenbolone, a significant elevation of the c-Ha-ras expression was observed. Enhanced expression of c-myc was detected in SHE cells transformed by benzo(a)pyrene or trenbolone.

DNA patterns in parathyroid disease predict postoperative parathyroid hormone secretion.

Flow cytometric analysis of the nuclear deoxyribonucleic acid (DNA) content of parathyroid glands excised from patients with hypercalcemic hyperparathyroidism has identified three distinct DNA patterns. The most frequent pattern showed a high percentage of cells with tetraploid DNA, which indicated an increase in the G2 and M phase of the cell cycle. Thirty-four patients were found to have abnormal tetraploid DNA content. One patient had a normal diploid pattern, and seven were found to have an aneuploid DNA population in their excised parathyroid glands. This unexpected finding of aneuploid DNA appears to be an unique feature of these endocrine glands because they have no histologic or clinical characteristics of malignant change. All patients have remained normocalcemic and clinically well after excision of only grossly enlarged glands. Postoperative parathyroid hormone (PTH) levels were correlated in 17 patients with DNA analyses of biopsy specimens from 30 normal-sized glands which were left in situ. Seven patients with elevated PTH postoperatively had high tetraploid or aneuploid DNA in all 13 glands from which biopsy specimens had high tetraploid or aneuploid DNA in all 13 glands from which biopsy specimens had been taken. In 10 patients with normal PTH levels, six had normal diploid patterns, whereas four had high tetraploid DNA in their gland biopsy specimens. DNA content present in biopsy specimens of normal-sized, in situ glands was predictive (p less than 0.042) of parathyroid gland secretory activity. These findings suggest that the stimulus for parathyroid gland hyperfunction often affects more than a single enlarged gland and persists after clinical cure, as shown by a more rapid cell turnover in some remaining glands and continued hypersecretion of hormone.

Structure determination of Cryptococcus neoformans serotype A-variant glucuronoxylomannan by 13C-n.m.r. spectroscopy.

A series of polysaccharides was derived by physical and chemical methods from an antigenic, O-acetyl-containing, glucuronoxylomannan (GXM), isolated from the growth medium of Cryptococcus neoformans (CDC B2550) serotype A-variant having composition ratios of Man:Xyl:GlcA:OAc = 10:4:3:6. 13C-N.m.r. spectra of derivatives provided new structural evidence for GXM. Treatment of GXM with Li in ethylenediamine gave a xylomannan (XM, with Man:Xyl = 5:2). Smith degradation of XM gave a mannan (M). Ultrasonic treatment of GXM gave GXM-sonicated (GXMS). Treatment of GXM with 3-(3-dimethylaminopropyl)-1-ethylcarbodiimide.HCl and then with NaBH4 gave reduced GXMS (RGXMS), or with aq. trifluoroacetic acid gave partially acid-hydrolyzed GXMS. Periodate oxidation of GXM and NaBH4 reduction of the product gave a polyalcohol-mannan (PM). Treatment of GXMS, RGXMS, and PM with NH4OH at pH 11 gave the respective O-deacetylated analogs. Comparison among the 13C-n.m.r. spectra of GXM, the various derivatives, and reference monosaccharides allowed the following conclusions: M is (1----3)-alpha-D-mannopyranan; XM consists of the M backbone with 91% of the Xyl on nonadjacent Man residues as 2-O-beta-D-Xylp substituents and with 9% as 4-O-D-Xylp substituents on other Man residues. GXM consists of the XM structure, but with non-D-xylosylated Man residues substituted with 2-O-beta-D-GlcpA substituents and with 6-O-acetyl groups distributed approximately equally on Man residues that have other substituents and those that have none. The molecular mechanics program MM2 was used to estimate the relative energies of anomeric orientations of the typical glycosidic linkage in M. The results suggest that 6'-OH----O-2 H-bonding is significant in the minimal-energy orientation of M, with phi = -36 degrees and psi = 51 degrees, and that two other glycosidic orientations may be important in the 2-O- or 6-O-substituted derivatives of M.

Structural variability in the glucuronoxylomannan of Cryptococcus neoformans serotype A isolates determined by 13C NMR spectroscopy.

Cryptococcus neoformans, the etiologic agent of cryptococcal meningoencephalitis, produces glucuronoxylomannan (GXM) as the major capsule component. Purified GXMs obtained from eight serotype A isolates of C. neoformans were treated by ultrasonic irradiation and then O-deacetylated prior to their comprehensive chemical analysis by GLC, GLC-MS, and 13C NMR spectroscopy. The average xylose: mannose: glucuronic acid molar ratio of the eight isolates is 1.96 +/- 0.25: 3.00: 0.58 +/- 0.10. Methylation analyses and 13C NMR spectroscopy show a general structure for GXM that is comprised of a linear (1----3)-alpha-D-mannopyranan substituted with beta-D-GlcpA and with beta-D-Xylp at O-2. Variable quantities of unsubstituted (1----3)-alpha-D-Manp were observed between the eight isolates studied. In several isolates some of the (1----3)-alpha-D-Manp residues are disubstituted with beta-D-GlcpA at O-2 and with beta-D-Xylp at O-4; this type of substitution was not previously thought to occur in serotype A isolates. Heterogeneity, between isolates, in the disposition of the substituents along the mannopyranan backbone was revealed by 13C NMR spectroscopy. The eight isolates, and three isolates previously studied, were each assigned to one of four distinct groups based on the 13C NMR chemical shifts of the anomeric carbons. Six of the eleven isolates gave identical spectra (Group I). The six major anomeric resonances from Group I were assigned to specific glycosidic linkages present in GXM. The remaining five isolates gave more complex spectra that are indicative of additional linkages and comprise the remaining three groups. Three of these five isolates contain substantial amounts of linkages previously thought to be distinctive of serotypes B and C, i.e., Manp residues that are 4-O-glycosylated with beta-D-Xylp. Methylation analyses only predicted an average repeating unit, whereas 13C NMR spectroscopy demonstrated that GXM from each isolate may be categorized into four groups by the occurrence of distinct sequences of carbohydrate residues.